Analysis device for combination libraries

Abstract

An analytical device is disclosed that includes a high density of wells arrayed thereon.

Description

[Technical Field]

[0001] 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 of up to 10 million compounds. [Background technology]

[0002] Combination libraries are well-known in the literature and often utilize beads. Each of these beads contains many 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 the evaluation of the structure 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 due to the attachment of the linker connecting 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.

[0003] 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. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] While the use of cleavable linkers can help avoid structural interference problems imposed by beads and / or linkers, the scaling up of the number of individual wells on analytical instruments to accommodate larger libraries introduces yet another problem. If adjacent wells are too close together, some of the test solution in one well may spill out, contaminating the test solution in the adjacent well. Either such spill can alter the results by resulting in either a false affirmation or dilution of the reported activity of the active compound. The former can occur when the test compound in the solution is active and some of the solution "spilles" into a test well containing an inactive compound. As a result of this spill, the well containing the inactive compound now contains the active compound, leading to a false report that the active compound has activity in that well. The latter can occur when spillage from the well containing the inactive compound contaminates the well containing the active compound, reducing the concentration of the active compound so that the reported activity, when reported in a dose-dependent manner, is less than the actual activity.

[0005] This spillage problem becomes particularly real when analytical instruments have a large number of wells that are very close together. To maintain a workable size for the instrument, the well density is increased to the point where aqueous solution in one well can spill and contaminate adjacent wells. At this density, the reliability of the analytical results decreases due to the decreasing reliability of individual wells as the well density increases. This presents a difficult situation for technicians: either use an analytical instrument with wells separated by a distance that can no longer accommodate the desired well density, or tolerate spillage that reduces the reliability of the data produced during analysis.

[0006] Furthermore, each well in the analyzer contains a target, which is the intended binding site of the test compound. The target is preferably located in or near the center of the well. However, if the target is a viable cell, after deposition, the cell may migrate to the corners of the well, where cell visualization becomes more difficult. Since analytical results are often measured by cell visualization, a failure to adequately visualize the cells is a significant drawback to the analysis's ability to convey reliable information about cell activity. [Means for solving the problem]

[0007] In light of the above, it is beneficial to provide an analytical device that prevents spillage and, when appropriate, prevents the movement of the target when placed in a well.

[0008] In one embodiment, the disclosure provides an analytical apparatus including high-density wells configured to prevent partial spillage of an aqueous solution from a first well to a second well. In one embodiment, the disclosure provides an analytical apparatus that obstructs the migration of targets, such as viable cells, located within the wells. For example, obstructing target migration can reduce the risk of the target moving to locations within the well where it is difficult to reliably detect the resulting biological effects of soluble compounds absorbed into the cells.

[0009] Therefore, in one embodiment of the apparatus, an analytical apparatus (1) is provided which includes high-density wells (2) aligned above itself, Each of the aforementioned wells (2) is a) 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), b) A partition (3) separating adjacent wells (2) from each other, wherein each partition has a length of at least about 10 microns from the nearest edge of the first well (2) to the nearest edge of the second well (2'), and the second well (2') is the nearest adjacent well from the first well (2), and at least the surface portion of the partition (3) includes a hydrophobic and water-repellent layer (4) incorporated therein, including its surface or extending from its surface.

[0010] In each embodiment, the well (2) of the apparatus comprises one or more beads (6), each containing a number of copies of a single compound released to the bead in a dose-dependent manner. In each embodiment, the floor wall (8) comprises a target capture 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.

[0011] In one embodiment, one or more of the beads further include an mRNA capture component.

[0012] In another embodiment of the apparatus, an analyzer (1) is provided which includes high-density wells (2) aligned above itself. Each of the aforementioned wells (2) is a) A floor wall (8) and side wall (7) comprising one or more beads (6) and one or more targets (16) in an aqueous solution (17), wherein one or more beads (6) in each well (2) comprises numerous copies of a single compound releasedly bound to the bead (6) in a dose-dependent manner, and each of the beads (6) comprises an mRNA capture component, the floor wall (8) and side wall (7), b) A partition (3) that separates adjacent wells (2) from each other, each having a length of at least about 10 microns from the nearest edge of the first well (2) to the nearest edge of the second well (2'), wherein the second well (2') is the nearest adjacent well from the first well (2), and includes a partition (3), The floor wall (8) includes a target capture element (5) that captures the target (16) and prevents the target from moving within the well (2) after it has been placed. At least the surface portion of the partition (3) includes a hydrophobic and water-repellent layer (4) which is incorporated therein or extends upward therefrom and is substantially free of the aqueous solution.

[0013] In yet another embodiment of the apparatus, an analytical apparatus (1) is provided which includes a number of wells (2) aligned above itself, each of the wells (2) is a) A floor wall (8) and side wall (7) comprising one or more beads (6) and one or more targets (16) in an aqueous solution (17), wherein one or more beads (6) in each well (2) comprises numerous copies of a single compound releasedly bound to the bead (6) in a dose-dependent manner, and each of the beads (6) comprises an RNA capture component, the floor wall (8) and side wall (7), b) A partition (3) that separates adjacent wells (2) from each other, wherein the distance from the nearest edge of the first well (2) to the nearest edge of the second well (2') is at least about 10 microns in length, and the second well (2') is the nearest adjacent well from the first well (2), The floor wall (8) includes cell-capturing elements (5) that capture mammalian cells and prevent the movement of cells within the well (2) after the cells have been placed. At least the surface portion of the partition (3) includes a hydrophobic and water-repellent layer (4) which is incorporated therein or extends upward therefrom and is substantially free of the aqueous solution. The upper surface of the aqueous solution (17) in each well (2) is covered with a hydrophobic fluid (18).

[0014] In one preferred embodiment, the device includes at least 10 wells per square millimeter, and preferably a well density of at least about 1,000 to 10,000,000 wells per device. For example, the device may include at least 1,000 wells, or at least about 100,000 wells, or at least about 100,000 wells, or at least about 1,000,000 wells.

[0015] In other preferred embodiments, each of the partitions (3) is about 20 microns in length from the closest edge of the first well (2) to the closest edge of the second well (2'), and the second well (2') is the closest neighboring well from the first well (2). In each embodiment, the preferred range of the length of the partition (3) is at least about 10 microns to about 30 microns, and preferably about 15 microns to about 25 microns.

[0016] In one embodiment, a single well (2) contains a target or multiple copies of that target (16), optionally in the presence of an aqueous solution (17). In one embodiment, the target (16) is a mammalian cell, the aqueous solution (17) is a growth medium for that cell, and the viability of the cell in the solution is maintained. In one embodiment, the mammalian cell is a human cell.

[0017] In one embodiment, the target (16) is a mammalian cell, and the target capture element (5) includes a compound (including a polymer) that binds to or complexes with the cell to prevent movement of the cell within the well.

[0018] In one embodiment, a method for suppressing spillage in an analyzer device having a high density of wells each containing an aqueous solution is provided, the method comprising a) mm 2A step of providing a density of wells on the apparatus of at least 10 wells per unit, wherein the edge of each well is placed at least about 10 microns from the nearest edge of the nearest adjacent well, thereby aligning the wells on the apparatus to provide partitions (3) between the wells, b) The step of applying a biocompatible hydrophobic water-repellent film or layer (4) to at least a portion of the partition (3) such that the transfer of a portion of the aqueous solution in one well (2) to an adjacent well (2) is inhibited by placing the apparatus (1) or a substance containing it on it.

[0019] In one embodiment, a method is provided for preventing the movement of a target (16) placed near the center of the bottom surface of a well (2), the method comprising the step of applying a target capture element (5) in a sufficient amount to prevent the movement of the target (16).

[0020] An apparatus suitable for performing analysis for a combination library is provided. This apparatus may include an analyzer. This analyzer may include at least 10,000 wells on the top surface of the analyzer. Each of these at least 10,000 wells may include a floor and sidewalls configured to hold one or more beads and one or more targets in an aqueous solution. The analyzer may include a surface partition separating a first well of the at least 10,000 wells from a second well of at least 10,000 wells.

[0021] The distance along the top surface of the analyzer from the nearest edge of the first well to the nearest edge of the second well may be approximately 10 microns (μm) to approximately 50 μm.

[0022] The second well may be the well closest to the first well.

[0023] Each of the surface partitions may include at least a portion of a hydrophobic layer.

[0024] The hydrophobic layer may be configured to limit the spillage of the aqueous solution from the first well to the second well.

[0025] The analyzer may have a top surface area. The density of at least 10,000 wells on the top surface area is in square millimeters (mm²). 2 Each well may have at least 10 wells.

[0026] Each well may have a well diameter ranging from approximately 30 μm to approximately 250 μm. Each well may have a well depth ranging from approximately 30 μm to approximately 400 μm.

[0027] Density is mm 2 At least 10 wells per mm 2 Each well may contain approximately 400 balls.

[0028] Density is mm 2 Approximately 40 wells per unit, mm 2 Each well may contain approximately 150 balls.

[0029] The distance along the top surface from the nearest edge of the first well to the nearest edge of the second well may be approximately 10 μm to approximately 30 μm.

[0030] The apparatus may further include mammalian cells maintained in an aqueous growth medium for mammalian cells. The aqueous growth medium may be configured to maintain the viability of the mammalian cells in solution.

[0031] The aqueous growth medium may be maintained in at least one of at least 10,000 wells.

[0032] Mammalian cells can be human cells.

[0033] The target capture element may contain poly-D-lysine.

[0034] The distance along the top surface from the nearest edge of the first well to the nearest edge of the second well may be approximately 15 μm to approximately 25 μm.

[0035] The analytical apparatus may have at least approximately 100,000 wells on its upper surface.

[0036] The hydrophobic layer may contain a biocompatible hydrophobic substance selected from polyethylene, polypropylene, ethylene-propylene block copolymer, polytetrafluoroethylene, (trichloro)octadecylsilane (OTS), amorphous fluorinated polymer, and polydimethylsiloxane (PDMS).

[0037] At least one floor of at least 10,000 wells may further contain a target capture element that captures mammalian cells.

[0038] At least a portion of the floor of at least one of the at least 10,000 wells may be hydrophilic.

[0039] Drawings illustrating specific embodiments of each analytical apparatus of the present invention are provided. These apparatuses include necessary and optional components. These components in these drawings are numbered for ease of reference, and components common to multiple drawings share the same number. It should be understood that each component described herein is non-limiting and provided for illustrative purposes only. Equivalents of individual components are included within the scope of the present invention. [Brief explanation of the drawing]

[0040] [Figure 1A] This is a schematic cross-sectional view showing a part of one embodiment of the apparatus (1) of the present invention. [Figure 1B] This is a schematic cross-sectional view showing a part of one embodiment of the apparatus (1) of the present invention. [Figure 2A] This is a cross-sectional view showing a part of the apparatus (1) described in Figures 1A and 1B. [Figure 2B]This is a cross-sectional view showing a part of the apparatus (1) described in Figures 1A and 1B. [Figure 2C] This is a cross-sectional view showing some other embodiments of the apparatus (1) described herein. [Figure 3] This figure shows an arbitrary embodiment of the present invention. [Figure 4] This figure shows one procedure for forming a hydrophobic and water-repellent layer (4) on a partition (3) of an analytical apparatus described herein.

[0041] 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 and hydrophobic layer (4) that forms part of the surface separating one well from the other wells. 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), a target (16), and a solution (17). The contents of the other wells (2) are omitted for clarity only, as shown in Figure 2A.

[0042] 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 and water-repellent layer (4) extending upward from at least a portion of the partition (3).

[0043] 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). [Modes for carrying out the invention]

[0044] Apparatus and methods for performing analysis on large 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.

[0045] The technical terms used herein are for the purpose of describing only specific embodiments 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 clearly indicates otherwise.

[0046] "Optional" or "ontionally" means that the event or situation described thereafter may or may not occur, and that the description includes instances in which the event or situation may occur and instances in which it may not occur.

[0047] The term "about," when used before a numerical specification such as temperature, time, quantity, concentration, or other values, including a range, indicates an approximate value that may vary by (+) or (-) 10%, 5%, or 1%, or any partial range or lower value between them. Preferably, the term "about" means that the dose may vary by + / - 10%.

[0048] "Comprising" or "comprises" is intended to mean that the composition and method described include the elements, but do not exclude other elements.

[0049] "Consisting essentially of," when used to define a composition or method, means excluding other elements that have some essential importance to the combination for the stated purpose. Therefore, any composition that becomes essential from the elements defined herein does not exclude other substances or processes that do not substantially affect the basic and novel properties of the claimed invention.

[0050] "Consisting of" means excluding trace elements of other contaminants and anything beyond the substantial steps of the method. Embodiments defined by each of these conversion terms are within the scope of the present invention.

[0051] The term "assay device" refers to a device capable of simultaneously analyzing multiple test compounds against a target. Such a device includes a number of wells, each individual well preferably containing multiple copies of a compound that are substantially identical. The device includes a substance that emits light through it. For example, light may be shone onto the device or light may be generated from within the device. In one embodiment, the light transmitted through the device is of a wavelength and intensity such that at least some of the cleavable bonds that attach each of the multiple copies of the substantially identical compound to the bead cleave from the bead, creating a solution of that compound at a certain concentration in the well. In one embodiment, the light transmitted through the device is fluorescence generated from molecules in a particular well, which are preferably not bound to the bead. Because the fluorescence is transmitted through the device, the fluorescence thus generated is detectable outside the device.

[0052] In one embodiment, the analyzer includes more than 1,000,000 wells, and preferably up to about 10,000,000 wells. In one embodiment, the analyzer includes about 10,000 to about 10,000,000 wells, and preferably about 50,000 to about 2,000,000 wells. In one embodiment, the size of the apparatus is up to about 10,000 square millimeters.

[0053] The term "target" refers to a biological substance or other substance whose binding affinity of a test compound to the target and / or the biological effect of that binding is to be evaluated. Exemplary targets include monoclonal or polyclonal antibodies, fragments of monoclonal or polyclonal antibodies, mammalian cells, DNA, RNA, siRNA, proteins (e.g., fusion proteins, enzymes, cytokines, chemokines, etc.), viruses, and the like. In a preferred embodiment, the target is a mammalian cell, such as a human cell.

[0054] The term "target capturing element" refers to a biocompatible layer or membrane of a compound or mixture of compounds. In one embodiment, this layer or membrane binds to or complexes with the target on the bottom surface of a well with sufficient strength to prevent the target from moving within the well. In other embodiments, the target capturing element is a biocompatible layer or membrane that does not interfere with the integrity of the target in a suspension or solution. In other embodiments, the complex of the target and the target capturing element is 1 × 10⁻¹⁶ -3 Dissociation constant (K) less than μmol / μL d ) is defined by. In one embodiment, when a large number of cells are recruited into a single well, the target capture element also inhibits cell aggregation.

[0055] The term "releasably bound" means that a compound bound to a bead can be released by applying a stimulus that breaks that bond. Such bonds are sometimes referred to as "cleavable" bonds. The appropriate stimulus for releasing the compound depends on the bond used. There are many examples of such bonds and appropriate stimuli to break them in the art. Non-exclusive examples of cleavable bonds include those released by changes in pH, enzyme activity, oxidative changes, redox reactions, ultraviolet light, infrared light, ultrasound, and changes in magnetic fields. A comprehensive overview of such cleavable bonds and the corresponding stimuli required to break them is provided in Taresco et al., "Self-Responsive Prodrug Chemistries for Drug Delivery," Wiley Online Library, 2018, onlinelibrary.wiley.com / doi / full / 10.1002 / adtp.201800030.

[0056] The term "compound," which is interchangeable with "test compound," refers to a compound being evaluated for its binding affinity to a target and / or the biological effects of that binding. Such compounds are typically part of a structure-activity relationship (SAR) analysis because they are related to a specific target. Analyzing which compounds bind to a target and which do not provides meaningful data to those skilled in the art regarding the effects of structural changes in that compound. Similarly, evaluating the biological effects (or activity) of such binding provides further information to those skilled in the art regarding what structural differences alter these biological effects.

[0057] The term "substantially the same" as used in reference to compounds means that the majority of compounds on the bead are identical. In one embodiment, at least 80%, preferably at least 90%, and more preferably at least 95% of the compounds are identical. Compounds that are not identical are typically the result of incomplete reactions on the bead, where these compounds become starting materials or intermediates to the final product. Such compounds are thought to lack sufficient structure to interact meaningfully with the target.

[0058] The term "fluid" refers to a liquid or a fluid powder.

[0059] The term "releasably bound to said bead(s)(6) in a dose-dependent manner" means that the compound is bound to the bead via a cleavable linker, and since the cleavage is titrable, the amount of compound released is controllable. In one embodiment, the amount of compound released by the cleavable linker is evaluated by linking multiple copies of a companion marker, such as a fluorescent compound, bound to the same or different beads by the same cleavable linker. Upon binding to the bead, an incleavable quenching molecule arrives in close proximity to the fluorescent compound to reduce or eliminate its fluorescence. A standardized plot of the amount of fluorescent compound cleaved from the bead by a cleavage factor (e.g., ultraviolet light of a predetermined wavelength and intensity) against the fluorescence intensity is created over a set period. The ultraviolet light is equally irradiated onto the test bead with the cleavable test compound and the bead with the cleavable fluorescent compound. As evidenced by the standardized plot of fluorescence intensity, the degree of cleavage of the fluorescent compound correlates with the amount of test compound released. Thus, if the amount of the released test compound can be controlled and correlated with the amount and concentration of the test compound in the solution, the amount of solution per test well can be determined.

[0060] The term "biocompatible" refers to a substance that is compatible with each component used in the device, including but not limited to beads, targets, target capture elements, compounds, mRNA, and adopted aqueous solutions. If the target is viable cells, the biocompatible substance must maintain the cell's viability during use. Similarly, for proteins, polypeptides, antibodies, DNA, and mRNA, the biocompatible substance must preserve the functional properties of these components. Device

[0061] The ability to analyze very large combination libraries of compounds is limited by the overall size constraints of the instrument and the density of wells on the instrument. As well size decreases, the ability to accommodate more wells per square millimeter improves. However, such improvement is limited because well integrity requires the existence of the shortest possible distance between adjacent wells. For example, if wells are too close together, some of the aqueous solution in one well may spill into the other, making the evaluation of both wells questionable. Generally, the shortest distance between wells is at least about 50 microns, which ensures that spillage from one well to another is substantially reduced / prevented. However, such a separation distance is at odds with high well density.

[0062] In the apparatus described herein, the well design allows the shortest distance between wells to be as short as approximately 10 microns and approximately 5 microns, while maintaining the integrity of the wells by preventing spillage through the hydrophobic and water-repellent surfaces or protrusions between the wells. This allows for a significantly larger number of wells per square millimeter. Therefore, in each embodiment, the separation interval of the wells may be less than 50 microns, less than 40 microns, less than 30 microns, or less than 20 microns, including portions thereof, within any of the values ​​or ranges described, with each having a shortest separation distance of approximately 5 microns, or approximately 10 microns, or approximately 15 microns.

[0063] The respective diameters of the wells also control the density of the wells on the device. For example, a device having wells with a diameter of about 40 microns can tolerate a significantly higher density than a device with wells having a diameter of about 150 microns. For practical purposes, the devices described herein have a high density of wells, such as those having at least 10 wells per square millimeter of the device surface including the wells.

[0064] Finally, the device of the present invention should be sized for easy use by those skilled in the art. For example, a conventional 96-well plate is about 128 mm × 85 mm (or about 7.4 inches × 3.3 inches). These plates provide a well density of 0.00885 wells per mm 2 In contrast, the devices described herein can have up to about 400 wells per mm, and preferably, 2 at least 10 wells per mm, and more preferably, 2 from about 40 wells per mm to 2 about 150 wells per mm well density. In each embodiment, the wells have a well diameter of about 60 to 150 microns. Overall, a well density of about 200 wells per mm 2 would provide over 2,100,000 wells if sized to be compatible with a conventional 96-well plate. However, many different device sizes are possible with a preferred maximum size from not exceeding about 12 inches (300 mm - X axis) to not exceeding about 12 inches (300 mm - Y axis). The high well density devices described herein provide an exceptionally high processing capacity for combinatorial libraries. 2

[0065] ​Moving on to Figures 1A and 1B, an outline is provided showing an exemplary portion of the surface of apparatus 1 having a thickness of approximately 1 mm (100), each of the indicated wells (2) having a maximum diameter (105) of approximately 150 microns (measured along the longest axis), a well (2) depth (110) of approximately 150 microns, and a distance of at least 20 microns from the nearest edge of one well to the nearest edge of the second well, which is its nearest adjacent well.

[0066] More generally, the apparatus 1 in Figures 1A and 1B has a top and bottom thickness (100) of at least about 0.1 mm and contains a number of wells (2) on its surface. Each well (2) has a diameter (105) of about 30 to about 250 microns, and preferably about 50 to about 150 microns. Each well (2) has a depth (110) of about 30 to about 400 microns, and preferably about 150 microns. This is 2.65 × 10 when the diameter of the well is about 150 microns and the depth is about 150 microns. 6 It provides a volume in a well of cubic microns or 0.00265 milliliters.

[0067] The apparatus described herein may include, but are not limited to, many biocompatible materials, including cyclic olefin polymers (COP) commercially available from Zeon Specialty Materials, Inc. (San Jose, California, USA), cyclic olefin copolymers (COC) commercially available from many suppliers such as Polyplastics USA, Inc. ("Farmington Hills, Michigan, USA"), polyimides commercially available from many suppliers such as Putnam Plastics (Dayville, Connecticut, USA), polycarbonates commercially available from many suppliers such as Forster Corporation (Punam, Connecticut, USA), polydimethylsiloxane commercially available from Edge Embossing (Medford, Massachusetts, USA), and polymethyl methacrylate commercially available from Parchem Fine & Specialty Chemicals (New Rochelle, New York, USA).

[0068] The apparatus of the present invention is well known in the art and can be readily prepared by a thermal embossing method which includes pressing a pattern onto a polymer that has been softened by heating it to a temperature just above its glass transition temperature. A subsequent cooling step of the polymer provides high-density wells in the apparatus described herein. Alternatively, injection molding techniques can be used, which are well known in the art. Furthermore, it is possible to laser etch a solid block of biocompatible polymer to introduce a desired number of wells having appropriate size, volume, and shape, as well as a desired well density.

[0069] Figures 1A and 1B show a portion of a partially formed apparatus (1) including a number of wells (2) and partitions (3) separating the wells (2) from one another (see Figures 2A to C for enlarged views of the partitions (3)). In one embodiment, each partition (3) is located at least about 10 microns in length from a first well (2) to its nearest adjacent well (2'). This shortest distance between wells ensures well integrity so that each well (2) contains a uniform aqueous solution (without spillage) and that each well (2) contains one or more beads, the beads containing multiple copies of the same test compound bound thereto. In a preferred embodiment, the partitions (3) have lengths measured from the nearest adjacent well ranging from about 5, 10, or 20 microns, and more preferably from about 29 microns to less than 50 microns.

[0070] When creating wells (2) by thermal embossing with partitions (3) approximately 10 microns in length as described above, the thermoplastic polymer sheet is heated to a temperature slightly above the glass transition temperature as described above. Preferably, a stamp containing many circular prongs uniformly distributed on the surface at the desired density is selected. Each prong is sized to have a diameter and depth correlated with the size of the wells (2) described above. The distance between any two adjacent prongs is at least approximately 10 microns (i.e., the partitions (3) are at least approximately 10 microns thick). The stamp is sized so that the portion containing the prongs fits within the upper surface of the sheet. Sufficient force is applied to the stamp to ensure that the entire length of the prongs is pressed into the sheet. The required force depends on the degree of softness of the sheet and can be readily determined by those skilled in the art. Once the sheet has cooled, the prongs are removed to provide a sheet containing the wells (2) and partitions (3) at this point, as shown in Figures 1A and 1B.

[0071] Alternatively, the partially formed apparatus (1) shown in Figures 1A and 1B can be prepared by conventional injection molding using two mold halves: one (male mold half) having projections corresponding to the stamp projections, and the other (female mold half) forming the base portion of the apparatus. The two mold halves are placed side by side facing each other to form a cavity that takes the shape of apparatus (1) shown in Figures 1A and 1B. Injection of a monomer or reactive oligomer composition into this cavity and subsequent polymerization provides apparatus (1) at this point, including wells (2) and partitions (3) as shown in Figures 1A and 1B.

[0072] In one embodiment, after thermal embossing or mold forming, a silicon dioxide coating may be applied to the upper surface of the apparatus (1), including the bottom surface of the well (2) (i.e., the floor wall of the well (2), see Figure 2A) (8), by conventional sputtering techniques. Preferably, the thickness of the silicon dioxide layer is about 0.5 to about 100 nanometers, and more preferably about 10 to 50 nanometers. The silicon dioxide coating provides a reaction layer that binds both the hydrophobic biocompatible layer (4) and the target capture elements (5) to be formed.

[0073] Figures 2A, 2B, and 2C show different embodiments of the apparatus (1) at different stages of construction. For example, Figure 2A shows the apparatus (1) having a well (2) with a biocompatible hydrophobic water-repellent layer (4) defining the sides (7) and bottom (8), as well as the top surface of the partition (3). The first well (2) shows a target capture layer (5) and a bead (6).

[0074] Figure 2B further includes the target (16) in the aqueous solution (17) within the well (2). Figure 2C shows an alternative configuration of the biocompatible hydrophobic water-repellent layer (4) from that disclosed in Figure 2A. In Figure 2C, the water-repellent layer (4) is formed only over a portion of the partition (3), which can be formed by laser etching the water-repellent layer (4) after formation in order to shorten the length of the partition (4).

[0075] Regarding the structural specifications of the apparatus (1), after applying a silicon dioxide coating to the top surface of the apparatus (1), including the bottom surface (8) of the wells (2), each partition (3) is modified to include a biocompatible hydrophobic water-repellent layer (4) that prevents the aqueous solution (17) from spilling from one well to the other, as shown in Figure 4. This water-repellent layer (4) includes biocompatible hydrophobic water-repellent materials such as polyethylene, polypropylene, ethylene-propylene block copolymer, polytetrafluoroethylene, (trichloro)octadecylsilane (OTS), amorphous fluorine polymers (such as CYTOP®), and polydimethylsiloxane (PDMS).

[0076] The biocompatible water-repellent layer (4) is created by conventional coating techniques. For example, as shown in step 1 of the process in Figure 4, one such technique involves coating a disk (24) with a solution of a biocompatible water-repellent substance dissolved in a suitable solvent (e.g., ethanol) compatible with the apparatus. The disk (24) is rotated (not shown) to create a thin film (23) of solution about 1 to 5 microns thick. The rotation is stopped and the top surface of the apparatus (1) is placed on / in the thin film (23), as shown in step 2 of Figure 4. The apparatus (1) is removed from the disk (24) within about 1 to 5 minutes, as shown in step 3, and allowed to dry to form a water-repellent layer (4) about 1 to 5 microns thick.

[0077] In the alternative embodiment, the water-repellent biocompatible layer (4) is formed by injection molding to the desired thickness. Since the addition of the water-repellent biocompatible layer (4) is added to the depth of each well, it should be understood that the total depth of the wells mentioned above refers to the depth after the formation of the water-repellent layer (4).

[0078] The application of the target capture (layer) element (5) to the bottom of the well (2) is achieved according to steps 4 to 5 in Figure 4. In step 4, the target capture element (5) is poly-D-lysine (PDL), although it is used for illustrative purposes only. For example, a sufficient amount of PDL is dissolved in an aqueous solution to achieve a concentration of approximately 0.1 mg / mL. PDL is commercially available from many suppliers. One preferred supplier of PDL is ThermoFisher Scientific, 10010 Mesa Rim Road, San Diego, California, USA, as catalog number A389040. Other examples of target capture elements (5) include fibronectin (ThermoFisher Scientific, catalog number 33016015) and vitronectin (Sigma Aldrich, catalog number 5051).

[0079] As shown in step 4 of Figure 4, the partially formed apparatus (1) without the PDL target capture element (5) is immersed in a container containing the PDL solution. This immersion is continued for about 1 hour. As shown in step E of Figure 4, the apparatus (1) is removed and dried. The hydrophobic coating on the top surface of the apparatus (1) prevents the deposition of PDL on its surface, thereby providing the target capture element on the bottom surface (8) of the well (2) and possibly on the side walls (7) of the well (2).

[0080] The target capture element (5) is either biocompatible with the bottom surface (8) of the well (2) and adheres to the target (17) at the site of deposition to prevent the target from moving once deposited, or it is biocompatible with the target (1) when the target (1) is in solution or in suspension. Preferably, the overall properties of the target capture element (5) are hydrophilic, but hydrophobic regions are permitted. In one embodiment, the target capture element (5) is selected to adhere to the bottom surface (8) of the well (2) and the target (17) deposited thereon. The target capture element (5) includes substances such as poly(amino acids), DNA, RNA, siRNA, antibodies, antibody fragments, proteins, and polypeptides. A specific target capture element (5) is selected based on the adopted target (16), and such selections are well known to those skilled in the art. In one embodiment, the target (16) is a mammalian cell such as a human HeLa cell, and the target capture element (5) is a D-lysine polymer (PDL). Approximately 1×10 9 From approximately 1 x 10 14 A D-lysine polymer having 1 lysine residue is preferred.

[0081] When the hydrophobic biocompatible layer (4) is used in combination with the target capture element (5), the apparatus (1) described herein enables a very high well density per square millimeter, as well as the maintenance of reproducible detection of cells deposited in the wells (2) using electromagnetic energy detection means (e.g., light). The presence of the hydrophobic biocompatible layer (4) described herein prevents or eliminates spillage of aqueous solution from adjacent wells.

[0082] The presence of the target capture element (5) helps prevent problems associated with the movement of targets accumulated near the center of the bottom of well 2 or to the corners of the bottom of well 2. When such movement occurs, the reliability of the irradiation and reading of electromagnetic energy irradiated onto and recovered from target 5 decreases.

[0083] Preferably, the target capture element (5) is bound to the target (1) deposited on the surface (8) by non-covalent interactions including electrostatic, hydrophilic (e.g., hydrogen bonding), hydrophobic, and van der Waals forces. Such binding can be measured by an equilibrium dissociation constant (Kd, sometimes KD) in which smaller values ​​correlate to stronger binding interactions. In one embodiment, the target capture element (5) is bound to the target (1) with a sufficient dissociation constant to prevent the target (1) from moving within the well (2). Preferably, the binding of the target to the target capture element is about 1 × 10⁻⁶. -3 Not exceeding, and more preferably about 1 × 10 ―5 Provides a Kd that does not exceed μmol / μL.

[0084] The above process provides a method for forming an analytical apparatus (1), the apparatus comprising a number of wells (2). This method, a) A step of heating a biocompatible thermoplastic material to a temperature just above its glass transition temperature in order to soften the material, b) A step of applying a stamp to the surface of the heated material, wherein the stamp comprises a number of prongs, each prong being sized to have a diameter and depth correlated to the size of the well to be formed, and the distance between any two adjacent prongs being at least 10 microns, c) Applying sufficient pressure to the stamp to ensure the entire length of the prongs is submerged in the sheet, followed by the step of releasing the pressure to provide wells (2) having a bottom (8) and sides (7), each having a partition (3) that separates each well from an adjacent well (2), d) Optionally, a step of applying a layer of silicon dioxide to the exposed surfaces of the partition (3) and well (2), e) A step of applying a layer of biocompatible hydrophobic material (4) to the partition (3), f) The step of applying a layer of target capture elements (5) to the bottom surface of the well (2), This provides a device (1) that can prevent the aqueous solution (17) from spilling from one well (2) to an adjacent well (2), while also preventing the target accumulated in the well (2) from moving within the well (2).

[0085] In another embodiment shown in Figure 3, the outer edge (28) of the apparatus (1) extends slightly upward to allow for the addition of a layer of hydrophobic fluid (18) with a density less than that of water. This layer (18) provides additional protection against spillage and prevents contamination of the wells (2) by contaminants such as dust and pollen that could affect the test results. The hydrophobic fluid 18 is biocompatible and has a density of less than 0.99 grams per cubic centimeter at 25°C so that the fluid forms a layer over the aqueous solution. One preferred hydrophobic fluid 18 is silicone oil, which is available from many distributors such as SigmaAldrich, Inc., St. Louis, Missouri, USA. The hydrophobic fluid 18 can be applied in any way, including by a dispenser that is placed over the apparatus (1) and sprays a mist of the fluid in a manner that does not cause any spillage of the aqueous solution (17) from one well (2) to the other well (2). One means for providing a hydrophobic fluid layer (18) is provided in U.S. Patent Application No. 16-774875 (Patent Attorneys Case No. 057698-503F01US), entitled “Caps for Assay Device.” As shown in Figure 3, a method is provided for preventing spillage and evaporation, comprising the steps of providing an arbitrary wall (28) in the apparatus and placing a hydrophobic liquid (18) over a filled well (2).

[0086] The following examples are provided for illustrative purposes only and do not constitute any limitation to the claimed invention. All temperatures are in Celsius unless otherwise stated, and all conditions are at atmospheric pressure unless otherwise stated. In these examples, the following abbreviations have the following meanings: mL = milliliter mm = millimeter mm 2 = square millimeters OTS = Trichloro(octadecyl)silane PMMA = Polymethyl methacrylate rpm = revolutions per minute μL = microliter μm = micron

[0087] <Example 1: Formation of device (1)> A sheet of thermoplastic PMMA measuring 76mm (X-axis) x 50mm (Y-axis) x 1mm (Z-axis) (available from Lucite International Cassel Works, Bilingham, UK) is heated to a temperature slightly above its glass transition temperature (Tg), approximately 125°C, to soften the plastic. 2 Select a stamp containing many circular prongs uniformly arranged in four rows on the surface at a density of approximately 40 prongs per row. Each row of prongs is approximately 50 mm long and 7 mm wide.

[0088] Each prong has a diameter of approximately 150 μm and a depth of approximately 150 μm from base to end. The distance between any two adjacent prongs is approximately 20 μm. The stamp is sized so that each row of prongs fits within the upper surface of the sheet. Sufficient force is applied to the stamp to ensure that the entire length of the prongs is embedded within the upper surface of the sheet. The required force depends on the degree of softness of the sheet and can be readily determined by those skilled in the art. Once the sheet has cooled, the prongs are removed to provide a partially formed apparatus (1) with wells (2) and partitions (3), as shown in Figure 1.

[0089] A thin film of silicon dioxide (SiO2) is coated onto an apparatus (1) having wells (2) and partitions (3) by conventional sputtering techniques well known in the art. The sputtering process is continued until a silicon dioxide film with a thickness of approximately 30 nanometers is formed. The purpose of using this film is to enhance the adhesion of both the hydrophobic layer (4) and the target capture element (5) to the apparatus (1).

[0090] The next steps for preparing apparatus (1) are shown in Figure 4.

[0091] Figure 4 shows the formation of a water-repellent element (3) on the upper surface of a partially formed apparatus (1) with a silicon dioxide layer in place. Specifically, a rotatable disk (24) is placed on a spinner and a solution of OTS in ethanol at a concentration of approximately 25 micromoles is applied to it. The spinner is started and rotated at a speed of approximately 1,000 rpm. The spin is continued until the solution (23) is uniformly deposited on the disk. Typically, the spin continues for less than 1 minute and then stops, and the thickness of the solution (23) is approximately 0.1 microns to approximately 2 microns.

[0092] In step 2 of Figure 4, the upper surface of the partially formed apparatus (1) is placed in the solution (23) on the disk (24), which is stationary at this point, and is maintained there for about 5 minutes. In step 3 of Figure 4, the partially formed apparatus (1) is removed from the disk and dried to form a water-repellent layer (4) with a thickness of about 1 to 2 microns.

[0093] Figure 4, step 4 shows the formation of target capture elements (6) on the bottom surface of well (2). In Figure 4, step 4, the container (25) is filled with a poly-D-lysine solution (26) obtained from ThermoFisher Scientific, 10010 Mesa Rim Road, San Diego, California, USA, catalog number A389040. For example, dissolve enough PDL in the aqueous solution to achieve a concentration of approximately 0.1 mg / mL. Immerse the partially formed apparatus (1), without the PDL target capture elements (5), in the container containing the PDL solution (26). Continue this immersion for approximately 1 hour. Remove the apparatus (1) and dry it as shown in Figure 4, step 5. The hydrophobic coating on the top surface of apparatus (1) prevents the deposition of PDL on its surface, thereby providing target capture elements (4) on the bottom surface (8) of well (2) and possibly on the side walls (7) of well (2).

[0094] The above example is provided for illustrative purposes only and is not limiting. Other techniques may be used to form apparatus (1).

Claims

1. 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.

2. The apparatus includes a well density of 10 to 400 wells per square millimeter. The apparatus according to claim 1.

3. The aforementioned target is maintained in an aqueous solution. The apparatus according to claim 1.

4. 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.

5. The mammalian cells mentioned above are human cells. The apparatus according to claim 4.

6. The aforementioned target capture element contains poly-D-lysine. The apparatus according to claim 5.

7. 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.

8. The apparatus has a well density of 10 to 400 per square millimeter. The apparatus according to claim 7.

9. The aforementioned target is maintained in an aqueous solution. The apparatus according to claim 7.

10. 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.

11. The mammalian cells mentioned above are human cells. The apparatus according to claim 10.

12. The aforementioned target capture element contains poly-D-lysine. The apparatus according to claim 11.

13. The bead further comprises an mRNA capture component. The apparatus according to claim 1.

14. The apparatus includes a well density of at least 10 wells per square millimeter. The apparatus according to claim 1.

15. The apparatus has a well density of 10 to 400 wells per square millimeter. The apparatus according to claim 14.

16. The aforementioned target is maintained in an aqueous solution. The apparatus according to claim 13.

17. 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.

18. The mammalian cells mentioned above are human cells. The apparatus according to claim 17.

19. The aforementioned target capture element contains poly-D-lysine. The apparatus according to claim 18.

20. 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 density of the wells on the aforementioned apparatus is mm ２ The steps include aligning the wells on the apparatus such that each well has at least 10 wells, 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), including method.

21. The bottom of the well further contains a sufficient amount of target-capturing elements to inhibit the movement of the target. The method according to claim 20.

22. The method further includes the step of introducing one or more beads (6) each containing multiple copies of a single compound that are releasably bound to one or more beads, The compound can be released in a dose-dependent manner. Each of the one or more beads (6) includes an RNA capture component. The method according to claim 20.

23. The further step includes introducing a target into each of the aforementioned wells, The method according to claim 20.

24. A method for forming an analytical apparatus (1), wherein the apparatus includes a plurality of wells (2), and this method is To soften the material, the biocompatible thermoplastic material is heated to just above its glass transition temperature. A step of applying a stamp to the heated surface of the material, wherein the stamp comprises a plurality of prongs, each prong having a diameter and depth correlated to the size of the well (2) to be formed, and the distance between any two adjacent prongs is at least 10 microns and less than 50 microns; The steps include applying sufficient pressure to the stamp to ensure the entire length of the prongs is submerged in the sheet, and then removing it to provide wells (2) having partitions (3) that separate each well from adjacent wells (2), and having bottom surfaces (8) and sides (7), Optionally, the step of applying a layer of silicon dioxide to the exposed surfaces of the partition (3) and the well (2), The steps include applying a layer of biocompatible hydrophobic material (4) to the partition (3), The steps include providing an extending wall (28) to the outer edge of the analytical device (1), The steps include applying an additional hydrophobic fluid layer (18) inside the extending wall (28) over the well (2) and the biocompatible hydrophobic material (4), wherein the additional hydrophobic fluid layer (18) is configured to prevent contamination of the well (2) by contaminants, The steps include applying a layer of target-capturing elements (5) to the floor wall (8) or side wall (7) of the well (2) to capture targets (16) containing cells and to suppress the aggregation of the targets (16), Includes, This provides a device (1) that can prevent the aqueous solution (17) from spilling from one well (2) to an adjacent well (2), while also preventing the target accumulated in the well (2) from moving within the well (2). method.

25. An apparatus suitable for performing analysis for combinatorial libraries, The aforementioned device is An analytical apparatus comprising at least 10,000 wells on the upper surface of the analytical apparatus, wherein each of the at least 10,000 wells comprises a floor and side walls configured to hold one or more beads and one or more targets in an aqueous solution, A surface partition that separates the first well of the at least 10,000 wells from the second well of the at least 10,000 wells, Equipped with, The distance along the upper surface of the analyzer from the nearest edge of the first well to the nearest edge of the second well is between 10 microns (μm) and 50 μm. The second well is the adjacent well closest to the first well. Each of the aforementioned surface partitions includes at least a portion of a hydrophobic layer, The hydrophobic layer is configured to limit the spillage of the aqueous solution from the first well to the second well. The analytical apparatus further comprises one or more extending walls and an additional hydrophobic fluid layer disposed above the well and the hydrophobic layer, wherein the additional hydrophobic fluid layer is located within the one or more extending walls and is configured to prevent contamination of the well by contaminants. The floor wall (8) or side wall (7) of the well includes a target capture element (5) that captures a target (16) containing cells, inhibits the movement of the target within the well after the target is placed in the well, and suppresses the aggregation of the target. The analytical apparatus has an upper surface region, and the density of the at least 10,000 wells on the upper surface region is in square millimeters (mm²). ２ Each well has at least 10 wells. Each of the aforementioned wells has a well diameter of 30 μm to 250 μm and a well depth of 30 μm to 400 μm. Device.

26. The density of the rose is mm ２ At least 10 wells per mm ２ Each well contains 400 balls. The apparatus according to claim 25.

27. The density of the rose is mm ２ From 40 wells per unit, mm ２ Each well contains 150 units. The apparatus according to claim 26.

28. The distance along the upper surface from the nearest edge of the first well to the nearest edge of the second well is between 10 μm and 30 μm. The apparatus according to claim 25.

29. The apparatus includes mammalian cells maintained in an aqueous growth medium for mammalian cells. The aqueous growth medium is configured to maintain the viability of the mammalian cells in the solution. The aqueous growth medium is maintained in at least one of the at least 10,000 wells. The apparatus according to claim 25.

30. The mammalian cells mentioned above are human cells. The apparatus according to claim 29.

31. Furthermore, it has a target capture element containing poly-D-lysine, The apparatus according to claim 29.

32. The distance along the upper surface from the nearest edge of the first well to the nearest edge of the second well is 15 μm to 25 μm. The apparatus according to claim 28.

33. The analytical apparatus has at least 100,000 wells on its upper surface. The apparatus according to claim 25.

34. The hydrophobic layer comprises a biocompatible hydrophobic material selected from polyethylene, polypropylene, ethylene-propylene block copolymer, polytetrafluoroethylene, (trichloro)octadecylsilane (OTS), amorphous fluoropolymer, and polydimethylsiloxane (PDMS). The apparatus according to claim 25.

35. The floor of at least one of the at least 10,000 wells further comprises a target capture element for capturing the mammalian cells. The apparatus according to claim 29.

36. At least a portion of the floor of at least one of the at least 10,000 wells is hydrophilic. The apparatus according to claim 25.

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