Transfer dispenser for assay apparatus with bead size exclusion function

Abstract

A transfer dispenser for an assay device is disclosed. The dispenser allows for the transfer of a single assay component to a single well of the assay device, thereby ensuring that the assay performed in each well contains only a single component. The dispenser includes multiple shafts, each shaft having a variable cross-sectional width and capable of releasably accommodating a single assay component having a width greater than a minimum cross-sectional width in the shaft. Each dispenser is mounted on the assay device such that each shaft is aligned with a single well on the assay device, and when a single assay component is released from the dispenser, only a single assay component is captured in a single well.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 63 / 197,972, filed Jun. 7, 2021, and U.S. Provisional Application No. 63 / 273,389, filed Oct. 29, 2021, the entire contents of which are incorporated herein by reference.

[0002] [Technical Field] The present disclosure describes a transfer dispenser for an assay device. The dispenser enables feeding a single assay component into a single well within the assay device for each use. Thereby, when the assay being performed requires the presence of a single unit of the above components, it is ensured that each assay performed in each well contains only such a single unit.

Background Art

[0003] Combinatorial libraries are well known in the literature and often utilize beads. Each bead contains multiple copies of a single compound attached to the bead by a linker. Additionally, beads typically contain a reporting element such as DNA that enables 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 the assay. Therefore, the biological data generated by the assay may be compromised due to the potential that the bound compound cannot effectively bind to the selected target. This can be due to physical interference arising from the bead or steric interference due to the binding of the linker connecting the compound to the bead. Regarding the latter, this binding can inhibit the ability of other potent compounds to bind appropriately to the target, resulting in assay results that may be weaker than the actual potency of the compound. Further, when the target is a cell and penetration of the compound into the cell is required as part of the assay, the likelihood that the compound remaining bound to the bead will penetrate the cell is low.

[0004] One option to address this problem involves the use of a cleavable linker that releases the compound from the beads by cleaving under appropriate stimulation (e.g., light). The compound, upon entering a solution such as a test well, freely orients itself to exert maximum potency in the assay. Furthermore, the release of these compounds can be controlled in such a manner that the amount of compound released is controlled to provide significant dose-dependent data. See, for example, Patent Document 1 (U.S. Patent Application Publication No. 2019 / 0358629, or U.S. Patent No. 10828643). The entire contents of Patent Document 1 are incorporated herein by reference.

[0005] A typical combinatorial library uses thousands of beads, each containing multiple copies of the same test compound. Such beads can be prepared by the well-known split / pool synthesis method. In some cases, the identification of a compound on a bead is recorded by a reporter molecule, such as DNA. In other cases, the identification of each reaction step performed on each bead is recorded by the addition of a DNA segment corresponding to that step, thereby generating a DNA strand unique to each compound. Typically, each well contains a single bead and other assay components, such as a single mammalian cell. If a given well in the assay apparatus shows a positive "hit" (active compound), the DNA is recovered, amplified, and sequenced. The resulting sequence is a collection of the specific reaction steps used to synthesize the compound, allowing synthetic chemists to confirm the structure of the active compound.

[0006] One option for increasing the amount of information generated by an assay is to increase the number of wells in the assay instrument (e.g., a high-throughput instrument). Generally, assay instruments with tens or hundreds of wells, in contrast to those with tens or hundreds of wells, provide more information about which structures provide activity for a particular target.

[0007] Furthermore, to accommodate aqueous solutions and other assay components, the size of the wells must be much larger than the assay components themselves, such as beads. Therefore, adding only a single small bead to a single well becomes a significant challenge. If, by chance, two or more beads containing different compounds are added to a single well, the ability to assess which compound is active (or if both are active) becomes problematic at best. When assay instruments contain thousands or millions of individual wells, the ability to add only one bead to each well becomes a major challenge. If the assay requires the addition of two different components (e.g., a single bead and a single cell in a single well), the method of adding a single assay component to each well becomes even more complex.

[0008] Furthermore, commercially available beads do not have a uniform size. Rather, these beads usually exhibit a Gaussian curve (bell curve), and the reported size of these beads is the average of that curve. This means that a group of beads reported to have a diameter of 20 microns may contain a subset of beads with a diameter significantly smaller than 20 microns. The volume of a sphere is given by the formula "4 / 3 × π × R". 3 Because this is based on the principle that a bead with a radius of 70% of the average radius will occupy only 34% of the space of an average-sized bead. Therefore, two small beads may occupy the same cavity, which can limit the value of the transfer device.

[0009] To date, a dispenser with multiple cavities has been described in Patent Document 2 (U.S. Patent No. 11027272), the entirety of which is incorporated herein by reference. Patent Document 2 discloses that one method to avoid the above problem is to exclude small beads from a group of beads. In other words, the Gaussian curve can be cut to a size such that small beads are removed. This can be done, for example, by size exclusion techniques. However, this requires the beads to be further processed before use, and in some cases, the small beads may become unsuitable for use.

[0010] Therefore, the need remains to provide a dispenser for adding a single assay component to a single well of a high-throughput assay instrument. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] U.S. Patent Application Publication No. 2019 / 0358629 (U.S. Patent No. 10828643) [Patent Document 2] U.S. Patent No. 11027272 [Overview of the project]

[0012] One or more of the following features may be present in any feasible combination.

[0013] In one embodiment, a dispenser 1 is provided, comprising a top surface 2 and a bottom surface 3, and a plurality of shafts 4 extending through the dispenser 1, each of which has a first opening (upper opening) 5 and a second opening (bottom opening) 6 smaller than the first opening 5. In some embodiments, the second opening 6 is preferably located at or near the bottom of the dispenser 1. However, as shown in Figures 5-7 and 11-12, the second opening 6 may be located away from the dispenser 1 or the tip (e.g., the bottom) of the shaft 4, or it may be located inside the shaft 4. The first opening 5 is configured to allow assay components to be placed inside or on the shaft 4, while the second opening 6 of the shaft 4 is configured to allow smaller assay components of a certain diameter or less to pass through the shaft 4 and exit the dispenser 1. As intended, the shaft 4 holds / captures only a single assay component, such as a bead 11, in a reversible, releaseable, or transferable manner. This is because smaller assay components will pass through the shaft 4, while larger assay components with a width or diameter exceeding the width or diameter of the shaft 4 will not fit onto or inside the shaft 4.

[0014] In one embodiment, the dispenser 1 is configured to be attached (fitted, coupled, engaged) to an assay apparatus 20 having a plurality of wells 21, and when attached, each shaft 4 of the dispenser 1 is aligned with a single well 21 of the assay apparatus 20. When dispensed, the assay components move from the dispenser 1 into the assay apparatus 20, and a single assay component is captured in a single well 21.

[0015] In one embodiment, the shaft 4 of the dispenser 1 is configured to hold only a single assay component 10. The assay component 10 may be a bead 11 containing multiple identical compounds reversibly linked by a cleavable linker. The bead 11 may optionally also include a DNA reporter that records either the structure of the test compound (test compound) bound to the bead 11, or the synthesis steps used to prepare the test compound.

[0016] In one embodiment, the assay component 10 includes mammalian cells, such as human cells, which are essential for the assay to be performed.

[0017] In one embodiment, a system for dispensing beads (e.g., beads 11) into an assay apparatus (e.g., assay apparatus 20) comprises an assay apparatus having a plurality of wells (e.g., wells 21) and a dispenser (e.g., dispenser 1) having a plurality of shafts (e.g., shaft 4). Each of the shafts has a variable (non-constant) cross-sectional width and is capable of releasing a single assay component having a width greater than the smallest cross-sectional width in the shaft. The dispenser is fitted (mounted) onto the assay apparatus such that each shaft is aligned with a single well on the assay apparatus, and when the single assay component is released from the dispenser, only the single assay component is captured in the single well.

[0018] In one embodiment, the first shaft among the plurality of shafts has a frustoconical or hourglass-shaped contour.

[0019] In one embodiment, the first shaft among the plurality of shafts has a circular cross-section, and the cross-sectional width of the first shaft is the diameter of the circular cross-section.

[0020] In one embodiment, the first shaft among the plurality of shafts includes a first portion whose width gradually decreases in the height direction of the first shaft, and a second portion whose width gradually increases in the height direction of the first shaft.

[0021] In one embodiment, the first shaft among the plurality of shafts includes a first portion having a first width and a second portion having a second width.

[0022] In one embodiment, the first shaft among the plurality of shafts has a portion where the reduction rate of the cross-sectional width gradually decreases in the height direction of the first shaft.

[0023] In one embodiment, the first shaft among the plurality of shafts has a portion where the increase rate of the cross-sectional width gradually decreases in the height direction of the first shaft.

[0024] In one embodiment, the system further includes a channel (flow path) (e.g., channel 17) disposed under the shaft, and the channel has a height greater than the minimum cross-sectional width of the shaft. The channel is configured to collect one or more second assay components having a second width smaller than the minimum cross-sectional width of the shaft through which the assay component passes, and to send the second assay components to a second dispenser including a plurality of second shafts.

[0025] In one embodiment, the system further includes the second dispenser, each of the plurality of second shafts has a second variable cross-sectional width, and the minimum second cross-sectional width of each of the plurality of second shafts is smaller than the minimum cross-sectional width of the shaft. Each of the plurality of second shafts is configured to releasably accommodate a single second assay component having a second width greater than the minimum second cross-sectional width within the second shaft.

[0026] In one embodiment, the dispenser includes an outlet through which one or more third assay components are dispensed, and the third assay component has a third width that exceeds the opening width of the corresponding shaft opening.

[0027] In one embodiment, the single assay component is beads or cells.

[0028] In one embodiment, the dispenser includes a plurality of shafts. Each shaft has a variable cross-sectional width and is configured to releasably accommodate a single assay component having a width greater than the minimum cross-sectional width within the shaft. The dispenser is fitted (attached) onto or above the assay device such that each shaft is aligned with a single well on the assay device in such a manner that only the single assay component is retained within a single well when the single assay component is dispensed from the dispenser.

[0029] In one embodiment, a first shaft of the plurality of shafts has a frustum-shaped or hourglass-shaped profile.

[0030] In one embodiment, a first shaft of the plurality of shafts includes a first portion in which the width gradually decreases in the height direction of the first shaft and a second portion in which the width gradually increases in the height direction of the first shaft.

[0031] In one embodiment, a first shaft of the plurality of shafts has a circular cross-section, and the cross-sectional width of the first shaft is the diameter of the circular cross-section.

[0032] In one embodiment, a first shaft of the plurality of shafts includes a first portion having a first width and a second portion having a second width.

[0033] In one embodiment, the first shaft among the plurality of shafts has a portion in which the cross-sectional width decreases at a rate that gradually decreases in the height direction of the first shaft.

[0034] In one embodiment, the dispenser further comprises a channel located beneath the plurality of shafts, the channel having a height greater than or equal to the minimum cross-sectional width of the plurality of shafts. The channel is configured to collect one or more second assay components having a second width smaller than the minimum cross-sectional width of the shaft through which the assay components pass, and to dispense the second assay components to a second dispenser comprising the plurality of second shafts.

[0035] In one embodiment, the dispenser is provided with an outlet from which one or more third assay components are dispensed, and the third assay component has a third width that exceeds the opening width of the corresponding opening of the shaft.

[0036] In one embodiment, the single assay component is a bead or a cell.

[0037] The above and other functions of the subject matter disclosed herein can be better understood by referring to the following drawings, detailed description, and claims. [Brief explanation of the drawing]

[0038] The following detailed explanation and attached drawings will make it easier to understand the above features and other characteristics.

[0039] [Figure 1] A top view of dispenser 1 is shown. Dispenser 1 has a top surface 2, a bottom surface 3, and a shaft 4 that penetrates dispenser 1. One shaft 4 of an exemplary embodiment is shown in three dimensions. [Figure 2A]A shaft 4 sized to fit a single assay component is shown. In the illustrated example, the assay component is a substantially spherical bead 11 according to an exemplary embodiment. [Figure 2B] The insertion of a bead 11 into a shaft 4 according to an exemplary embodiment is shown. In this embodiment, the bead 11 is caught inside the shaft 4 in a portion of the shaft 4 where the width, i.e., diameter, is narrowed so as to prevent further passage of the bead 11. [Figure 2C] This demonstrates that, in order to capture the bead 11 according to an exemplary embodiment, it is sufficient for the bead 11 to be partially inserted into the shaft 4. [Figure 2] This indicates that small beads 12 having a width or diameter smaller than the narrowest part of the shaft 4 are removed. In this case, the small beads 12 exit through the second opening 6 at the bottom of the shaft 4. [Figure 3A] The diagram shows the filling of an empty dispenser 1 with assay components. The illustrated sealing cap 30, which has an inlet 31 and an outlet 32, is used in conjunction with a funnel-shaped cone 40 to deliver beads 11 into the shaft 4 of the dispenser 1 according to an exemplary embodiment. [Figure 3B] The image shows the filling of an empty dispenser 1 with assay components, particularly the beads 11 delivered into the shaft 4 of dispenser 1 according to an exemplary embodiment. Excess beads that do not fit into the shaft 4 exit through the sealing cap 30 and are recycled to another dispenser. For example, beads 11 exceeding the width of the first opening 5 may be guided through the exit 32 along the positive x-axis. Excess beads from another dispenser may be analyzed and / or re-guided to a shaft of a different size than shaft 4. [Figure 3C] This shows that small beads 12 that are not held within shaft 4 are sent from shaft 4 to channel 17. The small beads 12 may be collected individually or sent to a smaller shaft 54 ​​that can hold some or all of the small beads 12. Figure 3C shows a downward-sloping channel 17 through which the small beads pass. [Figure 3D] This shows that the small beads 12, which are not held within the shaft 4, are fed from the shaft 4 to the channel 17. Figure 3D shows the gently sloping, relatively flat channel 17 through which the small beads pass. [Figure 3E] This describes a size exclusion mechanism for obtaining beads with a specific width range in two stages, utilizing valve opening and closing control. In the first stage, beads with a width greater than a second threshold are excluded or removed, and in the second stage, beads with a width smaller than the first threshold are excluded or removed. [Figure 3F] This describes a size exclusion mechanism for obtaining beads with a specific width range in two stages. In the first stage, beads with a width greater than a second threshold are excluded or removed, and in the second stage, beads with a width smaller than the first threshold are excluded or removed. [Figure 3G] This describes a size exclusion mechanism for obtaining beads with a specific width range in two stages. In the first stage, beads with a width greater than a second threshold are excluded or removed, and in the second stage, beads with a width smaller than the first threshold are excluded or removed. This results in beads with a width between the first and second thresholds. [Figure 4A] The diagram shows the dispenser 1 integrated with or attached to the assay apparatus 20 in order to deliver the beads 11 from the shaft 4 to the wells 21 of the assay apparatus 20. An optional locking mechanism 23 facilitates the alignment of the dispenser 1 with respect to the assay apparatus 20. The dispenser 1 and the assay apparatus 20 do not need to be tightly pressed against each other, as long as they are locked in place. According to the exemplary embodiment, any gap 22 may exist, as long as it is smaller (narrower) than the width of the beads 11 or other assay components. [Figure 4B] This shows an inverted assembly structure of Figure 4A, as well as the delivery of beads 11 or other assay components from dispenser 1 to well 21 of assay apparatus 20, according to an exemplary embodiment. [Figure 5]An alternative embodiment is shown in which the narrowest part of the shaft 4 is located inside the hourglass-shaped contour of the shaft 4. In the example in Figure 5, the rate at which the width or diameter of the shaft 4 decreases in the height direction is constant. [Figure 6] Another example shows a shaft 4 with an hourglass-shaped contour. In the example in Figure 6, the narrowest part of the shaft 4 in terms of width or diameter is not located at the end of the shaft 4. In the example in Figure 6, the rate at which the width or diameter of the shaft 4 decreases in the height direction of the shaft 4 gradually decreases. [Figure 7] Another example shows a shaft 4 with an hourglass-shaped contour. In the example in Figure 7, the narrowest part of the shaft 4 in terms of width or diameter is not located at the end of the shaft 4. In the example in Figure 7, the rate at which the width or diameter of the shaft 4 decreases gradually increases in the height direction of the shaft 4. [Figure 8] The diagram shows a shaft 4 having a concave contour. [Figure 9] The diagram shows a shaft 4 having a convex contour. [Figure 10] The diagram shows a shaft 4 comprising multiple regions (parts or sections) each having a constant or nearly constant different cross-sectional width. In Figure 10, the shaft 4 is shown to include two regions that are different from each other. [Figure 11] The diagram shows a shaft 4 comprising multiple regions (parts or sections) each having a constant or nearly constant different cross-sectional width. In Figure 10, the shaft 4 is shown to include three distinct regions. [Figure 12] The diagram shows a shaft 4 having an hourglass-shaped outline and one or more inflection points. [Figure 13A] The diagram shows a shaft 4 having a zigzag contour, which alternately includes regions where the cross-sectional width changes and regions where the cross-sectional width is constant or approximately constant. [Figure 13B] The shaft in Figure 13A is shown together with the beads. [Figure 13C] The shaft in Figure 13A is shown with beads of a different size. [Figure 13D]The shaft in Figure 13A is shown with beads of a different size.

[0040] It should be noted that the drawings are not necessarily to scale. The drawings are intended to show only typical embodiments of the subject matter disclosed herein and should not be considered to limit the scope of this disclosure. Those skilled in the art will understand that the structures, systems, apparatus, and methods specifically described herein and shown in the accompanying drawings are non-limiting and exemplary embodiments, and that the scope of the invention is defined solely by the claims. [Modes for carrying out the invention]

[0041] Dispensers for loading assay components into an assay apparatus 20 capable of assaying a library of test compounds (test compounds) produced by combinatorial chemistry techniques, as shown in Figures 4A and 4B, are disclosed. However, before describing these embodiments in more detail, the following terms are first defined. Unless otherwise defined, terms used herein have their generally accepted scientific meanings.

[0042] For ease of reference, the numerous devices and figures used herein are summarized below.

[0043] [Dispenser] As shown in Figures 2A, 2B, 2C, 2D, 3A, and 5, the dispenser 1 delivers assay components, such as beads 11, to the assay apparatus 20. In this specification, beads 11 may be referred to in the singular or plural form. The dispenser 1 comprises a top surface 2, a bottom surface 3, and one or more shafts (channels, recesses, cavities) 4 that penetrate the dispenser 1. Figure 1 is an enlarged view showing the dispenser 1 with one shaft 4 according to one embodiment. The shaft 4 may have a variable (non-constant) cross-sectional width, i.e., diameter. In some embodiments, the shaft 4 may have a circular cross-section, and the cross-sectional width may be the diameter. In an example where the shaft 4 has a circular cross-section, the diameter 7 of the shaft 4 may be narrower at the second opening 6 compared to the first opening 5, as shown in Figure 1. In the illustrated embodiment, the shape of the shaft 4 is a substantially frustoconical shape with the base of the cone removed (note that the drawings are not necessarily to scale). In Figures 3A and 3B, the depth of the shaft 4, i.e., the height h1, indicates how far the shaft 4 extends along the y-axis. On the other hand, the cross-sectional width of the shaft 4, i.e., the diameter, can be measured along multiple different xz planes. In other embodiments, the shaft 4 may have an elliptical cross-section, and the cross-sectional width of the shaft 4 may be along the minor axis.

[0044] As will be explained later in Figures 5 to 12, the shaft 4 should not be interpreted as being limited to a frustoconical shape. Furthermore, the terms “width” or “section width” may refer to either the minor axis or the major axis, depending on the context and / or which entity (e.g., shaft 4 or bead 11) is being referred to. For example, a statement that the width or section width of shaft 4 is greater than the width or section width of bead 11 may be interpreted as meaning that the minor axis of shaft 4 is greater than the major axis of bead 11.

[0045] [beads] The beads 11 are preferably approximately spherical, and each bead contains multiple copies of the same unique compound compared to other beads. If the beads 11 are spherical, their diameter and height are identical. If the beads 11 are not spherical or have different dimensions along multiple different axes (e.g., major axis, minor axis, height), the beads 11 are captured or held within the shaft 4 as long as the first dimension of the bead 11 exceeds the minor axis of the shaft 4 and the second dimension of the bead 11 exceeds the major axis of the shaft 4. For example, as shown in Figures 2A-2C, 3A, 5, and 11, the beads 11 are an example of assay components.

[0046] [Assay device] As shown in Figures 4A to 4B, the assay apparatus 20 is a high-throughput assay apparatus that includes multiple wells 21 in which the assay is performed using multiple copies of a single test compound.

[0047] [Sealing cap] As shown in Figures 3A and 3B, the sealing cap 30 is a cap sized to fit onto the dispenser 1. The sealing cap 30 includes an inlet port (intake port) 31 as an inlet. The inlet port (intake port) 31 delivers the beads 11 to the shafts 4 of the dispenser 1, thereby bringing a single bead 11 to each shaft 4. The sealing cap 30 may have an outlet port (outlet) 32 on the opposite side of the inlet port (intake port) 31 to collect any beads 11 that were not captured by the shafts 4. Such beads 11 are fed into the outlet 32 ​​along the positive x-axis. The sealing cap 30 allows the beads 11 to flow into the shafts 4. Once the beads 11 are captured (deposited) in the shafts 4, the sealing cap 30 may be removed. Furthermore, any excess beads 11 that exit through the outlet 32 ​​or a second opening (bottom opening) 6 of the shafts 4 may be collected for further use. In one embodiment, as shown in Figures 3C to 3D, one or more small beads 12, each having a width smaller than the minimum width within the shaft 4, may flow through the shaft 4 into the channel (lane) 17 below the shaft 4.

[0048] [cell] The cells are mammalian cells, such as mouse cells, pig cells, or primate cells (including human cells). The cells may be used in the assay apparatus 20 to evaluate the biological activity of the test compound.

[0049] The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the invention. Where used herein, the singular form includes the plural form unless otherwise specified in the context.

[0050] "Optional" or "optional" means that the event or situation described afterward may or may not occur, and that the description includes both cases in which the event or situation occurs and cases in which it does not occur.

[0051] The term "approximately" is used before a numerical specification that includes a range, such as temperature, time, quantity, or concentration, to indicate an approximate value that may vary by 10%, 5%, or 1% before or after the specified value, or any partial range or value that falls within these ranges.

[0052] For example, the term "approximately," when used in reference to a quantity, means that the quantity may vary by up to 20%. The diameter of the beads may be interpreted as being 20% ​​or 10% above or below the stated average diameter.

[0053] "To include," "to possess," or "to contain" is intended to mean that the composition and method include the listed elements but does not exclude other elements.

[0054] When used to define compositions and methods, "essentially consisting of" means excluding other elements that are essentially important to the combination for the purposes described. Thus, compositions essentially consisting of the elements defined herein do not exclude other materials or steps that do not substantially affect the basic and novel properties of the claimed invention.

[0055] "Consists of" means excluding elements of other components in trace amounts and substantial method steps. Embodiments defined by each of these terms are within the scope of the present invention.

[0056] The term "assay apparatus" refers to an apparatus capable of simultaneously assaying multiple test compounds against a target within a single well. The assay apparatus comprises multiple wells, each preferably containing assay components (assay components, assay parts), such as beads, that provide multiple copies of substantially the same compound. The assay apparatus may have a large number of wells, for example, up to 2,000,000 or more. In one embodiment, the number of wells ranges from 5,000 to about 2,000,000. In one embodiment, the well density on the assay apparatus is at least 10 wells per square millimeter, and the number of wells is at least about 50,000.

[0057] The term "assay component" refers to a micron-sized, micron-shaped component used when performing a particular assay. In one embodiment, the assay component is a bead. In another embodiment, the assay component is a mammalian cell. In yet another embodiment, the assay component includes both beads and cells.

[0058] The term "beads" refers to beads 11, which are well known in the art for use in combinatorial chemistry. In one embodiment, the surface of the beads 11 contains multiple identical test compounds bound together via cleavable linkers. The beads 11 may also contain DNA barcodes that record the structure of the test compounds or the synthesis steps used to synthesize the compounds, and / or mRNA capture components that may be combined with the DNA barcodes. These DNA barcodes are bound to the beads 11 by either cleavable or non-cleavable linkers. If the barcodes are bound via cleavable linkers, it is preferable that the cleavable linkers used with the DNA barcodes are cleaved by a mechanism different from the mechanism required to release the test compounds from the beads.

[0059] In another embodiment, the beads 11 contain multiple copies of the same reporter molecule. An example of a reporter molecule is a fluorescent molecule bound to the beads 11 via a cleavable linker. Preferably, the reporter molecule is bound using the same cleavable linker used to bind the test compound to the beads 11. When used in this manner, the beads 11 may also contain a quencher molecule (not shown) bound in close proximity to the fluorescent molecule on the beads 11 to attenuate the resulting fluorescence. Typically, the quencher molecule is bound to the beads by a non-cleavable bond or by a cleavable bond that is cleaved by a mechanism different from the cleavable linker used to bind the fluorescent molecule to the beads.

[0060] Alternatively, the quencher is bonded to the beads by the same linker used to bond the test compound to the beads. In this embodiment, the fluorescent compound is bonded to the linker by a non-cleaving bond, or by a cleavable bond that is cleaved by a mechanism different from that of the cleavable linker used to bond the quencher to the beads.

[0061] During an assay, it can be essential to know the extent to which the test compound is released from the beads 11 in response to a stimulus that cleaves a cleavable bond. When the beads 11 are used with a reporter molecule, this can be determined by measuring the change in fluorescence generated by cleaving the fluorescent compound from the quencher against a standard curve.

[0062] For example, if a reporter molecule and a test compound are bound to beads by the same cleavable linker, the release of the test compound due to a stimulus that cleaves the cleavable linker will also release the reporter molecule in the same quantifiable manner. In this way, the reporter molecule and the quencher are separated, and the resulting change in fluorescence correlates with the amount of test compound released. See, for example, Patent Document 1 (U.S. Patent Application Publication No. 2019 / 0358629, or U.S. Patent No. 10828643). The entire contents of Patent Document 1 are incorporated herein by reference.

[0063] Alternatively, if the quencher molecule and the test compound are bound to the beads by the same cleavable linker, the release of the test compound due to a stimulus that breaks the cleavable linker will also release the quencher in the same quantifiable manner. In this way, the reporter molecule and the quencher are separated, and the resulting change in fluorescence correlates with the amount of test compound released.

[0064] In another alternative embodiment, the quencher molecule, test compound, and reporter compound are all bound to the beads 11 by the same cleavable linker, and all of these are cleaved from the beads 11 by the applied stimulus. In this way, the reporter molecule and the quencher molecule are separated from each other in the aqueous environment of the assay. This results in a change in fluorescence that correlates with the amount of test compound released.

[0065] Beads 11 are typically in polymer form. Numerous types of beads 11 are commercially available, including, for example, amino-functionalized beads, carboxyl-functionalized beads, and magnetic beads with functional groups. These beads 11 come in a variety of sizes, such as approximately 0.1 microns to over 50 microns. See, for example, Spherotech, Inc., Lake Forest, Illinois, USA, and Agilent, Inc., Santa Clara, California, USA. These beads are readily functionalized to contain test compounds and / or reporter molecules using conventional chemistry well known in the art. Beads with a nominal diameter of approximately 25 microns include both smaller and larger beads, with a number average of approximately 25 microns.

[0066] In one embodiment, the assay component is viable mammalian cells, such as human cells. These cells are used in an assay to evaluate the biological activity (if any) of a given test compound. Assays using mammalian cells are well known in the art. Suitable cells include cancer cells, insulin-expressing beta cells, neurons, and the like.

[0067] The term "test compound" refers to a compound that is releasably bound to the beads 11 and, upon release, is tested for biological activity in an assay performed in well 21 of the assay apparatus 20.

[0068] The term “releaseable binding” means that the test compound bound to bead 11 can be released by applying a stimulus that breaks the bond. Such bonds are sometimes referred to herein as “cleavable” bonds. This technique has a wealth of examples of cleavable bonds and suitable stimuli to break them. Non-limiting examples of cleavable bonds include bonds released by pH changes, enzyme activity, oxidative changes, redox reactions, UV 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 “ADVANCED THERAPEUTICS 1:1800030, Stimulus-Responsive Prodrug Chemistry for Drug Delivery” (Taresco, V., Alexander, C., Singh, N., Pearce, AK, 2018, onlinelibrary.wiley.com / doi / full / 10.1002 / adtp.201800030), the entire contents of which are incorporated herein by reference.

[0069] The term "shaft" refers to a shaft (recess, cavity, or hole) 4 that penetrates from the top surface 2 to the bottom surface 3 of the dispenser 1. The shaft 4 has a size and / or shape such that it captures and holds one or more assay components, such as beads 11, as described herein. The size of the shaft 4 correlates with the size of the assay component 10 such that only one assay component, such as a single bead 11, is reliably captured by a single shaft 4. The shaft 4 captures a single assay component, and as a result, a single component can be reliably transferred to a single well 21 in the assay apparatus 20. Therefore, the obtained assay result may be directly attributable to, correlated with, or caused by the compound present in that single assay component. On the other hand, if multiple assay components are transferred to a single well 21 simultaneously, the assay result cannot be directly attributable to any of the individual assay components or any of the corresponding compounds present on the individual assay components. Thus, by transferring or guiding only a single assay component to a single well 21 at a time, assay results for a specific compound can be confirmed, and consequently, the accuracy, reliability, and efficiency of the assay are greatly improved. In one embodiment, the capture / release mechanism of the shaft 4 is gravity-assisted based on the diameter decreasing at the second opening 6 compared to the first opening 5.

[0070] The assay component capture mechanism is based on the diameter 7 of the shaft 4 and the rate of decrease of the shaft 4's diameter (in the height direction of the shaft 4) relative to the height h1 of the shaft 4, which determines how far the assay component can advance downward from the shaft 4. In one embodiment, the first opening 5 is at least about 110% of the average width of the assay component, such as the beads 11. The height h3 of the shaft 4 is set at at least about 110% of the average width of the assay component, such as the beads 11, to eliminate the possibility of two or more assay components being fitted and held in the shaft 4 at the same time. If two or more assay components are held in the shaft 4 at the same time, two or more assay components may be transferred to a single well 21 simultaneously, potentially resulting in uncertain and / or unreliable assay results. In one embodiment, the second opening 6 is the narrowest opening, width, or diameter in the shaft 4, and is about 70% of the average size, i.e., average width, of the assay component. In another embodiment, the second opening 6 is about 80%, about 85%, or about 90% of the width or diameter of the assay component 10. Using the aforementioned relative dimensions of the first opening 5 and the second opening 6, the shaft 4 can hold assay components having a sufficient range of widths or diameters. Furthermore, by utilizing these relative dimensions, the amount of dead space or irrelevant space within the shaft 4 after assay components have already been captured can be reduced. Reducing the amount of dead space or irrelevant space will prevent or reduce the possibility of additional assay components becoming stuck or stagnant on top of already captured assay components or in the gaps left after capture of already captured assay components. Otherwise, if the relative difference in width or diameter between the first opening 5 and the second opening 6 is too large, i.e., exceeds a predetermined threshold ratio, a wide range of beads having various widths or diameters may be captured by the shaft 4, but a large amount of unused space may remain within the shaft 4 after the beads have been captured.

[0071] Various shafts 4 are shown in Figures 5 to 12 and Figures 13A to 13D. The shafts 4 shown in any of Figures 5 to 12 and Figures 13A to 13D can be implemented in combination with any of Figures 1, 2A to 2D, 3A to 3G, and 4A to 4B. In Figure 5, the shaft 4 may have an hourglass shape. In particular, the shaft 4 may have a first portion (first section) between a first opening 5 and a second opening 6 in which the width or diameter of the cross-section gradually decreases in the height h1 direction of the shaft 4 (e.g., the negative direction of the y-axis), and a second portion (second section) between a second opening 6 and a third opening 15 at the bottom of the shaft 4 in which the width or diameter of the cross-section gradually increases in the height h1 direction of the shaft 4 (e.g., the negative direction of the y-axis). The second opening 6 may be located inside the shaft 4. In Figure 5, the second opening 6 represents the boundary between the first and second parts, and the second opening 6 may be a cross section having the smallest width or diameter compared to any other cross section within the shaft 4. In one embodiment, as shown in Figure 5, the second part is directly connected to the first part without any intervening part. However, in another embodiment, an intervening part may be connected between the first and second parts. This intervening part may have a constant width along the height h1 direction (e.g., the negative direction of the y-axis), or it may have a variable (non-constant) width, for example, gradually decreasing or increasing, or alternating between decreasing and increasing. The second opening 6 may be equidistant from the first opening 5 and the third opening 15, or it may be closer to either the first opening 5 or the third opening 15. In other words, the height h3 of the first part may be the same as or different from the height h4 of the second part. In Figure 5, the first rate at which the width or diameter of the cross-section of the shaft 4 gradually decreases in the height h1 direction is constant in the first part, and / or the second rate at which the width or diameter of the cross-section of the shaft 4 gradually increases in the height h1 direction is constant in the second part. The first rate (decrease rate) may be the same as or different from the second rate (increase rate). Such an hourglass shape can promote adhesion of the beads 11 to the shaft 4.The width of the third opening 15 may be smaller or larger than the width of the first opening 5.

[0072] In Figure 6, the shaft 4 may have a different hourglass shape than that shown in Figure 5. Unlike the shaft 4 shown in Figure 5, in the shaft 4 of Figure 6, in the first portion, the first rate at which the width or diameter of the cross-section gradually decreases in the height h1 direction of the shaft 4 (e.g., the negative y-axis direction) is variable (non-constant), and / or, in the second portion, the second rate at which the width or diameter of the cross-section gradually increases in the height h1 direction of the shaft 4 (e.g., the negative y-axis direction) is variable (non-constant). The first portion is located between the first opening 5 and the second opening 6 and has a height h3, and the second portion is located between the second opening 6 and the third opening 15 and has a height h4. Height h3 may be the same as or different from height h4. In one embodiment, as shown in Figure 6, the second portion is directly connected to the first portion without any intervening portion. However, in another embodiment, an intervening portion may be connected between the first portion and the second portion. This intervening portion may have a constant width along the height h1 direction (for example, the negative y-axis direction), or it may have a variable (non-constant) width, for example, gradually decreasing or increasing, or alternating between decreasing and increasing. The rate of change of the first ratio (decrease rate) may be negative (gradually decreasing) in the height h1 direction (for example, the negative y-axis direction) of the shaft 4. This means that the width or diameter of the cross-section decreases gradually and slowly as one approaches the second opening 6 from the first opening 5. The rate of change of the second ratio (increase rate) may be positive (gradually increasing) in the height h1 direction (for example, the negative y-axis direction) of the shaft 4. This means that the width or diameter of the cross-section increases gradually and sharply as one approaches the third opening 15 from the second opening 6. The shaft 4 in Figure 6 may have a concave contour. The width of the third opening 15 may be smaller or larger than the width of the first opening 5.

[0073] In Figure 7, the shaft 4 may have a different hourglass shape than those in Figures 5 and 6. In the shaft 4 of Figure 7, in the first portion, the first rate at which the width or diameter of the cross-section gradually decreases in the height h1 direction of the shaft 4 (e.g., the negative y-axis direction) is variable (non-constant), and / or, in the second portion, the second rate at which the width or diameter of the cross-section gradually increases in the height h1 direction of the shaft 4 (e.g., the negative y-axis direction) is variable (non-constant). The first portion is located between the first opening 5 and the second opening 6 and has a height h3, and the second portion is located between the second opening 6 and the third opening 15 and has a height h4. Height h3 may be the same as or different from height h4. In one embodiment, as shown in Figure 7, the second portion is directly connected to the first portion without any intervening portion. However, in another embodiment, an intervening portion may be connected between the first portion and the second portion. This intervening portion may have a constant width along the height h1 direction (for example, the negative y-axis direction), or it may have a variable (non-constant) width, for example, gradually decreasing or increasing, or alternating between decreasing and increasing. The rate of change of the first ratio (decrease rate) may gradually increase in the height h1 direction (for example, the negative y-axis direction) of the shaft 4. This means that the width or diameter of the cross-section decreases gradually and sharply as one approaches the second opening 6 from the first opening 5. The rate of change of the second ratio (increase rate) may gradually decrease in the height h1 direction (for example, the negative y-axis direction) of the shaft 4. This means that the width or diameter of the cross-section increases gradually and gently as one approaches the third opening 15 from the second opening 6. The shaft 4 in Figure 7 may have a convex contour. The width of the third opening 15 may be smaller or larger than the width of the first opening 5.

[0074] In Figure 8, shaft 4 may have a concave contour, similar to the first portion of shaft 4 shown in Figure 6. The shaft 4 in Figure 8 may exhibit a variable (non-constant) rate of decrease in the width or diameter of the cross-section in the height h1 direction of shaft 4 (e.g., the negative y-axis direction). The rate of change of the variable (non-constant) rate of decrease may be negative (gradually decreasing) in the height h1 direction of shaft 4 (e.g., the negative y-axis direction). This means that the width or diameter of the cross-section decreases gradually and slowly as one approaches the second opening 6 from the first opening 5 in the negative y-axis direction.

[0075] In Figure 9, the shaft 4 may have a concave contour, similar to the first portion of the shaft 4 shown in Figure 6. The shaft 4 in Figure 9 may exhibit a variable (non-constant) rate of decrease in the width or diameter of the cross-section in the direction of the height h1 of the shaft 4 (e.g., the negative direction of the y-axis). The rate of change of the variable (non-constant) rate of decrease may gradually increase in the direction of the height h1 of the shaft 4 (e.g., the negative direction of the y-axis). This means that the width or diameter of the cross-section decreases gradually and sharply as one approaches the second opening 6 from the first opening 5 in the negative direction of the y-axis.

[0076] In Figure 10, the shaft 4 may have a first portion having a height h3 and a constant or substantially constant diameter or width w2, and a second portion having a height h4 and a constant or substantially constant diameter or width w3. The diameter or width w3 of the second portion is smaller than the diameter or width w2 of the first portion. The height h3 of the first portion may be smaller than or larger than the height h4 of the second portion. Alternatively, the height h3 of the first portion may be equal to the height h4 of the second portion. In one embodiment, as shown in Figure 10, the second portion is directly connected to the first portion without any intervening portion. However, in another embodiment, an intervening portion may be connected between the first and second portions. This intervening portion may have a constant width along the height h1 direction (e.g., the negative direction of the y-axis), or it may have a variable (non-constant) width, for example, gradually decreasing.

[0077] In Figure 11, the shaft 4 may further include a third portion having a height h5, in addition to the first and second portions shown in Figure 10. The diameter or width w4 of the third portion is constant or substantially constant. The diameter or width w4 of the third portion is greater than the diameter or width w3 of the second portion. The diameter or width w4 of the third portion may be greater than or less than the diameter or width w2 of the first portion. Alternatively, the diameter or width w4 of the third portion may be equal to the diameter or width w2 of the first portion. The end of the third portion may be a third opening 15. In one embodiment, as shown in Figure 11, the third portion is directly connected to the second portion without any intervening portion. However, in another embodiment, an intervening portion may be connected between the second and third portions. This intervening portion may have a constant width along the height h1 direction (e.g., the negative direction of the y-axis), or it may have a variable (non-constant) width, for example, gradually increasing.

[0078] In Figure 12, the shaft 4 may have a different hourglass shape than those in Figures 5 to 7. The shaft 4 in Figure 12 may have a variable (non-constant) first ratio (decrease rate) in which the width or diameter of the cross-section gradually decreases in the height h1 direction (e.g., the negative y-axis direction) of the first portion, and / or a variable (non-constant) second first ratio (increase rate) in which the width or diameter of the cross-section gradually increases in the height h1 direction (e.g., the negative y-axis direction) of the second portion. The first portion is located between the first opening 5 and the second opening 6 and has a height h3, and the second portion is located between the second opening 6 and the third opening 15 and has a height h4. Height h3 may be the same as or different from height h4. In one embodiment, as shown in Figure 12, the second portion is directly connected to the first portion without any intervening portion. However, in another embodiment, an intervening portion may be connected between the first portion and the second portion. This intervening portion may have a constant width along the height h1 direction (e.g., the negative y-axis direction), or it may have a variable (non-constant) width such that it gradually decreases or increases, or decreases and increases alternately. In the first portion, the contour of the shaft 4 may have an inflection point 61. From the first opening 5 to the inflection point 61, the rate of change of the variable (non-constant) first proportion (decrease rate) may gradually increase in the height h1 direction (e.g., the negative y-axis direction) of the shaft 4. This means that as you approach the inflection point 61 from the first opening 5, the width or diameter of the cross-section decreases gradually and sharply. However, from the inflection point 61 to the second opening 6, the rate of change of the variable (non-constant) first proportion (decrease rate) may gradually decrease in the height h1 direction (e.g., the negative y-axis direction) of the shaft 4. This means that as you approach the second opening 6 from the inflection point 61, the width or diameter of the cross-section decreases gradually and gently.

[0079] In the second part, the contour of the shaft 4 may have a second inflection point 62. From the second opening 6 to the inflection point 62, the rate of change of the variable (non-constant) second proportion (increase rate) may gradually increase in the direction of the height h1 of the shaft 4 (e.g., the negative direction of the y-axis). This means that as you approach the inflection point 62 from the second opening 6, the width or diameter of the cross section increases gradually and sharply. However, from the inflection point 62 to the third opening 15, the rate of change of the variable (non-constant) second proportion (increase rate) may gradually decrease in the direction of the height h1 of the shaft 4 (e.g., the negative direction of the y-axis). This means that as you approach the third opening 15 from the inflection point 62, the width or diameter of the cross section increases gradually and gently. The width of the third opening 15 may be smaller or larger than the width of the first opening 5.

[0080] In Figures 13A to 13D, the shaft 4 may have a zigzag contour in which regions with varying cross-sectional widths and regions with constant or approximately constant widths alternate. In particular, the shaft 4 may have a first opening 5 having a cross-sectional width w2. Directly below the first opening 5 (in the negative y-direction), there may be a first region 71 in which the cross-sectional width gradually decreases from w2 to w3. The first region 71 may extend by a height h2 in the negative y-direction. Directly below the first region 71 (in the negative y-direction), there may be a second region 72 in which the cross-sectional width w3 remains constant or approximately constant. The second region 72 may extend by a height h3 in the negative y-direction. Directly below the second region 72 (in the negative y-direction), there may be a third region 73 in which the cross-sectional width gradually decreases from w3 to w4. The third region 73 may extend by a height h4 in the negative y-direction. Directly below the third region 73 (in the negative y-direction), there may be a fourth region 74 where the cross-sectional width w4 remains constant or approximately constant. The fourth region 74 may extend by a height h5 in the negative y-direction. Directly below the fourth region 74 (in the negative y-direction), there may be a fifth region 75 where the cross-sectional width gradually decreases from w4 to w5. The fifth region 75 may extend by a height h6 in the negative y-direction. Directly below the fifth region 75 (in the negative y-direction), there may be a sixth region 76 where the cross-sectional width w5 remains constant or approximately constant. The sixth region 76 may extend by a height h7 in the negative y-direction and terminate at the second opening 6. In some embodiments, the values ​​of heights h2, h3, h4, h5, h6, and h7 may be equal to each other, or at least some of these values ​​may be different. However, in some embodiments, the values ​​of heights h2, h3, h4, h5, h6, and h7 may be approximately equal to each other. For example, the ratio of the maximum to the minimum value among the values ​​of heights h2, h3, h4, h5, h6, and h7 may be 2 or less, or 1.5 or less. Furthermore, the angles of the first region 71, the third region 73, and the fifth region 75 with respect to the y-axis may be equal to each other, or at least one of these angles may be different. For illustrative purposes, in Figure 13C, the angle of the first region 71 with respect to the y-axis is shown as reference numeral θ.In some embodiments, the angles of the first region 71, the third region 73, and the fifth region 75 with respect to the y-axis may be less than 45 degrees or less than 60 degrees. In some embodiments, the angles of the first region 71, the third region 73, and the fifth region 75 with respect to the y-axis may be 30 degrees or more and 60 degrees or less. In some embodiments, the angles of the first region 71, the third region 73, and the fifth region 75 with respect to the y-axis may be 15 degrees or more and 75 degrees or less. Six regions are shown in Figures 13A to 13D, but the number of regions is arbitrary.

[0081] The above configuration assumes that the inclinations of the first region 71, the third region 73, and the fifth region 75 are constant, but in some alternative embodiments, at least a portion of the inclinations of the first region 71, the third region 73, and the fifth region 75 may not be constant. In other words, the rate of decrease in the cross-sectional width along the negative direction of the y-axis in the first region 71, the third region 73, and / or the fifth region 75 may be variable (non-constant), as shown, for example, in Figures 6 to 9 and Figure 12.

[0082] In Figure 13B, bead 11 can contact the side wall at the boundary (intersection) between the first region 71 and the second region 72 and be held (fixed) to the side wall. Bead 11 does not need to contact the shaft 4 at other positions. In Figure 13C, a smaller bead 81 can contact the side wall at the boundary (intersection) between the third region 73 and the fourth region 74 and be held (fixed) to the side wall. Bead 81 does not need to contact the shaft 4 at other positions. In Figure 13D, a smaller bead 91 can contact the side wall at the boundary (intersection) between the fifth region 75 and the sixth region 76 and be held (fixed) to the side wall. Bead 91 does not need to contact the shaft 4 at other positions. Thus, Figures 13B to 13D show beads of various sizes that can remain in a single position within the shaft 4. Since each of the beads 11, 81, and 91 can be held (fixed) within the side wall of the shaft 4 at only a single position in the y-axis direction, i.e., only at the boundary between two regions, the beads 11, 81, and 91 can be released into the assay well without clogging the shaft 4 when the shaft 4 is moved.

[0083] The shaft 4 may have contours other than those explicitly shown in Figures 2A-2D, 5-12, and 13A-13D. Any combination of features illustrated or described with respect to the features described above may be contemplated. For example, the shaft 4 may include one or more parts having a constant width or diameter, as shown in Figures 10-11, and one or more parts having a variable (non-constant) width or diameter, as shown in Figures 5-9, 12, and 13A-13D. As another example, the shaft 4 may include one or more parts having a variable (non-constant) width or diameter such that the rate of change of the width or diameter is constant, and one or more parts having a variable (non-constant) width or diameter such that the rate of change of the width or diameter increases or decreases. One or more references to the shaft 4 may refer to any contours illustrated and described with respect to Figures 2A-2D, 5-12, and 13A-13D. In Figures 6 to 9 and Figure 12, the rate of reduction in the cross-sectional width of multiple different regions in the negative direction of the y-axis may be variable (non-constant).

[0084] While it is preferable to use substantially spherical assay components with a uniform longest axis throughout, other shapes may be used. One such non-spherical shape useful herein is a solid, rotatable orbiform with a uniform width throughout. Yet another shape useful herein is elliptical. Preferred shapes having an elliptical cross-section include ellipsoids with a major-axis-to-minor-axis ratio greater than about 1 and less than about 1.5, preferably less than about 1.2. As used herein, the term “axis” refers to the longest axis within the assay component.

[0085] [Dispenser] The ability to assay very large combinatorial libraries of compounds typically requires delivering a single assay component, such as a bead 11, into a single well 21 of the assay apparatus 20. In practice, an assay apparatus 20 can incorporate approximately 2 million or more wells 21. As shown in Figures 3A, 3B, 4A, and 4B, the diameter of the wells 21 of these assay apparatuses 20 is significantly larger than the diameter or width of the shaft 4 of the dispenser 1. Such a size difference makes adding a single assay component, such as a bead 11, into a single well 21 a technically challenging undertaking.

[0086] [Beads as assay components] In this embodiment, the assay component is a bead 11. These beads are preferably spherical or substantially spherical, and preferably have a diameter of about 0.5 microns or more and about 100 microns or less. Figure 1 shows a dispenser 1 having a plurality of shafts 4.

[0087] More generally, the dispenser 1 in Figure 1 has a top-to-bottom thickness 8, preferably at least about 0.1 mm to about 5 mm, and includes a plurality of shafts 4. The thickness 8 may be any value or a partial range within the above range (including upper and lower limits). The dispenser 1 comprises one of a plurality of biocompatible materials. Examples of biocompatible materials include, but are not limited to, cycloolefin polymers (COP) sold under the trade name ZEONEX by Zeon Corporation (Tokyo, Japan), cyclic olefin copolymers (COC) sold by many suppliers such as Polyplastics USA (Farmington Hills, Michigan, USA), polyimides sold by many suppliers such as Putnam Plastics (Dayville, Connecticut, USA), polycarbonates sold by many suppliers such as Foster Corporation (Putnam, Connecticut, USA), polydimethylsiloxane sold by Edge Embossing (Medford, Massachusetts, USA), and polymethyl methacrylate sold by Parchem Fine & Specialty Chemicals (New Rochelle, New York, USA).

[0088] The dispenser 1 described herein can be readily manufactured by a hot embossing method well known in the art. In such a hot embossing method, a sheet of thermoplastic polymer is used, which is heated to a temperature slightly above its glass transition temperature to soften the plastic. A stamp is selected that includes a number of protrusions arranged on the surface in a desired pattern. Each prong is sized to have a width or diameter and depth that correlates with the size and shape of the shaft 4 as described above. In some embodiments, the prongs may include or be similar to a frustum or truncated cone, but may be any desired shape as long as the cross-sectional width is variable. Using the prongs, the dispenser 1 can be produced or prepared such that the second opening 6 is narrower than the first opening 5, or has different cross-sectional widths. The stamp is made sized so that the entire prong fits into the sheet to a predetermined depth. Sufficient force is applied to the stamp so that the prong of the desired length sinks into the sheet and penetrates it. The force required at this time depends on the degree of softness of the sheet and can be easily determined by those skilled in the art. Once the sheet has cooled, the prongs are removed, and the sheet including the shaft 4 is obtained as shown in Figure 1.

[0089] Alternatively, the dispenser 1 in Figure 1 may be manufactured by conventional injection molding using two mold halves, namely, one half (male half) having protrusions corresponding to the stamp protrusions and the other half (female half) forming the base of the device. The two mold halves are positioned side by side to form a shaft 4 in the shape of the dispenser 1 shown in Figure 1. A monomer or reactive oligomer composition is injected into this cavity and subsequently polymerized to obtain the dispenser 1 including the shaft 4, as shown in Figure 1.

[0090] In this embodiment, the dispenser 1 may be manufactured together with the assay device 20 to ensure proper alignment between the shaft 4 and the well 21.

[0091] Figure 2A shows a spherical bead 11 positioned above the shaft 4, configured to be sized so that the bead 11 fits completely inside the shaft 4. Figure 2B shows the spherical bead 11 inside the shaft 4. On the other hand, Figure 2C shows a spherical bead 11 with a diameter larger than the upper opening (first opening) 5, where part of the bead 11 is located inside the shaft 4 and the rest of the bead 11 is located outside the shaft 4. In this case, there is a sufficient volume of bead 11 inside the shaft 4 to firmly hold the bead 11 within the shaft 4. Figure 2D shows the removal of a small bead 12 having a diameter smaller than the narrowest diameter of the shaft 4, and the small bead 12 exits from the bottom of the shaft 4. In other words, in Figure 2D, the small bead 12 is not held or trapped inside the shaft 4. Thus, loading or trapping a single bead 11 into the shaft 4, along with the size exclusion of the small bead 12, can be achieved seamlessly in a single step without manual intervention. Further details of the configuration in which the small beads 12 are not held or captured are shown in and described in relation to Figures 3C and 3D.

[0092] Referring back to Figures 2A and 2B, the diameter of the first opening 5 is larger than the diameter of the spherical beads 11, so that the beads 11 are located inside the shaft 4. In one embodiment, the diameter of the first opening 5 is in the range of 150% or less of the diameter of the spherical beads 11, or 150% or less of the average diameter of the spherical beads 11. Thus, in such embodiments, the diameter of the first opening 5 may be large enough so as not to leave excessive empty space, in order to reduce or eliminate the possibility of a second bead becoming completely or partially trapped on already trapped beads, or in gaps in the shaft 4 after beads have already been trapped or held inside the shaft 4. Furthermore, since the second bead is blocked by the already trapped beads, it cannot cross the space already occupied by the beads and cannot exit through the shaft 4. For example, if the first opening 5 had a diameter of 1000% (e.g., 10 times) the average diameter of the spherical beads 11, the probability of multiple beads becoming stuck or trapped inside the shaft 4 could exceed an acceptable threshold probability. At the same time, the diameter of the first opening 5 must be large enough to adequately distribute beads 11 of different sizes within the shaft 4; otherwise, an excessive number of beads 11 will not be retained within the shaft 4. By using a shaft 4 with a variable (non-constant) width or diameter, and spherical or elliptical beads 11, it becomes possible for smaller beads to simply pass through the shaft 4 rather than two beads being retained within the shaft 4 at the same time. As a result, two beads will never fill a single well 21 simultaneously.

[0093] [Load] The loading of beads 11 onto the shaft 4 of dispenser 1 can be achieved by any of the many processes known in the art. As shown in Figure 3A, the sealing cap 30 has an inlet port 31 for introducing beads 11 and an outlet port 32 for collecting excess beads 11. The sealing cap 30 is sized to fit over dispenser 1 and is positioned on dispenser 1 such that both the inlet port 31 and the outlet port 32 are aligned over dispenser 1.

[0094] In a preferred embodiment, the sealing cap 30 is sized and shaped to fit onto the dispenser 1 by any number of known features. These features include a connecting projection extending upward from the dispenser 1, the connecting projection passing through a hole in the body of the sealing cap 30. Alternatively, a clip or other locking device / configuration that snaps and locks both the dispenser 1 and the sealing cap 30 into a fixed configuration may be attached to either the dispenser 1 or the sealing cap 30. The specific locking mechanism is not important.

[0095] The funnel-shaped cone 40 is sized such that the narrower end of its cone fits into the inlet port 31 of the sealing cap 30. The wider end of the funnel-shaped cone 40 allows for the addition of beads 11. The beads 11 are delivered onto the surface of the dispenser 1 through the inlet port 31, either alone or in fluid, and move from the inlet port 31 towards the outlet port 32 (for example, along the positive x-axis). The beads 11 may be poured into each shaft 4 until each shaft 4 holds a single bead 11, and any excess beads are recycled through the outlet port 32 for recycling to another dispenser 1. Excess beads may exceed a threshold width or diameter (for example, the width or diameter of the first opening 5), thereby preventing them from entering any of the shafts 4 through the first opening 5. In one embodiment, excess beads may be assayed and / or redirected to a shaft of a different setting having a larger width or diameter compared to shaft 4. Therefore, excess beads can be automatically transported and processed in a continuous iteration or stage according to their size, with little to no manual intervention.

[0096] As shown in Figures 3A and 3B, the height h1 of the shaft 4 indicates the distance the shaft 4 extends along the y-axis. The height h1 is sufficient to hold a single bead 11, but is limited to a height that reduces the amount of dead space or irrelevant space within the shaft 4. This can reduce the possibility of further beads getting stuck or accumulating on or in the gaps after the beads 11 have been captured. In one embodiment, the height h1 may be between 0.5 and 2 times the average diameter of the beads 11. In another embodiment, the height h1 may be between 0.5 and 1.25 times the average diameter of the beads 11. In yet another embodiment, the height h1 may be between 0.75 and 1.25 times the average diameter of the beads 11.

[0097] In one embodiment, one or more shafts 4 may have a frustum or truncated cone shape. The shaft 4 may have an inclination angle (base angle) measured between the first opening 5 and the side surface. This inclination angle may be in the range of about 50 degrees or more and 80 degrees or less with respect to the top surface 2 of the dispenser 1. The smaller the inclination angle, the greater the rate of change of the width or diameter of the shaft 4 in the height h1 direction. If the rate of change exceeds a threshold, the amount of empty space remaining after one bead 11 has already remained or been fixed inside the shaft 4 may increase the possibility of further beads remaining on the one bead 11 or in the remaining gaps of the shaft 4. Therefore, the inclination angle may be selected to exceed a threshold angle (e.g., 30 degrees). At the same time, the inclination angle should be selected so that beads with a sufficiently and not excessively narrow distribution range can be guided into the shaft 4. For example, if the inclination angle is too close to 90 degrees, the rate of change of the width or diameter of the shaft 4 in the height h1 direction will be minimized, and only beads with a narrow range of sizes will be guided into the shaft 4. Therefore, the tilt angle may be selected to be less than a threshold angle (e.g., 85 degrees).

[0098] In one embodiment, the funnel-shaped cone 40 is tilted so that the beads 11 flow partially horizontally and fill the shaft 4. In another embodiment, the dispenser 1 is slightly inclined so that the side adjacent to the inlet port 31 of the sealing cap 30 is higher than the side adjacent to the outlet port 32 of the sealing cap 30. Such a configuration can be achieved by applying a slight downward slope of at least about 1 degree from the inlet port to the outlet port. The downward slope is preferably at least about 1 to about 10 degrees, more preferably about 1 to about 5 degrees (or any value or partial range within that range (including lower and upper limits)). This allows the beads 11 to cross the downward slope at a suitable speed. This speed is preferably such that relatively small beads 11 can pass through the shaft 4, while relatively large excess beads 11 can be sent through the outlet port (recovery port) 32 and captured, while the beads can be easily captured by the shaft 4 in the dispenser 1. The conical size and contour of shaft 4 are designed to remove small beads present on these shafts in a manner that allows two beads 11 to be captured simultaneously in a single well 21. As shown in Figure 3B, this process allows a single bead 11 having a width d1 (e.g., indicating that the long axis or both axes have a width d1) to be captured within the single shaft 4 of dispenser 1.

[0099] As shown in Figure 3C, smaller beads 12, each having a width or diameter d2 smaller than the narrowest width or diameter m1 of the shaft 4, are guided out of the shaft 4. In one embodiment, the smaller beads 12 are guided from the shaft 4 into a channel 17. The channel 17 may be a fluid channel, such as a microfluidic channel. In Figure 3C, the channel 17 is shown in a downward-sloping configuration so that gravity can push or assist the smaller beads 12 through the channel 17, but as shown in Figure 3D, the channel 17 may have a relatively flat and gentle slope. In some embodiments, the smaller beads 12 may be pushed or assisted along the channel 17 via fluid pressure and / or other forces (e.g., suction or vacuum) in addition to or as an alternative to gravity. As the smaller beads 12 pass through the shaft 4, the minimum height h2 of the channel 17 extending along the y-axis may be greater than or equal to the minimum width m1 of the shaft 4 so as to prevent the smaller beads 12 from getting stuck in the channel 17. In Figure 3D, smaller beads 12 can pass through the channel 17 and enter the receiving section (container) 18. From the receiving section 18, the smaller beads 12 can be fed into or guided into a funnel (tube, pipe, channel, conduit, or guide) 19. The funnel 19 may be implemented in a similar, or identical, manner to, the funnel-shaped cone 40. The minimum width or diameter of the funnel 19 may be such that all the small beads 12 can pass through the funnel 19 without getting stuck. The entry of beads from the receiving section 18 into the funnel 19 may be regulated (controlled) using a gate 13 or other similar mechanism. The gate 13 may be controlled mechanically and / or electrically. In other examples, the gate 13 may be rotated or switched to open at a fixed or variable time interval and remain closed at other time intervals. In some examples, the small beads 12 may pass directly from the channel 17 into the funnel 19 rather than being temporarily held in the receiving section 18. In other words, channel 17 may be directly connected to or in communication with funnel 19.

[0100] Once the small beads 12 enter the funnel 19, they may pass through or be guided to a pair of small shafts 54 having a minimum width or diameter m2 smaller than that of shaft 4. A subset of the small beads 12 may be trapped within the small shafts, while the remaining small beads may pass through even smaller shafts. In this manner, smaller beads 12 may be continuously or iteratively fed through smaller shafts with no or minimal manual intervention until all or nearly all beads are held within their individual shafts and ready to be placed into the wells to be assayed. During each iteration or cycle, smaller beads may be held within the shafts and / or assayed. Another advantage is that, if the amount of compound bound to the beads correlates or is proportional to the width or diameter of the beads, a continuous assay allows for further determination or confirmation of whether, or how, the amount of compound affects the assay results. In a purely illustrative and non-limiting configuration, the first iteration may include performing the assay on beads 11 having a width or diameter of at least 0.8 times and no more than 1 times the threshold width or threshold diameter. A second iteration may include performing the assay on small beads 12 having a width or diameter of 0.64 times or more and 0.8 times or less the threshold width or threshold diameter. Furthermore, a third iteration may include performing the assay on even smaller beads having a width or diameter of 0.512 times or more and 0.64 times or less the threshold width or threshold diameter. The receiving portion 18 and funnel 19 are shown only in Figure 3D, but the receiving portion 18 and funnel 19 may be implemented in the same or similar manner as in Figure 3C.

[0101] On the other hand, Figures 3E to 3G show a size exclusion mechanism for obtaining beads with a specific width range in two stages. In the first stage, beads larger than a second threshold width are excluded or removed through a spare channel. In the second stage, beads smaller than a first threshold width are excluded or removed. The first threshold width may be smaller than the second threshold width. Thus, beads between the first and second threshold widths are retained. Figure 3E shows an embodiment using the opening and closing of valves 24, 25, 26, and 27 during various processes such as bead loading or distribution. According to the embodiment in Figure 3E, beads larger than the second threshold width that are stuck in channel 55 and / or channel 56 are released, beads smaller than the first threshold width are released, and beads that are stuck in path 43 but not in channels 45, 46, 47, and 48 are released.

[0102] In Figures 3E to 3G, a funnel-shaped cone 40, identical or similar to that shown in Figure 3A, can house or accommodate beads 11, 41, and 51 having varying widths. For example, bead 11 has a width d1, bead 41 has a width d3 smaller than d1, and bead 51 has a width d4 larger than d1. The beads 11, 41, and 51 may be introduced into the first stage 52 via the funnel-shaped cone 40 or other mechanism or device for housing the beads. The beads 11, 41, and 51 can enter a channel 53 from the first stage 52. Once the beads 11, 41, and 51 enter the channel 53, they pass through a channel 55 or channel 56. Channels 55 and 56 allow the passage of beads smaller than a second threshold width m3, but prevent the passage of beads larger than a second threshold width m3. Channels 55 and 56 are shown in Figures 3E to 3G to have a width that gradually increases along the negative direction of the y-axis. In particular, channels 55 and 56 have a width m3 at their respective inlets and a width m4 at their respective outlets. Width m4 may be greater than width m3. Although the width of channels 55 and 56 is shown to gradually increase over the entire height of channels 55 and 56 (along the y-axis direction), in some alternative embodiments, the width of channels 55 and 56 may gradually increase only at the top of each channel 55 and 56 (e.g., the top half, top third, or top two-thirds in the y-axis direction), while the width of the rest of channels 55 and 56 remains constant or substantially constant. The inclination angle of channels 55 and 56 with respect to the x-axis may be between 30 and 85 degrees. In other alternative embodiments, channels 55 and 56 may have a constant or substantially constant width with respect to the negative direction of the y-axis. Although only two channels are shown in the figures, any number of channels may be provided.

[0103] Here, bead 51 may have a width d4 that exceeds the width m3 of channel 55. Therefore, bead 51 may be prevented from passing through channel 55. On the other hand, beads 11 and 41 may have widths d1 and d3, respectively. Widths d1 and d3 may both be smaller than the width m3. Therefore, beads 11 and 41 can pass through channel 56. Beads such as bead 51 that remain in or clog at least a portion of channels 55 and 56, such as the upper part of channels 55 and 56, can be discharged or removed by ultrasonic treatment, gravity, and / or fluid flow.

[0104] Beads 11 and 41 released through channel 56 can enter a second stage (second phase) 42. In the second stage (second phase) 42, beads 11 and 41 can cross path 43 and enter one of channels 45, 46, 47, or 48. Channels 45, 46, 47, and 48 may have a smaller width m1 (e.g., a first threshold) at the bottom of each channel 45, 46, 47, or 48. On the other hand, the larger width m5 at the top of channels 45, 46, 47, or 48 may be greater than or equal to width m3. Here, the width d1 corresponding to bead 11 may be greater than width m1, and the width d3 corresponding to bead 41 may be less than width m1. Thus, bead 11 may be held in channel 45, and bead 41 may pass through channel 45. As shown in Figures 4A and 4B, any beads held in a channel (e.g., bead 11) may be transferred and / or passed through an assay apparatus to be assayed. For example, as shown within the dashed rectangle in Figure 3F, the set of channels 45, 46, 47, and 48 may be removed and moved onto the assay apparatus. Although four channels 45, 46, 47, and 48 are shown in the figure, any number of channels may be implemented within the second stage 42.

[0105] In this way, beads between the first threshold width and the second threshold width may be retained for subsequent assays. For example, the first threshold width may be 9 microns or 8 microns, and the second threshold width may be 11 microns or 12 microns. Thus, the beads in dispenser 1 may have their respective widths within the threshold range of the target width. For example, the width range of the beads may be within 10 percent or 20 percent of a particular target width (e.g., 10 microns). In other embodiments, the width range of the beads may be within 2 percent or 5 percent of a particular target width. Such size control can be important for conducting reliable assays to ensure that a uniform or nearly uniform amount or concentration of the compound is compared across multiple wells. Since the beads contain chemicals and the surface area of ​​a bead is related to the square of its width, if a bead is twice as wide as another bead, it means that one bead contains four times as much of the chemical as the other bead. Thus, even small differences in bead size can result in large differences in the amount or concentration of the chemical on the beads. The embodiments shown in Figures 3E to 3G can be combined with the embodiments shown in Figures 3C to 3D to obtain, for example, a specific range of bead sizes continuously across multiple different iterations, as described above.

[0106] Figure 3E shows a valve controlled to open and close during several different processes. The valve includes a first valve 24 located at the outlet of the funnel-shaped cone 40, a second valve 25 located at the outlet of the collector 57, a third valve 26 located at the outlet of the path 43, and a fourth valve 27 located downstream of channels 45, 46, 47, 48. Any or all of the valves 24, 25, 26, 27 may be fluid valves or microfluidic valves and may be electronically and / or mechanically controlled. Any or all of the valves 24, 25, 26, 27 may be two-way valves.

[0107] While loading beads (e.g., beads 11, 41, 51) through the funnel-shaped cone 40, the first valve 24 may be opened to allow the flow of beads through the funnel-shaped cone 40. The second valve 25 and the third valve 26 may be closed. The fourth valve 27 may be opened to purge (remove) smaller beads and prevent the accumulation of smaller beads. While releasing beads (e.g., bead 51) that have accumulated in the channels 55, 56, the path 53, and the collector 57, the second valve 25 may be opened to allow the bead 51 to pass through the collector 57 and exit the collector 57. In some embodiments, the third valve 26 may be opened in the reverse direction (e.g., in the negative direction rather than the positive direction of the x-axis) in accordance with the fluid flow 301. Therefore, in the fluid flow 301, fluid backflush and / or back pressure may cause fluid to enter through channels 55, 56 and through the third valve 26, and the beads may be further guided through the second valve 25. In some embodiments, in addition to or instead of this, a fluid jet 58 may push the beads out of the collector 57. Note that the first valve 24 and the fourth valve 27 may be closed during bead discharge. During the process in which beads 41 smaller than the first threshold width are pushed out through the bottom surface 3, the fourth valve 27 may be opened so that a fluid flow from a fluid jet 68 or the like can push the beads 41 through the fourth valve 27 along the x-axis. In some embodiments, the third valve 26 may be opened in the reverse direction so that fluid back pressure can pass from path 43 through channels 45, 46, 47, 48 in the negative x-axis direction and further guide the beads out through the fourth valve 27 according to the fluid flow 302. Although the fluid flow 302 is shown only as passing through channel 48, the fluid from the fourth valve 27 may also pass through channels 47, 46, and 45. In some embodiments, as an addition or alternative, fluid jets 68 and / or fluid jet 49 may push beads through the fourth valve 27. In particular, the fluid from fluid jet 49 may flow through channels 45, 46, 47, and 48.During this time, the first valve 24 and the second valve 25 may be closed.

[0108] During the process of extracting the desired beads 11, beads trapped in channels 45, 46, 47, 48 may be recovered through a third valve 26. Thus, the third valve 26 may be opened. In some embodiments, optionally, the fourth valve 27 may be opened in the reverse direction so that the beads are pushed out through the path 43 and through the third valve 26 by backflushing and / or back pressure of fluid in the negative x-axis direction from the fourth valve 27, as well as the fluid flow 302. In some embodiments, removal may be performed additionally or alternatively by sonication and / or gravity.

[0109] Alternatively, if the desired bead 11 is to be held within channels 45, 46, 47, and 48, the aforementioned process of extracting the desired bead may be skipped. By holding the desired bead 11, the channels may be inverted, flipped, or replaced so that the desired bead 11 falls into the assay apparatus as shown in Figures 4A and 4B.

[0110] In some embodiments, the aforementioned operations for loading beads, releasing beads, extruding beads, and extracting beads of a desired size may be performed at predetermined time intervals. The predetermined time intervals may be fixed and / or periodic. For example, during a first time interval, beads may be loaded. During a second time interval following the first time interval, beads retained or trapped in channels 55, 56, pathway 53, and collector 57 may be extruded. During a third time interval following the second time interval, beads 41 may be extruded from the bottom surface 3. During a fourth time interval, beads 11 of a desired size may be extracted and inverted onto the assay apparatus 20, as shown in Figures 4A and 4B. In alternative embodiments, instead of being performed at time intervals, the aforementioned operations may be performed based on the concentration of beads detected in channels 55, 56, pathway 53, collector 57, bottom surface 3, and channels 45, 46, 47, 48. This detection may be performed via imaging, such as fluoroscopy.

[0111] Once all the beads from the funnel-shaped cone 40 have been processed, or once each of the channels in the second stage 42 has held a bead, channels 45, 46, 47, and 48 may be removed, disassembled and turned inside out, inverted, or moved into the assay apparatus, as shown inside the dashed rectangle in Figure 3F. Processing the beads from the funnel-shaped cone may require passing the beads through the channels of the first stage 52 or feeding the beads into the collector 57. Thus, any beads remaining in channels 45, 46, 47, and 48, and in the other channels of the second stage 42, can be transferred to the assay apparatus.

[0112] [Distribution] As described above, each of the shafts 4 of the dispenser 1 is pre-selected to align with a single well 21 of the assay apparatus 20. Additional beads that may be lodged on top of or clogged with beads already captured within the shaft 4 may be fed from the shaft 4 towards the outlet 32 ​​via air, fluid, or vacuum pressure along or near the top surface 2. As shown in Figures 4A and 4B, the distribution of beads 11 into the wells 21 is achieved by positioning the assay apparatus 20 on the dispenser 1 and ensuring that each shaft 4 is aligned with the corresponding well 21. The dispenser 1 and the assay apparatus 20 are preferably locked in place by a locking mechanism 23 that ensures the shafts 4 remain aligned with the wells 21 during distribution. Alignment may be confirmed by aligning a marker (not shown) on the assay apparatus 20 with a corresponding marker (not shown) on the dispenser 1. The mechanisms shown in Figures 4A and 4B may be implemented in conjunction with the relevant parts shown in other figures (Figures 1, 2A-2D, 3A-3G, 5-12, and 13A-13D, etc.).

[0113] In Figure 4A, an arbitrary small gap 22 may exist between the dispenser 1 and the assay apparatus 20, provided that the gap 22 is smaller than the width or diameter of the beads 11, preferably about 50% or less of the width or diameter of the beads 11. The height of the gap 22 (e.g., along the y-axis) may be adjustable using a locking mechanism 23 and may be controlled mechanically and / or electrically. For example, the locking mechanism 23 may be fixed in a rail on the assay apparatus 20 and / or shaft 4, and the relative position to which the locking mechanism 23 is fixed on the rail may be adjustable. Thus, the gap 22 can be adjusted based on a range or distribution of the width or diameter of the beads 11. In some exemplary embodiments, the assay apparatus 20 may be configured without a gap 22.

[0114] Once alignment is confirmed, the dispenser 1 can be placed on the assay device 20 simply by reversing the combination of the dispenser 1 and the assay device 20. In the case of gravity-based dispensing, force or energy may be required on the dispenser 1, for example, to release the beads 11 into the well 21. In one embodiment, the force or energy thus required is at least about 1 Newton or at least about 0.01 Joules. Such force or energy can be applied by any method (e.g., back pressure, vacuum or suction pressure, tapping, vibration, sonication, centripetal force, temperature change, etc.). Due to the size distribution of the beads, some beads may be more tightly bound within the dispenser than others, and force (additional force) may be required to separate them. The amount of force required to separate the beads may be inversely proportional to the width or diameter of the beads 11. Alignment of the shaft 4 and the well 21 ensures that a single bead 11 is captured in a single well 21, without the beads 11 flowing into a well other than the intended well.

[0115] [Cells as assay components] Another example of an assay component is cells used to evaluate the response to contact and / or uptake of a test compound during the assay. Cells may be added to the dispenser 1 in a similar manner to the beads 11, but with the following additions: In examples where the shaft 4 has a frustoconical contour, the shape of the shaft 4 is based on the inclination angle of the frustoconical, which can be evaluated by well known geometric principles. In one embodiment, the inclination angle may be less than 90 degrees with respect to the top surface 2 of the dispenser 1. In one embodiment, the inclination angle is in the range of about 30 degrees or more and about 85 degrees or less with respect to the top surface 2 of the dispenser 1. The length, depth, or height of the shaft 4 is based on the thickness of the dispenser 1 and is typically in the range of about 0.1 mm or more and about 5 mm or less. Since the diameter of human cells ranges from approximately 6 microns to approximately 50 microns or more, the shape of the shaft 4 that holds such cells is preferably in the range of approximately 3 microns or more and approximately 25 microns or less (or any value or partial range within that range) on the lower side, and preferably in the range of approximately 9 microns or more and approximately 75 microns or less (or any value or partial range within that range) on the higher side, and is selected according to the size of the cells and the angle of the frustum of the shaft 4. The selected cells need to be able to roll on the surface of the dispenser 1.

[0116] Otherwise, the cells are added to dispenser 1 in substantially the same manner as the beads 11, and subsequently to wells 21 of assay apparatus 20. In some cases, it is desirable to add multiple copies of the same cells to a single well 21 of assay apparatus 20. In such cases, these cells may be loaded one per dispenser into another dispenser that is structurally identical or similar to dispenser 1. The additional cells are added to wells 21 of assay apparatus 20 in the same manner as the initial cells. Similarly, other assay components 10 may be added in the same manner.

[0117] [system] In one embodiment, a system for dispensing beads into an assay apparatus is provided. The system is configured as follows: The system comprises a dispenser 1 having a plurality of shafts 4 and an assay apparatus 20 having a plurality of wells 21. Each shaft 4 has a variable (non-constant) cross-sectional width. Each shaft 4 is capable of dispensing only a single assay component (e.g., beads 11) having a width greater than the minimum cross-sectional width within the shaft. The dispenser 1 is mounted (fitted) on the assay apparatus 20 such that each shaft 4 is alignable to a single well 21 on the assay apparatus 20, so that when a single assay component is dispensed from the dispenser 1, only the single assay component is captured in a single well 21.

[0118] The present invention is not limited to the compositions, reagents, methods, systems, diagnostic methods, experimental data, etc., disclosed herein. Furthermore, the present invention is not limited to the preferred embodiments disclosed herein.

[0119] The subject matter described herein may be embodied in systems, apparatus, methods, and / or articles, depending on the desired configuration. The embodiments described above do not represent all embodiments that correspond to the subject matter described herein, but rather are merely examples that correspond to aspects relevant to the subject matter described herein. While some modifications have been described in detail above, other modifications or additions are also possible. In particular, further features and / or variations may be provided in addition to those described herein. For example, the embodiments described above may cover various combinations and subcombinations of the disclosed features, and / or combinations and subcombinations of some further features disclosed above. Furthermore, the logical flows shown in the accompanying figures and / or described herein do not necessarily require a specific order or sequence to achieve the desired result. Other embodiments may also be included in the following claims.

[0120] Furthermore, while certain features of the present invention may be disclosed in relation to only one of several embodiments, such features may be combined with at least one other feature of other implementations so as to be desirable and advantageous for any given or particular application. Moreover, to the extent that the terms “equip,” “have,” “include,” their variations, and other similar terms are used in either the detailed description or the claims, these terms are intended to be inclusive as open transition words without excluding additional or other elements.

[0121] Furthermore, the terms “example” or “exemplary” are used herein to mean that one aspect or one embodiment functions as an example or embodiment. No aspect or configuration described herein as “exemplary” should necessarily be construed as being preferable or advantageous to any other aspect or configuration. Rather, the use of the terms “example” or “exemplary” is intended to illustrate a concept in a concrete way. The term “or” as used in this application is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless otherwise specified or it is clear from the context, the phrase “X uses A or B” is intended to mean either of the natural inclusive substitutions. That is, the phrase “X uses A or B” satisfies the cases of X using A, X using B, and X using both A and B. Furthermore, countable noun elements used in this application and the appended claims should generally be construed to mean “one or more” elements unless otherwise specified or it is clear from the context that a singular form is being referred to.

[0122] The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit this disclosure. Where used herein, countable nouns include plural forms unless the context clearly indicates otherwise. Furthermore, where used herein, the terms “equip,” “have,” and “include” specify the presence of the described features, integers, steps, actions, elements, and / or components, but should be understood not to exclude the presence or addition of one or more other features, integers, steps, actions, elements, components, and / or groups thereof. Where used herein, the term “and / or” includes any combination of one or more of the relevant enumerated items.

[0123] Although at least one exemplary embodiment is described as using multiple units to perform an exemplary process, it is understood that the exemplary process may be performed by one or more modules.

[0124] The use of terms such as “first,” “second,” and “third” in this specification is not intended to indicate an order, but is provided to identify different structures, dimensions, or operations, and unless a specific order is explicitly specified herein, structures, dimensions, or operations may be performed in an order different from the specified order.

[0125] The approximate expressions used herein and throughout the claims may be applied to modify any quantitative expressions that may change to an acceptable extent without altering the underlying function of the subject matter. Thus, values ​​modified by terms such as “approximately,” “approximately,” and “substantially” are not limited to the specified exact values. In at least some examples, approximate expressions may correspond to the precision of an instrument used to measure a value. Throughout the specification and claims, scope limitations are combined and / or interchangeable, and such scopes, unless otherwise specified by context or wording, include all subscopes identified and contained therein.

[0126] In the above description and claims, a list of elements or features may follow phrases such as “at least one” or “one or more.” The term “and / or” may also be used in lists of two or more elements or features. Unless implicitly or explicitly contradicted by the context in which it is used, such phrases are intended to mean any of the enumerated elements or features individually, or any of the enumerated elements or features in combination with other enumerated elements or features. For example, the phrases “at least one of A and B,” “one or more of A and B,” and “A and / or B” shall mean “A alone, B alone, or a combination of A and B,” respectively. A similar interpretation applies to lists containing three or more items. The phrases “at least one of A, B, and C,” “one or more of A, B, and C,” and “A, B, and / or C” shall mean “A alone, B alone, C alone, A and B, A and C, B and C, or A and B and C.” Furthermore, the use of the term "based on" in the foregoing and in the claims is intended to mean "based on at least partially," and any features or elements not described are also permitted.

Claims

1. A system for distributing multiple beads into an assay device, An assay apparatus having multiple wells, A dispenser having multiple shafts, Each of the shafts has a variable cross-sectional width and is capable of releasing a single assay component having a width greater than the minimum cross-sectional width within the shaft. The dispenser is mounted on or above the assay apparatus such that, when the single assay component is released from the dispenser, only the single assay component is deposited in a single well, and each of the shafts is aligned with the single well on the assay apparatus. system.

2. The first of the plurality of shafts has a frustum-shaped or hourglass-shaped contour. The system according to claim 1.

3. The first shaft among the plurality of shafts has a circular cross-section, and the cross-sectional width of the first shaft is the diameter of the circular cross-section. The system according to claim 1.

4. The first shaft among the plurality of shafts comprises a first portion in which the width gradually decreases in the height direction of the first shaft, and a second portion in which the width gradually increases in the height direction of the first shaft. The system according to claim 1.

5. The first shaft among the plurality of shafts comprises a first portion having a first width and a second portion having a second width. The system according to claim 1.

6. The first shaft among the plurality of shafts has a portion in which the cross-sectional width decreases at a rate that gradually decreases in the height direction of the first shaft. The system according to claim 1.

7. The first shaft among the plurality of shafts has a portion in which the cross-sectional width decreases at a rate that gradually increases in the height direction of the first shaft. The system according to claim 1.

8. The system further comprises channels located beneath the aforementioned multiple shafts, The channel has a height greater than or equal to the minimum cross-sectional width of the plurality of shafts. The channel is configured to collect one or more second assay components having a second width smaller than the minimum cross-sectional width of the shaft through which the assay components pass, and to dispense the second assay components to a second dispenser having a plurality of second shafts. The system according to claim 1.

9. Further comprising the second dispenser, Each of the plurality of second shafts has a second variable cross-sectional width, and the second minimum cross-sectional width of each of the plurality of second shafts is smaller than the minimum cross-sectional width of the shaft. Each of the plurality of second shafts is designed to release a single second assay component having a second width greater than the second minimum cross-sectional width within the second shaft. The system according to claim 8.

10. The dispenser is provided with an outlet from which one or more third assay components are dispensed, and the third assay component has a third width that exceeds the opening width of the corresponding opening of the shaft. The system according to claim 1.

11. The single assay component is a bead or a cell. The system according to claim 1.

12. A dispenser having multiple shafts, Each of the shafts has a variable cross-sectional width and is capable of releasing a single assay component having a width greater than the minimum cross-sectional width within the shaft. The dispenser is mounted on or above the assay apparatus such that, when the single assay component is released from the dispenser, only the single assay component is held in a single well on the assay apparatus, and each of the shafts is aligned with the single well on the assay apparatus. Dispenser.

13. The first of the plurality of shafts has a frustum-shaped or hourglass-shaped contour. The dispenser according to claim 12.

14. The first shaft among the plurality of shafts comprises a first portion in which the width gradually decreases in the height direction of the first shaft, and a second portion in which the width gradually increases in the height direction of the first shaft. The dispenser according to claim 12.

15. The first shaft among the plurality of shafts has a circular cross-section, and the cross-sectional width of the first shaft is the diameter of the circular cross-section. The dispenser according to claim 12.

16. The first shaft among the plurality of shafts comprises a first portion having a first width and a second portion having a second width. The dispenser according to claim 12.

17. The first shaft among the plurality of shafts has a portion in which the cross-sectional width decreases at a rate that gradually decreases in the height direction of the first shaft. The dispenser according to claim 12.

18. The system further comprises channels located beneath the aforementioned multiple shafts, The channel has a height greater than or equal to the minimum cross-sectional width of the plurality of shafts. The channel is configured to collect one or more second assay components having a second width smaller than the minimum cross-sectional width of the shaft through which the assay components pass, and to dispense the second assay components to a second dispenser having a plurality of second shafts. The dispenser according to claim 12.

19. The dispenser is provided with an outlet from which one or more third assay components are dispensed, and the third assay component has a third width that exceeds the opening width of the corresponding opening of the shaft. The dispenser according to claim 12.

20. The single assay component is a bead or a cell. The dispenser according to claim 12.

21. The first shaft among the plurality of shafts includes a first portion in which the cross-sectional width gradually decreases in the height direction of the first shaft, and a second portion adjacent to the first portion in which the cross-sectional width remains constant throughout. The assay component corresponding to the first shaft is held on the first shaft at the boundary between the first portion and the second portion. The system according to claim 1.

22. Each of the single assay components within the shaft has a width between a first threshold width and a second threshold width. The system according to claim 1.

23. Each of the single assay components within the shaft has a width within the threshold range of the target width. The system according to claim 1.

24. The threshold range is 10 percent of the target width. The system according to claim 23.

25. The dispenser further comprises one or more spare channels. Beads with a width smaller than the second threshold width pass through the spare channel, and beads with a width larger than the second threshold width are held and sent to the collector. The aforementioned auxiliary channel is located upstream of the plurality of shafts. The system according to claim 22.

26. The width of the auxiliary channel is constant throughout the entire height of the auxiliary channel, or it gradually decreases in the height direction of the auxiliary channel. The system according to claim 25.

27. The width of the pre-channel includes the width of the upper opening of the pre-channel and the width of the bottom opening of the pre-channel, wherein the width of the fourth is smaller than the width of the fifth. The system according to claim 25.

28. The auxiliary channel further comprises valves located at the inlet and outlet, respectively. The system according to claim 25.

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