Reversible transfer devices and uses thereof

Abstract

This disclosure relates to reversible (attachable and detachable) transfer devices and methods that facilitate high-throughput effector assays. The assays disclosed herein provide for an assay device and a replaceable transfer device. The transfer device can be replaced on the assay device with any of several interchangeable replacement devices.

Description

Attorney Docket No.009775.00044\WO REVERSIBLE TRANSFER DEVICES AND USES THEREOF Technical Field

[0001] This disclosure relates to reversible (attachable and detachable) transfer devices and methods that facilitate high throughput effector assays. Related Application

[0002] This application claims the benefit of U.S. Provisional Application 63 / 699,739, filed on September 26, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Background

[0003] Despite research and technological advances in single-target analysis (e.g., single cell analysis), there continues to be a gap in knowledge between attributing functional changes occurring during an assay to molecular data achieved at the end of the assay, especially in high-throughput assays. For example, when the assay is a cellular assay, conventional single-cell analysis may provide information on the genetic profiles of an analyzed cell and / or a limited proteomic profile. Still, such analyses often fail to include information regarding the biological and chemical changes that occur during the assay, especially if these changes are transient in nature. Relating the functional characteristics of individual cells or other targets to their underlying changes in biological and chemical constitution during the assay would be incredibly valuable for understanding, modeling, and engineering certain areas of biology and the underlying disease etiology and pathology.

[0004] In addition, the ability to evaluate and characterize different targets originating from a population of targets, such as a heterogeneous population of cells, and subject each target to the same stimulus or perturbation may provide a useful stratification that will further elucidate how different targets express individual functionality in response to the same stimulation or perturbation. In this way, the data collected as to functionality changes that are unique to a given target may help translate the differential functionalities of different targets into specific classes (e.g., specific cancer cells, neurons, epithelial cells, adipose cells, and the like). Thus, the ability to relate a single target with at least one aspect of its changes in functionality during the assay is of exceptional importance.

[0005] Due to the diversity of the heterogeneous population of targets tested, there is an inherent difficulty in correlating data such as mRNA obtained from this multiplicity of targets (e.g., cells) and associating the biological or chemical components of that data to a givenAttorney Docket No.009775.00044\WO target and to a given nanowell. Specifically, the recovered components from each nanowell are pooled with the recovered components from the other nanowells used in the assay. In the case of nucleic acids, these are sent for sequence readouts (for nucleic acids) using state of the art versions of sequencers, techniques, and technology. The difficulty arises due to the multiplicity of assays conducted, coupled with the pooling of nucleic acids and / or biochemical components. This pooling requires that the data from a single piece of nucleic acid must be assigned to a specific effector and a specific target in a specific nanowell for that information to be useful.

[0006] To further complicate matters, in a heterogeneous population of thousands to millions of targets, the technician often does not know which targets are the same, similar, or different without doing destructive analyses of each of the targets. In doing so, the skilled artisan may now be aware of the character of the destroyed target, such as a cell, but cannot attribute that character to any other targets / cells in that population unless, of course, destructive analysis of such other cells indicates that they are similar or the same. However, as before, such a correlation cannot be made without destroying the targets. As such, destructive analysis limits the information generated from the target and provides no information as to what other targets in that population may be the same or related.

[0007] Taken together, there is a lack of technology that can associate biological and / or chemical changes that occur during functional changes in a target arising from interaction with an effector (e.g., a stimulus or perturbation). These functional changes encompass alterations in biology and chemistry, including the expression of proteins, peptides, hormones, cytokines, chemokines, and nucleic acid components. Further, to obtain meaningful information, there is a need to correlate a target’s response to a given effector to a specific assay well and to correlate that information from a given well with that obtained from other wells. Notably, the technician must be able to associate different changes in target functionality arising from exposing a different effector in each well with common targets or with the same effectors in each well but using a different target for each well.

[0008] Human tissues are comprised of a highly heterogeneous mixture of cells interacting and regulating each other. There are many instances that contacting a target with an effector will produce a specific response in that target based on the identity of the effector, regardless of whether the contact is initiated in vivo or in vitro. Such provides a predictable correlation between assay results and the corresponding results in vivo.

[0009] As such, there is a need to comprehensively understand individual targets, their molecular signatures, and their environment as a basis to assess the modulation of theirAttorney Docket No.009775.00044\WO functionality by an effector in the development of novel pharmaceutical compounds. In addition, changes in target functionality are a foundational cornerstone in understanding the precise biology of disease states, which, in turn, provides a reasoned approach to understanding the etiology and pathology of the disease, as well as how best to treat it.

[0010] High-throughput single-target multiomics have been bolstered by innovations in a myriad of technologies. Despite progress in this field, there remains a gap in knowledge as to the change in target functionality and / or one or more features of individual targets in response to exposure to an effector. And further, there is a lack of understanding regarding how the change in functionality and / or one or more features initiates or continues a cascade of biological events within a complex living system. Absent knowledge of the change in functionality and / or one or more features of the target in response to a given effector, the relevance of such a change is not likely to be understood. At a basic level, there is an ongoing need for an understanding of how to correlate a change in functionality and / or one or more features of a specific target subject to a given effector to the cascade of biological events flowing therefrom.

[0011] Fundamentally, understanding multiple aspects of target biology in a high- throughput screening assay requires the ability to load a single target into each of a multiplicity of wells in an assay device. In many cases, there is a further need to load a defined number of targets and effectors (or a combination of effectors) into a single well, which often have sizeable differences in volume, density, fragility, and the like. Target biology, however, is a complex phenomenon in which a perturbation can create a cascade of biological events, many of which are transient in nature. Certain functional changes can be observed and recorded, such as changes in cell morphology that occur during the assay. However, many other changes occur only at the molecular level and, as such, are typically unobserved. This means that the technician can observe morphological changes in the cell during an assay, but these are merely the consequences of the underlying unobserved molecular changes. As such, there is an ongoing need to provide for devices and methods that can capture molecular changes in the cells’ biology in real time. Then, the technician can associate the consequences observed in the cell during the assay with these underlying changes in cellular biology, thereby correlating cause and effect in real time.

[0012] However, there is an unmet need for assay devices comprising a multiplicity of nanowells that are used in combination with a transfer component that can deliver one or more specific targets and / or effectors (e.g., a single target and / or effector) to each of the nanowells used in assay devices, which transfer component is reversibly placed on and offAttorney Docket No.009775.00044\WO the assay device. Among other factors, the reversible delivery component used in conjunction with the assay device needs to allow access to the nanowells during the assay without disrupting the ongoing assay; or cause cross-contamination of the aqueous solution in one nanowell from contaminating other adjacent nanowells; or, when the interrupted assay is to be allowed to further proceed after assessing the nanowells, the interruption must not damage the assay or the assay components. Still further, the devices and methods must be able to correlate the results of changes in cellular functionality observed in thousands to millions of nanowells to a specific nanowell, as well as the specific perturbation agent used to initiate such changes. And all of this must be accomplished while conducting thousands to millions of assays simultaneously in individual nanowells. Summary

[0013] The assays disclosed herein provide for an assay device and a reversible transfer device that is attachable and detachable to the assay device. The reversibility of the transfer device allows for the interchangeability of the transfer device with a second device during the assay conducted on the assay device. Accordingly, the transfer device can be replaced on the assay device with any of a number of replacement devices that are likewise interchangeable, including exchanging a first transfer device with another device, such as a second transfer device, at any time during or after the assay. The interchangeability of such components facilitates the delivery of assay beads, cells, nutrients, etc., into the nanowells of the assay device. In some embodiments, the interchangeable replacement devices include different transfer devices, delivery devices, sealing devices, and the like that are reversibly attached to the assay device.

[0014] This reversible attachment also permits the methods disclosed herein whereby the technician can access the aqueous surface of the assay wells during the assay. In some embodiments, the technician can add beads, cells, nutrients, etc., into the nanowells, alter assay conditions, or extract a portion of the aqueous solution in the nanowells for testing.

[0015] The ability to remove a portion of the assay solution during the assay allows for methods that can assess changes in assay conditions, including changes in cellular functionality, during the assay, and correlate these changes to a specific individual nanowell and components thereof.

[0016] Also described herein are methods, devices, and components that facilitate the identification of specific cells from a population of cells, such as a population of heterogeneous cells, where each of the nanowells comprises a single cell taken from theAttorney Docket No.009775.00044\WO population of heterogeneous cells.

[0017] This disclosure provides, in part, for devices and methods that facilitate high- throughput assays and, in particular, to assays where the devices permit assessing one or more changes in target functionality due to contact with an effector during the assay. These devices and methods further allow the technician to correlate changes in dynamic and static information (as defined herein below) arising from contacting an effector with a target in an individual identifiable nanowell in the assay device, where these changes occurred.

[0018] Each of the following embodiments are directed to aspects of individual components or combinations thereof used in the methods described herein.

[0019] In some embodiments, there is provided a transfer device (1) comprising: a bottom surface (2), a top surface (3), and side walls (4) wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, a height of D from the floor and a volume defined thereby, V-1, and each of which is configured to receive a defined number of effector moieties and / or targets; and inlet and outlet ports (7 and 8).

[0020] In some embodiments, a mating means is employed to attach said transfer device (1) to a second device, such as an assay device (9). In some embodiments, said mating means is reversible.

[0021] In some embodiments, said mating means comprises an adhesive (16) contacting the top surfaces of both devices; or a clamp; or a latch, one or more compatible protrusions (4B) on the transfer device (1) that mate with one or more compatible indentations (14A) on a second device (e.g., assay device (9)); or one or more compatible indentations on the transfer device (1) that mate with one or more compatible protrusions which align with indentations.

[0022] In some embodiments, the adhesive is a two-sided adhesive that can form a gasket.

[0023] In some embodiments, the mating means permit the contacting surfaces of the transfer device (1) and a second device to align a partial or complete hemispheric groove (17B) on the transfer device (1) which aligns with a partial or complete hemispheric groove (17A) on the second device which forms an tubular channel (17) wherein said tubular channel (17) is adapted to fit an O-ring or other compatible material (18) after combining the transfer device (1) with the second device. Preferably, another mating means as discussed herein (e.g., protrusions and corresponding indentations, clips, clamps, adhesives, etc.) is used inAttorney Docket No.009775.00044\WO conjunction with the O-ring or other compatible material (18) within the tubular channel (17), so as to apply pressure to the O-ring or other compatibile material (18), thereby forming a watertight and / or airtight seal.

[0024] In some embodiments, the mating means employ an adhesive (16), such as a two-sided pressure-sensitive adhesive, which attaches to the top surfaces (3 and 13) of both the transfer device (1) and the second device (e.g., assay device (9)), respectively, thereby forming a unitary device (11). In addition, the thickness of said adhesive (16) in the unitary device (11) defines the gap (G) between the two devices.

[0025] In some embodiments, the mating means between the transfer device (1) and the assay device (9) comprises hemispheric or partial hemispheric indentations (17A & 17B) that are aligned to each other to form a tubular channel (17) suitable to fit an O-ring (18). In some embodiments, the O-ring (18) is sized to fit within the tubular opening (19), thereby providing for a watertight / airtight fitting. In some embodiments, the transfer device (1) and the assay device (9) comprise partially hemispheric indentations (17A and 17B) of sufficient size to hold the O-ring (18) in place while providing a small distance between the two top surfaces (3 and 13), which defines a gap (G). In some embodiments, the O-ring (18) is fitted slightly larger than the tubular opening (19 ) such that when the transfer device (1) and the assay device (9) are aligned and combined, the O-ring (18), optionally compressed, forms said gap (G) defined by the distance that the O-ring (18) prevents the transfer device (1) from contacting the assay device (9) which gap (G) has a volume (V-1). In addition, the O-ring (18) forms a watertight barrier but for the inlet and outlet ports (7 and 8), and where the inlet port (7), the gap (G), and outlet port (8) provide for microfluidic communication therethrough.

[0026] In some embodiments, the mating means comprises at least one indentation or at least one protrusion (4B) on the transfer device (1) which mates with a compatible protrusion or compatible indentation (14A), respectively, on an second device, such as an assay device (9), to form a unitary assay device (11). See, for example, FIG.2.

[0027] In some embodiments, the transfer device (1) described above comprises at least one protrusion (4B). In some embodiments, the protrusion or protrusions (4B) extend vertically upward from the top surface (3) of the transfer device (1) to terminate in a portion of the top surface extending above the majority of the top surface (3).

[0028] In some embodiments, the transfer wells (5) of the transfer device (1) describedAttorney Docket No.009775.00044\WO above are enclosed in the area bound by said protrusion or protrusions (4B) or said indentation or indentations. Stated differently, the protrusion (4B) from or indentation into the top surface of the wall 4 is continuous along the entirety of the top surface of the encompasses the transfer wells (5) by a single continuous protrusion wall (4D) or trench, which may be a closed shape around the transfer wells, such shape may be, for example, circular, rectangular elliptical or oval. Alternatively, the protrusion (4B) or indentation can be a set of connected protrusion walls (4D) or trenches in said transfer device (1), which isolates the transfer wells (5) within the area defined by these protrusion walls (4D) or trenches.

[0029] In some embodiments, the protrusion or protrusions (4B) constitute a pillar or a set of pillars extending from said transfer device (1).

[0030] In some embodiments, a first set of protrusions (4B) constitutes a set of pillars that mate with a set of compatible indentations (14A) on a second device (e.g., assay device (9)). In some embodiments, when the protrusions (4B) comprise one or more pillars (4C), a second set of protrusions (4B) can be employed to form a continuous protrusion wall (4D) surrounding the transfer wells (5), thereby providing a separate mating means and a sealant means.

[0031] In some embodiments, the protrusion (4B) is a continuous protrusion that comprises a circular, oval, oblong, or other configuration (e.g., hourglass) that encompasses transfer wells (5) to be used in the transfer device (1). In some embodiments, the protrusions (4B) comprise a connected set of protrusion walls (4D) that form a polygon of any shape or size such as but not limited to a triangle, a rectangle, a trapezoid, a square, a pentagon, a hexagon, an octagon, and the like as well as any other configuration that is continuous or compatible with a set of indentations.

[0032] In some embodiments, the transfer device (1) is reversibly slidable with a second device in any one of the X, Y, or Z axes to form a unitary device (11).

[0033] In some embodiments, the transfer device (1) comprises one or more protrusions (4B) or indentations thereon which engage with indentations (14A) or protrusions on a second device, thereby allowing said devices to reversibly mate and, when mated, form a unitary device (11).

[0034] In some embodiments, the transfer device (1) comprises one or more indentations that extend vertically downward from the top surface (3) of the transfer deviceAttorney Docket No.009775.00044\WO (1) to form a cavity (4E) or a trench (4F) which terminates in bottom surface of the indentations which are above the bottom surface (2) of the transfer device (1).

[0035] In some embodiments, the transfer device (1) comprises said protrusion or protrusions (4B) which extend(s) laterally outward / upward from side wall (4) of the transfer device (1), wherein said protrusion (4B) forms a reversible mating mechanism with a second device comprising an indentation or indentations (14A) thereby forming a unitary device (11) when mated. See, e.g., FIGS.2-3 and 6.

[0036] In some embodiments, the transfer device (1) comprises said indentation or indentations (in addition or alternatively to the protrusion (4B)) which extend(s) laterally inward from side wall (4) of the transfer device (1) wherein said indentation or indentations in the side wall (4) of the transfer device (1) form a reversible mating mechanism with a second device comprising a compatible protrusion or protrusions (e.g., instead of, or in addition to, the indentation or indentations (14A)) thereby forming a unitary device (11) when mated.

[0037] In some embodiments, a transfer device (1) is reversibly attached to an assay device (9), to provide for a unitary assay device (11) wherein: a) said transfer device (1) comprises a bottom surface (2), a top surface (3), and side walls (4) wherein: said top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, and a volume, V-1, each of which are configured to receive only a defined number of effector moieties or targets; either at least one protrusion (4B) that extends outward from said transfer device (1); or at least one indentation that extends inward into said transfer device (1); and inlet and outlet ports (7 and 8) that are associated with the transfer device; b) an assay device (9) comprising a multiplicity of nanowells (15) each having a floor FL12, an opening diameter OD12, and a volume V-3, which, in combination, allow for conducting an assay in said nanowells (15), wherein said assay device (9) comprises either (i) at least one protrusion that extends outward from said assay device (9) and further wherein said top surface (13) of said assay device (9) is positioned such that it faces the top surface (3) of said transfer device (1); or (ii) at least one indentation (14A) that extends inward into said assay device (9); wherein said set of indentation(s) (14A) or protrusion(s) on the assay componentAttorney Docket No.009775.00044\WO mate with a corresponding set of protrusion(s) (4B) or indentation(s) on said transfer device (1) to form a conjoined unitary assay device (11); wherein said mating retains a gap (G) between the top surface (3) of said transfer device (1) and said top surface (13) of said assay component (9) wherein said gap (G) defines a volume (V-4) wherein said gap (G) has a vertical gap distance (D3) of about 500 microns or less; and further wherein said inlet and outlet ports (7 and 8) of said transfer device (1) are in fluid communication with an otherwise enclosed volume (V-4) thereby providing for microfluidic communication into said inlet port (7), through said volume (V-4) and out of said outlet port (8); provided that the opening diameter (OD1) of the transfer wells (5) of the transfer device (1) ranges from about 5% to about 60% of the opening diameter (OD12) of the nanowells (15) of the assay device (9) and further provided that upon mating of said assay device (9) and said transfer device (1), a unitary assay device (11) is provided wherein said transfer wells (5) align to said nanowells (15) on a one-to-one basis such that a projection of an opening of each transfer well (5) onto a corresponding nanowell (15) is encompassed by the area of said nanowell (15).

[0038] In some embodiments, the mating means between the transfer device (1) and the assay device (9) comprises hemispheric or partial hemispheric indentations (17A and 17B) that are alignable with each other to form a tubular opening (19) suitable to fit an O-ring (18). In some embodiments, the O-ring (18) is sized to fit within the tubular opening (19), thereby providing for a watertight fitting.

[0039] In some embodiments, the transfer device (1) and the assay device (9) comprise partial hemispheric indentations (17 and 17A) that are alignable with each other and are of sufficient size to hold the O-ring (18) in place while providing an opening gap (G) between the top surface (3) of the transfer device (1) and the top surface of the assay device (9) when the O-ring (18) is placed therein. In some embodiments, the O-ring (18) is fitted slightly larger than the tubular opening (19) such that when the transfer device (1) and the assay device (9) are combined and the partial hemispheric indentations (17A and 17B) are aligned, then the O-ring (18) can be inserted. When the transfer device (1) and the assay device (9) are aligned, the O-ring (18) can be inserted therebetween by applying a modest amount of inward pressure using, e.g., a clamp. Using the appropriate pressure, gap (G) is formed, and the size of that gap (G) is defined by the distance that the O-ring (18) prevents the transferAttorney Docket No.009775.00044\WO device (1) from contacting the assay device (9). In addition, the O-ring (18) forms a watertight barrier in gap (G) but for inlet and outlet ports (7 and 8). When so constructed, inlet port (7), gap (G), and outlet port (8) form a microfluidic channel as depicted by the arrows in FIG.2.

[0040] In some embodiments, gap (G) can be formed by appropriately sizing the protrusions (4B or 14B) and indentation (4A or 14A) such that gap (G) is formed by use of a protrusion (4B or 14B) that is slightly longer than the indentation (4A or 14A). When the protrusion or protrusions (4B or 14B) encompass or encompass the transfer wells (5), this results in a gap (G) which is watertight but for inlet and outlet ports (7 and 8). The height of said gap (G) is predicated on the excess length of protrusions (4B or 14B) which extend beyond the depth of indentations (4A or 14A). In some embodiments, the protrusions (4B or 14B) on said top surface (3) have an overview configuration that is circular, elliptical, oval, polygonal, or any other configuration that encompasses transfer wells (5). As above, when so constructed, inlet port (7), gap (G), and outlet port (8) form a microfluidic channel as depicted by the arrows in FIG.2.

[0041] In some embodiments, a protrusion (4B or 14B) that encompasses transfer wells (5) can be formed using a sealant such as an adhesive (16) which can include a thin film of solidified glue, or a 2-sided pressure-sensitive layer, or an O-ring (18) or a similar component placed inside the open tubular space (19) formed on the top surfaces of the transfer device (1) and the top surface (13) of the assay device (9) to seal gap (G) but for the inlet and outlet ports (7 and 8). Likewise, a gasket or a similar device can be used as the sealing means. In some embodiments, the sealing means employed are reversible.

[0042] In some embodiments, said indentation or protrusion (4B) on said transfer device (1) is continuous with the top surface (3) to form a surface feature continuous wall (4D) in a closed shape, such as a circle, oval, or oblong configuration. In other embodiments, said indentation (14A) on said assay device (9) corresponds to the protrusion wall (4D) that are connected to form a polygon or other similar configuration.

[0043] In some embodiments, the transfer wells (5) are positioned on the transfer device (1) to align with the nanowells (15) of the assay device (9) once the two components are coupled. In some embodiments, the transfer wells (5) are substantially smaller than the nanowells (15) such that the contents of a transfer well (5) can be reliably transferred to the nanowell (15). In some embodiments, the area of the opening (OD1) of the transfer well (5)Attorney Docket No.009775.00044\WO ranges from about 10 to about 60 percent of the area of the opening (OD12) of the nanowell (5) when the two are aligned. In some embodiments, the entirety of the opening (OD1) of the transfer well is within the area (OD12) of the nanowell (15).

[0044] In some embodiments, a unitary device (11) comprises a transfer device (1) and a second device, such as an assay device (9). In some embodiments, the two components of the unitary device (11) are reversibly attached to each other. In all cases, the inlet and out ports (7 and 8) are maintained on the transfer device (1).

[0045] In some embodiments, a sliding mechanism is used to form a unitary device (11) whereby a second device couples to a transfer device (1) simply by sliding the two components together until the mating means are aligned thereby forming a unitary device (11). In some embodiments, this mating means is reversible. In some embodiments, the sliding mechanism is complemented by a reversible alignment component whereby, when so aligned, the transfer wells (5) of the transfer device (1) and the nanowells (15) of the formed unitary device (11) are aligned. The alignment component can include a set of markers or a locking mechanism that locks when the two devices are aligned with the transfer wells (5) being placed completely under the nanotubes described herein. In some embodiments, the locking mechanism is reversible. In some embodiments, the locking mechanism is either integral to or in addition to the alignment mechanism. In either case, the locking mechanism maintains the alignment of the transfer wells (5) with the nanowells (15) of the assay device (9) is retained during the delivery of the effector or the target.

[0046] In some embodiments, the locking mechanism is a container in which the aligned unitary device (11) is inserted. In some embodiments, at least one pair of opposite side walls of the container is adjustable and can be adjusted inward toward each other to apply compressive pressure on the opposing side walls of the unitary device in contact therewith. In some embodiments, both pairs of opposing side walls of the container are adjustable inward. In some embodiments, the floor and / or the ceiling of the container can be adjusted inward toward the other, thereby applying vertical pressure on the unitary device (11), which, when an O-ring (18) is employed, can result in compression of the O-ring (18). In some embodiments, a combination of some or all of the compression elements described herein are used.

[0047] In some embodiments, the locking mechanism comprises a latch or a series of latches that lock the two devices together. In some embodiments, the locking mechanismAttorney Docket No.009775.00044\WO comprises a pop-up on a slidable member and a hole on the opposing slidable member such that when aligned, the pop-up and the hole are also aligned, resulting in the pop-up expanding into the hole to lock the position. In such cases, the pop-up is reversible by merely pressing down.

[0048] In some embodiments, the locking mechanism combines the alignment aspect with locking the device into a fixed set of X, Y, and Z coordinates, thereby ensuring that the transfer wells (5) remain aligned with nanowells (15). In such cases, the locking mechanism cradles the unitary device (11) while also ensuring that the transfer wells (5) are aligned under the nanowells (15).

[0049] In some embodiments, the unitary device employs a release that allows these two devices to be slidably disengaged. In some embodiments, the slidable engagement / disengagement can be achieved in any of the X axis, Y axis, and Z axis. In one embodiment, slidable engagement includes a tongue (29) and groove (29A) configuration to form a unitary device (11) as per FIGS.7A and 7B.

[0050] Alternatively, a unitary device can also be formed as per FIGS.7A-7C. These figures illustrate a transfer device (1) comprising transfer wells (5), inlet port (7), outlet port (8), a tongue (29A) located at both opposite ends of the transfer device (1) and a protrusion wall (4D) that extends upward but only on the proximal side of transfer device (1) to a height equal to gap (G) and connects each opposite end having tongue (29A) but, again, only on the proximal side of transfer device (1). FIG.7B illustrates an assay device (9) having nanowells (15), a groove (29A) that mates with a tongue (29), which is slidably engageable. In addition, the distal side of the assay device also has a protrusion wall (4D), the height of which matches that of the gap (G). As illustrated, a one-sided pressure-sensitive adhesive (16) is used to generate a fluid retention area under the position of the nanowells (when faced downward).

[0051] In some embodiments, an alignment mechanism can be used after the unitary device is formed. This alignment mechanism ensures that the transfer wells (5) of the transfer device (1) align over the nanowells (15) of the assay device, as well as ensures that the water-tight nature of the microfluidic channel is not compromised. When so used, the alignment mechanism is reversible and, once the assay is complete, the alignment mechanism is reversed, and the transfer device (1) can be removed from the assay device (9) merely by sliding the two apart. In some embodiments, alignment of the transfer wells (5) of a transferAttorney Docket No.009775.00044\WO device (1) under the nanowells (15) of an assay device (9) can be achieved by applying a first colored surface (13) that circumscribes the nanowells (15) with a thin layer (e.g., a nanolayer) of, e.g., a yellow dye. The corresponding area around the transfer wells (5) on the top surface (3) of the transfer device (1) can be colored with a thin layer (e.g., a nanolayer) of, e.g., a blue dye. Alignment is ensured when a visibly green signal is observed by light passing through the otherwise transparent devices. All of these steps can be conducted robotically.

[0052] In some embodiments, the transfer device (1) comprises an inlet port (7) and an outlet port (8) on the same surface. In some embodiments, the same surface is the top surface (3).

[0053] In some embodiments, the transfer device (1) is a standalone device. That is to say that the transfer wells (5) of said device (1) can be loaded with an effector(s) or target(s) which can be monitored without having an assay device attached thereto, such as in US Patent No.10,000,732. In some embodiments, a cap or lid (not illustrated) is employed to assist in the transfer of the effector(s) or targets to the transfer wells.

[0054] In some embodiments, the transfer device (1) is interchangeable with a second device, such as a replacement device. Such devices include a second transfer device, a sequestering device such as a cap or lid, a perturbation bead transfer device, a capture bead transfer device, a nutritional delivery device, and the like. The replacement device is configured to mate with the assay device (9), preferably, in a reversible manner. Stated differently, the second transfer device (1) is designed to deliver a further assay component to each nanowell (15). The opening diameter (OD1) and volume (V-1) of the transfer wells (5) are sized to hold the desired number of effectors or targets. In many cases, the nanowells (5) of the assay device (9) contain a single target and one or more effector moieties per transfer well (1), especially when the assay is a cellular assay. Transfer of a different assay components, which is done in multiple steps, may require that the opening diameter (OD1) and volume (V-1) of transfer wells (5) to be used in each step are necessarily different for each of these components used. In such cases, it would be beneficial to have a separate transfer device (1) loaded with a target or effector prepared beforehand to minimize time in combining the required assay components into the nanowells (15) and to ensure that the proper components were added.

[0055] For cellular assays, in addition to a single cell, other assay components are often used alone or in combination with the cell, such as a perturbation bead, a capture bead, spatialAttorney Docket No.009775.00044\WO indices, and the like. Hence, to conduct a cellular assay or other assays that also require the use of multiple assay components in the nanowell (5), a replacement device can be used. Such replacement devices are preferably interchangeably with the transfer device (1) and are reversibly attached and detached from the assay device (9). In some embodiments, these replacement devices that add additional assay components to the nanowells (15) can be preloaded such that upon removal of a prior transfer device (1), a further transfer device (1) is loaded onto the assay device (9) to form a unitary device (11). Once loaded, the delivery of an assay component required for the assay can be added to the nanowells (15) via replacing the initial transfer device (1) with a replacement device that can be a second transfer device (1).

[0056] In some embodiments, there is provided a sequential method for transferring a cell and an additional assay component into a nanowell (15) of an assay device (9), which method comprises: a) selecting a transfer device (1) comprising a bottom surface (2), a top surface (3), and side walls (4) wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, a height of D and a volume defined thereby, V-1, and each of which is configured to receive a single cell wherein said transfer device (1) further comprises inlet and outlet ports (7 and 8); b) selecting an assay device (9) wherein said assay device comprises a multiplicity of nanowells (15) each of which comprises an opening diameter (OD12) and a volume (V-4), wherein said nanowells (15) comprise a cell-adhering protein or peptide adhering to the interior surface of said nanowell (15); c) reversibly attaching said assay device (9) to said transfer device (1) to form a unitary device (11) wherein the top surface (3) of said transfer device (1) comprising transfer wells (5) is faced upward while the top surface of said assay device (9) is facing downward; d) aligning the nanowells (15) of said assay device (9) over the transfer wells (5) such that the opening diameter (OD12) of the nanowells (15) encompasses the entirety of the opening diameter (OD1) of the transfer wells, wherein said attachment further defines a gap (G) having a volume V-4 which is connected by said inlet port (7) and said outlet port (8), thereby defining a microfluidic channel over said transfer wells (5); e) creating a flow of an aqueous solution comprising a heterogeneousAttorney Docket No.009775.00044\WO population of effector moieties into the inlet port (7), through gap (G), and out of outlet port (8), wherein said flow rate is controlled to allow at least a portion of said moieties to deposit in said transfer wells (5); f) continuing the flow of said aqueous solution until at least a portion of said transfer wells (5) are populated with a single effector; g) terminating the flow of said aqueous solution; h) inverting the unitary device (11) whereby a single cell in a transfer well is transferred into the nanowell (15) aligned thereto, wherein said cell so deposited adheres to said protein or peptide in the nanowell; i) removing the transfer device (1) from the unitary device (11); j) attaching a second transfer device (1) to said assay device (9) wherein said transfer wells (5) of said second transfer device (1) are sized to accept an effector and further wherein said attachment further defines a gap (G) having a volume V-4 that is connected by said inlet port (7) and said outlet port (8) thereby defining a microfluidic channel over said transfer wells (5); k) repeating steps d) through i) to provide for an assay device comprising a multiplicity of nanowells (15) each comprising a single cell and a single effector; l) continuing the flow of said aqueous solution until at least a portion of said transfer wells (5) are populated with an effector; m) terminating the flow of said aqueous solution and reversing the positions of the assay device and the second transfer device, thereby transferring the effector into the nanowell (15) aligned with the transfer well (5).

[0057] In some embodiments, the interior of the assay wells can be a plasma surface treated to render the surface hydrophilic. In some embodiments, the plasma-treated surface is coated with a cell-adhering layer, such as poly-L-lysine, poly-D-lysine, collagen, fibronectin, and the like.

[0058] In some embodiments, the process is further repeated by optionally replacing the second transfer device (1) with a third transfer device (1) having one or more additional effectors, targets, perturbation beads, capture beads, location indices, and assay reagents which are to be delivered to the nanowells (15). In such cases, the effector transferred with the additional transfer device (1) is optionally a perturbation bead or a capture bead. Furthermore, said bead can be a magnetic bead such that it can be retained in the nanowell (15) when inverted simply by use of magnetic forces.Attorney Docket No.009775.00044\WO

[0059] In high throughput assays, after the assay components have been added into the nanowells (15), it is conventional to employ a protective cover (not illustrated) over the top of the aqueous assay solution in the each of the nanowells (15). This cover can be added directly to the top of the assay device (9) and can include an oil, a wax, or an epoxy, or other similar compatible materials. In some embodiments, an oil is employed to cover the nanowells (15), which can be added by use of a transfer device (1). Alternatively, a wax or epoxide can be placed over the top surface (15) of an assay device (9).

[0060] As to the use of an oil, at the completion of the assay, most of this oil is typically removed from the top of the nanowells (15) (e.g., by pipetting) leaving a fine film of oil remaining over the aqueous assay solution and on the top surface (13) of the assay device (9). When the oil employed is a volatile oil such as fluorocarbon oils, the fine film can be allowed to simply evaporate, thereby exposing the aqueous assay solution in each nanowell (15). However, when the oil used is insufficiently volatile, it is also conventional to remove this thin film of oil by adding a detergent, which transfers the thin oil film into a clear suspension of oil droplets in the water solution. Suffice it to note that in assays where static functional information is adequate, the addition of a detergent will break the oil layer by forming an emulsion. In such cases, the addition of a detergent will also lyse cells and adversely alter effectors and / or targets that are susceptible to damage or destruction by detergents. Hence, this procedure is typically available only once, and once employed in an assay using detergent-susceptible components, the assay is complete.

[0061] In some embodiments, this disclosure provides for methods for reversibly applying and removing a cover over the nanowells (15) in an assay device (9) part of a unitary device (11) wherein said nanowells (15) of said assay device (9) comprise an aqueous assay composition. In such methods, the unitary device (11) must first be disassembled into the assay device (9) and the transfer device (1) (or replacement device). Once disassembled, the cover is exposed, which allows for removal and replacement as needed while not disturbing any of those assay components in the aqueous assay composition, which are susceptible to detergents. Specifically, this disclosure is predicated, in part, by the discovery that the confinements means provided by the unitary devices (11) described herein found between the top surface (13) of the assay device (9) and the top surface (3) of the transfer device (1) after mating allows for a cover to be included therein which cover is maintained over the aqueous assay composition in the nanowells (15). The cover is removable, which then exposes the aqueous assay composition. At such times that the cover is removed, theAttorney Docket No.009775.00044\WO technician can access this composition, thereby permitting an assessment of functional changes in the target, including chemical, biological, and genetic changes that were previously unavailable.

[0062] In addition, as different assay components are often required in an aqueous assay composition, the interchangeability of the transfer devices allows for an assembly line approach whereby a first transfer device (1) delivering, e.g., cells to the nanowells (15) of the assay device (9) can be readily removed and a second transfer device (1) can be preloaded with the additional assay component to be included and then attached to the assay device (9). The fact that the transfer device (1) includes both the inlet port (7) and the outlet port (8) as well as gap (G) formed by attaching a cap or lid thereto, permits using the microfluidic channel formed by this combination to preload the additional assay component into a separate transfer device (1) for subsequent attachment to the assay device (9) and delivery of the additional assay component into each of the nanowells (15).

[0063] As to specifics, when the cover is a volatile oil, then such can be formed after addition of aqueous assay composition, including the assay components, into the nanowells (15) of the assay device (9) using one or more transfer devices (1). When completed, the transfer device (1) used to deliver the last assay component to the nanowells (15) can now be used to deliver a volatile oil composition to cover the aqueous assay compositions in the nanowells (15). Alternatively, a separate transfer device (1) can be used in place of the transfer device (1) previously used to deliver the last assay component. The assay is then initiated and continued until such time or times that sampling of the aqueous assay composition in one or more nanowells (15) is desired. To access the nanowells (15), the technician can remove a majority of the volatile oil using conventional techniques, such as pipetting and draining, which leaves an oil film. This film is allowed to evaporate under the ambient assay conditions in use. In some embodiments, when the oil layer is relatively thin, there is no need to remove some of the oil from that layer. Rather, exposure to ambient assay conditions is sufficient to remove all of the oil, including the oil film.

[0064] In some embodiments, ambient assay conditions include a temperature of from about 15º to 45 ºC and an oil having a vapor pressure of from about 2 to about 15 mm of Hg at 37 ºC. As used herein, an oil is considered “volatile” when it evaporates under such ambient assay conditions without the need for applying external heat. In some embodiments, the oil has a thickness of less than about 500 microns. In some embodiments, after removing a portion of the oil to form an oil film, this film has a thickness of about 20 microns or less,Attorney Docket No.009775.00044\WO often about 15 microns or less. Due to its volatile nature, the oil film evaporates, leaving exposed aqueous solutions in the nanowells, which can now be accessed for sampling, the addition of reagents, nutrients, and the like. As desired, an oil layer is reestablished, preferably by attaching a replacement device with inlet (7) and outlet ports (8) and applying the oil layer using these ports.

[0065] Once sampling is completed and samples analyzed to generate data corresponding to each nanowell, the data from each nanowell (15) is correlated back to that nanowell as described above. For example, the perturbation bead or capture bead can bind to those components that are to be scrutinized. In the case where the target is a cell, such components can be extracellularly generated proteins, peptides, cytokines, chemokines, and the like found in the aqueous composition. Alternatively, the assay conditions can be modified during this step to include additional reagents, additional effector moieties, changes in pH, temperature, salt or buffer concentrations, and the like.

[0066] In some embodiments, the oil used is a hydrophobic fluorocarbon oil. In some embodiments, this oil can be retained on top of the aqueous layer even if it has a density greater than water as the surface tension attraction between the hydrophobic top surface of the assay device and the fluorocarbon oil offsets the density differential that would otherwise suggest that the oil should sink to the bottom of the nanowells. Suitable fluorocarbon oils include, by way of example only, Fluo-Oil 7500 / Fluo-Oil 40 / Fluo-Oil 135 / Fluo-Oil 200 (emulseo, Parc AMPERIS, Bâtiment BAYA, 8 rue Adrienne Bolland, 33600 Pessac, France), FC-40 / FC-70, Perfluorodecalin (MilliporeSigma, 400 Summit Drive, Burlington, MA 01803, United States). Likewise, suitable hydrophobic materials used as the top surface of the assay device include, by way of example only, wax, diamondoids (adamantane, dimantane, triadamantane, epoxy, and the like.

[0067] As to a cover made using a wax or an epoxy, these materials can be added onto the top surface of the assay device (9). For example, a wax cover can be pre-sized to fit over the top surface (13) of the assay device (9). Suitable waxes include microcrystalline waxes, paraffin wax, low-melting point silicone waxes (available from Genesse Polymers Corporation, Burton, Michigan, 48529, USA), and the like. For example, at a temperature close to the melting point, a low-melting-point silicon wax can be flowed through the inlet port (7) and onto the top surface (13) of the assay device (9), and there solidify over the top surface (13), including the top of the aqueous solution in the nanowells (15).Attorney Docket No.009775.00044\WO

[0068] Accordingly, in some embodiments, there is provided a unitary device (11) comprising an assay device (9) and a transfer device (1) wherein the assay device (9) comprises: a) a top surface (13) having a multiplicity of nanowells (15) facing upward wherein the nanowells (13) comprise an aqueous assay composition; and b) a transfer device having a top surface (3) positioned above said assay device (9) facing downward; c) confinement means extending above said top surface (13) of said assay device (9) and below the top surface (3) of said transfer device (1) that maintains a 3- dimensional space suitable to hold a water-insoluble cover therebetween; and d) a water-insoluble cover retained in said confinement means wherein said cover remains on the top surface (13) of the assay device (9), including the top surface of the aqueous assay solution in the nanowells (15), resulting in a protective covering over the nanowells (15).

[0069] In some embodiments, there is provided an assay device as described above, wherein said confinement means comprises an oil layer, a wax or an epoxy sheet which covers the top surface (13) of said assay device (9).

[0070] In some embodiments, the aqueous assay solution in said nanowells (15) comprises an effector and a target. In some embodiments, the target is a mammalian cell, and the effector is a perturbation compound reversibly bound to a perturbation bead, which comprises a plurality of substantially the same perturbation compound and a DNA barcode configured to identify a structure and / or a reaction step used to synthesize said compound. In some embodiments, the perturbation bead is coded with optically visible codes that identify the nanowell where the perturbation bead is located. In some embodiments, the optically visible code or codes for each nanowell are memorialized by an oligonucleotide or a set of oligonucleotides that are ligated together.

[0071] In some embodiments, the nanowells (15) in the assay device (9) are spatially indexed, which identifies each nanowell (15) by its X and Y axis coordinates.

[0072] In some embodiments, there is disclosed a method for reversibly applying and removing a cover positioned on a top surface (13) of an assay device (9) including over nanowells (15) found on said surface wherein said nanowells (15) comprise an aqueous assay composition including one or more detergent sensitive assay components, said method comprises: a) selecting an assay device comprising a hydrophobic surface (13) and aAttorney Docket No.009775.00044\WO multiplicity of nanowells (15) on said surface wherein said nanowells (15) comprise an aqueous assay solution including one or more detergent sensitive assay components and further wherein said top surface (13) of said assay device (9) comprises confinement means to maintain a volume of fluid over the top surface of the assay device (9) including over the nanowells (15); and b) adding a cover to said confinement means, which overlays the surface (13) of the assay device (9) and the aqueous assay solution in said nanowells (15), wherein said cover protects the contents of the individual nanowells; c) placing a cover over the assay device (9); d) initiating individual assays within each nanowell (15) comprises an aqueous assay solution; e) at one or more intervals during the assay procedure, remove the cover from said assay device (9), thereby exposing the aqueous assay composition in said nanowells (15); g) removing the cover, thereby exposing the aqueous assay composition; h) optionally i) modifying said aqueous assay composition by the addition of one or more assay components to said composition; or ii) removing components from said aqueous assay composition; or iii) removing an aqueous aliquot from said aqueous assay composition; and i) adding a cover over the top surface (13) of said assay device (9), including the top surface of said nanowells as per b) above.

[0073] In some embodiments, the method further comprises analysis of said assay components or the aliquot of aqueous assay composition removed from the nanowell (15) to assess functional changes in the target.

[0074] In some embodiments, said cover is an oil layer and a sufficient amount of the oil layer is removed from said surface (13), thereby leaving only a thin oil film on said surface (13). In some embodiments, said oil is a volatile oil and said thin oil film is maintained at ambient assay conditions to remove said oil film by evaporation.

[0075] In some embodiments, there is provided an assay method to screen a library of compounds to determine the effect of a perturbation compound from said library of perturbation compounds on a cell, which allows for reversible access to components of the assay in the nanowell (15) during the assay without disrupting the assay or the cell, said method comprising: a) selecting an assay device (9) comprising a multiplicity of nanowells (15) on saidAttorney Docket No.009775.00044\WO top surface (13) wherein said nanowells (15) comprise an aqueous assay composition and further wherein said top surface (13) of said assay device (9) comprises confinement means to maintain a volume of fluid over the top surface (13) of the assay device (9) including over the nanowells (15), wherein each nanowell (15) further comprises a perturbation bead which itself comprises a plurality of substantially the same, unique compound reversibly bound thereto, wherein said bead and said cell are maintained in an aqueous assay composition in said nanowells; b) adding a layer of a volatile oil to said confinement means, which layer overlays the top surface (13) of the assay device (9) and the aqueous assay solution in said nanowells (15), and optionally placing a lid or replacement device over the assay device (9); c) initiating the assay by releasing at least a portion of the compound from their respective perturbation bead, thereby allowing the free compounds to interact with the target or targets in the nanowell; d) at one or more intervals during the assay procedure, remove the lid or replacement device from the top surface (13) of said assay device (9) and optionally remove a portion of said exposed oil layer to provide for an oil film on said surface (13); e) maintain said assay device under assay conditions, thereby allowing said volatile oil or oil film to dissipate due to evaporation, thereby exposing the aqueous assay composition of each nanowell (15); f) identifying and accessing one or more nanowells (15) of interest without disrupting the cell and / or the nanowell (15) contents and removing an aqueous aliquot or one or more assay components from the nanowells (15) to assess functional changes in the cell; g) adding an oil layer over the top surface (13) of said assay device (9) including the top surface of said nanowells (15) and continuing the assay.

[0076] In some embodiments, one or more antibody-coated beads can be included in the nanowell, where each bead binds to a specific perturbation component expressed extracellularly by the cell due to contact with the effector. Alternatively, it can be a suitable antibody fragment or immunoglobulin fragment capable of binding to a desired target as well as other proteins or protein fragments. In some embodiments, the cells can be extracted from the nanowells and placed into a fresh medium or lysed in a fresh medium. The antibodies remaining in the assay solution can then be tested for the presence or absence of theAttorney Docket No.009775.00044\WO perturbation components, which are the binding partners of the antibodies used under conventional assay conditions. This allows the technician to evaluate changes in dynamic functionality while retaining the cell in fresh medium if one chooses to continue the assay, optionally in the presence of additional antibodies. In some embodiments, using antibodies to assess for extracellular expression of perturbation components, the use of an oil layer to isolate each nanowell from other nanowells is essential to prevent cross-contamination due to spillage. Brief Description of the Drawings

[0077] The drawings described herein are for illustration purposes only and are not intended to limit the scope of the present disclosure in any way. In order to facilitate an understanding of these drawings, the following reference numbers that are used in these drawings and are assigned to mean as follows:

[0078] For the sake of completion, the figures use the following reference numbers:Attorney Docket No.009775.00044\WO

[0079] FIG.1 illustrates a cross-section of a transfer device (1).

[0080] FIG.2 illustrates a cross-section of one example of a unitary device (11) which, in this case, comprises a mated pair of an assay device (9) with a transfer device (1), which are mated with protrusions (4B) on said transfer device (1) and compatible indentations (14A) on said assay device (9).

[0081] FIG.3 illustrates a cross-section of the device of FIG.2 having a mating means which can be an adhesive (16), a gasket, an O-ring (18), a plug, or an insert.

[0082] FIG.4A illustrates a cross-section of a unitary device (11) which comprises a transfer device (1) mated to an assay device (1) by interfacing with an O-ring (18) which, when so combined, the O-ring (18), optionally squeezed to maintain a water-tight fitting, defines the height of the gap (G). Mating, in this case, may be done by pressure, such as one or more clamps, holding devices, and the like. In this embodiment, a partially hemispheric groove is employed.

[0083] FIG.4B illustrates a cross-section of the reversibility of the device illustrated in FIG.4A by separating the transfer device (1) from the assay device (9).

[0084] FIG.4C illustrates a cross-section of a unitary device (11) which comprises a transfer device (1) mated to an assay device (1) by interfacing with an O-ring (18) which, when so combined, the O-ring (18), optionally squeezed to maintain a water-tight fitting, defines the height of the gap (G). Mating, in this case, may be done by pressure, such as one or more clamps, holding devices, and the like. In this embodiment, a triangular groove (17C) is employed.

[0085] FIG.4D illustrates the reversibility of the device of FIG.4C by separating the transfer device (1) from the assay device (9).

[0086] FIG.5A illustrates an overview unitary device (11) comprising a transfer deviceAttorney Docket No.009775.00044\WO (1) (depicted as the top component and including inlet port (7) and outlet (8), but without illustrating transfer wells (5)), using an adhesive (16) such as a two-sided pressure-sensitive adhesive as the mating means to connect the transfer device (1) to the assay device (9).

[0087] FIGS.5B-5D illustrate an exploded view of the individual components of the unitary device (11), which include the transfer device (1), the assay device (9), and the adhesive (16) (e.g., 2-sided pressure-sensitive adhesive). The inlet and outlet ports (7) and (8) are illustrated, whereas the transfer wells (5) and nanowells (15) are not.

[0088] FIG.6 illustrates a top view of a transfer device (1). Internal to side walls (4) are four connected protrusion walls (4D) that extend upward from the top surface (3) of the transfer device (1). These connected protrusions form a rectangular confinement means around the transfer wells (5) and are designed to mate with a corresponding set of indentations on the assay device (9) to form a unitary device (11), which encompasses transfer wells (5).

[0089] FIGS.7A and 7B illustrate a tongue and groove configuration for slidably engaging a transfer device (1) and an assay device (9) prior to formation of a unitary device (11).

[0090] FIG.7C illustrates the unitary device formed after slidably engaging these two components (1 and 9) into a unitary device (1). Detailed Description

[0091] This disclosure provides reversible transfer devices and methods that facilitate high-throughput effector assays and their combinations for use in such assays.

[0092] However, prior to describing the embodiments provided herein in more detail, the following terms are defined. Terms that are not defined herein have their accepted scientific meaning. Definitions

[0093] As used herein, the following terms have the following meaning. If a term is not defined, its generally accepted definition in the relevant technology shall govern.

[0094] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0095] As used herein, the term “optional” or “optionally” means that the subsequentlyAttorney Docket No.009775.00044\WO described event or circumstance can or cannot occur and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0096] As used herein, the term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations that may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or sub-value there between. Preferably, the term “about” when used with regard to a dose amount means that the dose may vary by + / - 5%.

[0097] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others.

[0098] As used herein, the term “consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0099] As used herein, the term “consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0100] As used herein, if the claims are to be construed for an inter partes proceeding in the United States only, the terms “preferable,” “preferred,” and “preferably” should be read out of any sentence. The terms are not to be used to limit claim construction in the United States.

[0101] As used herein, the term “effector moiety” or “effector” refers to any compound or population of elements or atoms, or an entity that can alter the functionality of a target when contacted with the target. Such an effector can comprise any one or more of the following: a compound, a cell, a bacteria, a virus, a fungus, a prion, an antibody, a chemokine, a cytokine, a hormone, an enzyme, a population of atoms such as radioactive atoms, toxic atoms, heavy metals, a nutrient, a reinvigoration or set of reinvigoration agents, and the like.

[0102] As used herein, the term “target” refers to any entity whose function, behavior, viability, and / or reproduction is potentially altered by an effector (to be assessed for alteration by the effector). Such targets include cells, antibodies, enzymes, chemokines, cytokines, and the like. In some embodiments, a cell is a bacterial cell, a viral cell, a fungal cell, or a plant,Attorney Docket No.009775.00044\WO animal or insect cell. In some embodiments, the cell is a mammalian cell and, preferably, a primate cell. In some embodiments, the cell is a human cell.

[0103] Non-limiting examples of effector moieties and targets that can be used in combination include the following:

[0104] As used herein, the term “compatible” as used with “protrusion” or “indentation” refers to its ability to properly mate with an indentation or protrusion on another device, thereby fitting one into the other. For the sake of completeness, if a protrusion and an indentation mate, then they are considered compatible, notwithstanding a failure to so indicate.

[0105] As used herein, the term “adhesive” refers to any manner of adhesive (16) such as glue, tape, including pressure-sensitive two-sided adhesive tape, or any solid, semi-solid or liquid material which results in adhesive layer connecting the surfaces of any two of an assay device (9), a transfer device (1), or a replacement device.

[0106] As used herein, the term “hemispheric groove” refers to a groove-like shape in a hemispheric form which encompasses a full 180° curve from one terminus to the other terminus. As used herein, the term “a partial hemispheric groove” refers to a groove that encompasses less than a full 180° curve from one terminus to the other terminus but, rather, contains a partial curve which is sufficient to cradle the O-ring (18) from either the bottom or top side of the partial groove, depending on orientation. Included within said term are grooves that approximate a hemisphere or partial hemisphere (17), which also hold an O-ring (18) and, as such, are functionally equivalent thereto. When so cradled, the O-ring (18) willAttorney Docket No.009775.00044\WO form a watertight seal and a gap (G) between the top and bottom surfaces. In some embodiments, the partial hemispheric groove (17) will span from about 90° to less than 180°.

[0107] Other suitable structures for use with an O-ring include polygonal structures, such as, but not limited to, triangular, rectangular (including squares), trapezoidal, pentagonal, and similar openings. Triangular openings (17C and 17D) are illustrated in FIGS.4C and 4D. In some embodiments, the triangular grooves (17C and 17D) have an angular opening sufficient to hold an O-ring (18) either directly or when squeezed. In some embodiments, the angular opening ranges from 30 to 150 degrees. In some embodiments, one of the said openings can be hemispheric or partially hemispheric, and the other can be polygonal.

[0108] As used herein, the term “a continuous protrusion” refers to a protrusion or protrusions (4B), which encompass the nanowells (15). In some embodiments, a continuous protrusion (4B) can be a single protrusion wall (4D) that encompasses the nanowells, such as in the form of a circle (cylinder), an ellipse, or an oval or other closed shape (e.g., in a plane parallel to a plane in which the nanowells are arranged).

[0109] As used herein, the term “encoded micro-components (EMC)” is micro- components that either individually or collectively define a code that is unique to that component or set of components. For example, a set of 20 different micro-components, each colored differently. Such differential coloring could be the same color with different color intensity, or different colors, or combinations of both. On each micro component, there is a symbol or image that is unique to that color and image. For example, a “$” could be printed on each of the 10 different colored micro-components. So, assuming that there are 40 different symbols (@, #, !, %, $, ^, &, *, a square, a triangle, a house, a car, etc. and also assuming that only two (2) micro-components are used per well, then the number of combinations becomes very large (e.g., [20 x 19] x 40 = over 15,000 different combinations exist. If only a single color and a single code are used in combination, then the number of possible different combination of symbols are used, the number is in excess of 10,000,000.

[0110] During or after the manufacture of these micro-components, each component is coded for its unique color or color intensity with a unique oligonucleotide strand, which is reversibly attached to the micro-component, and each symbol is coded with a unique oligonucleotide strand that is attached to the oligonucleotide strand for the color. For example, the oligonucleotide strand from the color can terminate in a GGGGGGGGG group,Attorney Docket No.009775.00044\WO whereas the oligonucleotide strands for the symbol is initiated with a CCCCCCCCC and terminated with a TTTTTTTTTT group. When used in a well, the poly G and poly C group will ligate to each other, whereas the poly T group is available to ligate to a poly A group on mRNA. In doing so, both the code for the color and the code for the symbol are bound to mRNA collected from a cellular target after lysing. Prior to assay completion, a picture of the X-, Y-indexed assay wells, each containing a unique oligonucleotide that, when sequenced, will determine the specific combination of micro components in a well that can be identified by the picture and then correlated to that indexed well. The mRNA collected associated with that oligonucleotide is then correlated to that specific well. This methodology allows for assigning individual assay wells to a specific EMC or set of EMCs by virtue of the sequenced oligonucleotide.

[0111] In some embodiments, instead of all being C’s, one can split the oligonucleotide into a hybridizing region and a barcode extension region. For example, CCCCCCCCC can be used to capture on GGGGGGGGG, and the rest of the oligonucleotide can be a unique barcode like ATCAG, which decodes a symbol. The same can apply to the G oligo, such as AGTACGGGG, in which AGTAC encodes the color. This allows for decodable information in both strands when extended. If the decodable information is not included with the hybridizing regions, it cannot be decoded because hybridization by itself does not code for either the color or the symbol.

[0112] As used herein, the term “nanowell” refers to assay wells having a maximum linear dimension of no greater than 1 mm and a volume of no more than 1 µL (1 microliter), and the term “maximum linear dimension” refers to the distance along the longest axis for that shape. For example, the diameter of an ellipse would be the semi-major diameter, and the diameter of a square would be from the top corner of the square to the opposite lower corner of the square.

[0113] As used herein, the term “multiplicity of nanowells” refers to any device or surface that comprises at least 96 wells; or at least 1,000 wells; or at least 10,000 wells; or at least 100,000 wells. Typically, the upper limit of wells is up to the size of the device or surface, the number of wells per unit area, and the minimal distance between wells to mitigate against spill-over. In general, assay devices comprising a multiplicity of nanowells can contain up to 5,000,000 wells per device. In some embodiments, the number of nanowells in said multiplicity of nanowells is between about 1,000 to 5,000,000, or between about 10,000 and 3,000,000.Attorney Docket No.009775.00044\WO

[0114] As used herein, the term “oil” refers to a fluorocarbon or hydrocarbon-based material that is a liquid at temperatures from at least from about 0º to about 180ºC. In the case of hydrocarbon-based oils, these comprise carbon and hydrogen atoms wherein the carbon atoms can be joined by single bonds, double bonds, and triple bonds, and further wherein the carbon atoms are found in acyclic, cyclic, fused cyclic ring, and multiring systems, some of which are aromatic. In some embodiments, adventurous amounts (e.g., less than 3 percent and preferably less than about 1 percent of the non-hydrogen atoms in said oil can be nitrogen, oxygen, or sulfur atoms.

[0115] As used herein, the term “fluorocarbon oil” refers to oils comprising primarily C-F bonds throughout. When each of the hydrogen atoms on the carbon atoms is replaced by a fluoro atom, then the oil has a formula CF3(CF2)nCF3. Such oils are commercially available from a number of sources and have a density that generally ranges from above 1 g / cc at 4°C to about less than 2 g / cc at 20 °C. Examples of commercially available fluorocarbon oils for use herein include, by way of example only, Fluo-Oil 7500 / Fluo-Oil 40 / Fluo-Oil 135 / Fluo-Oil 200 (emulseo, Parc AMPERIS, Bâtiment BAYA, 8 rue Adrienne Bolland, 33600 Pessac, France), FC-40 / FC-70 (MilliporeSigma, 400 Summit Drive, Burlington, MA 01803, United States). Fluorocarbon oils generally exhibit lower boiling points than hydrocarbon oils of the same carbon length, with boiling points increasing in both cases with increased carbon length. See “Physical-Chemical Properties and Estimated Environmental Fate of Brominated and Iodinated Organic Compounds,” Organic Bromine and Iodine Compounds, p.1506, January 2003.

[0116] As used herein, the term “thin film of oil” refers to an oil film that adheres to the top surface of the assay device after removal of most of the oil layer by conventional means such as draining, pipetting, pumping, microfluidics, capillary action, and the like. In general, the thin film of oil is typically no more than about 20 microns in depth; preferably no more than 10 microns in depth, and more preferably no more than 5 microns in depth.

[0117] As used herein, the term “confinement means” refers to any structure or configuration that allows for a fluid layer to be retained on the top surface of the assay device. In general, the top surface will have a perimeter of side walls that extend sufficiently upward to define a confinement volume within the perimeter. The perimeter can comprise a continuous wall, such as in a cylinder or an elliptical / oval surface configuration, a combination of walls, such as in a polygon. Alternatively, a scalloped or inward-sloping region can also define a confinement volume.Attorney Docket No.009775.00044\WO

[0118] As used herein, the term “aqueous assay solution” refers to the aqueous solution found in a nanowell, which comprises all the components required for the assay. The term “components” refers to all non-aqueous material needed for an assay including, but not limited to, a target, an effector, nutrients, one or more beads such as a perturbation agent, a capture bead, chemical reagents such as covalent bond cleaving reagents, buffers, antibodies, encoded micro-components (“EMC”), and the like.

[0119] As used herein, the term “aligned” or “alignment” refers to functional alignment such that the compatible components are sufficiently aligned so as to retain their functional use. For example, protrusions (4B) are functionally aligned to indentation (13A) if such can mate with each other despite a minor variability in the alignment.

[0120] As used herein, the term “hydrophobic surface” as it relates to the assay device (9) means that at least the top surface (3) of the assay device (9) is composed of hydrophobic material that has a static water contact angle (Θ) of greater than 90°, preferably greater than about 100°, more preferably greater than about 110° or about 120°. In some embodiments, the hydrophobicity of the material comprising the surface of the assay device (9) is sufficient to create a sufficient surface tension interaction between it and a heavier-than-water fluorocarbon oil to retain the oil above the water in the aqueous assay solution as described above.

[0121] As used herein, the term “reversible” refers to the ability to attach and detach a transfer device as described herein to a second device, such as an assay device, by mating the two devices in a manner wherein the mated pair can be separated without damage to either of the two devices. Examples of suitable mating structures include tongue and groove, protrusions and indentations, temporary magnetic attraction; hooks and loops (e.g., Velcro®), removable adhesives such as pressure sensitive adhesives having low tack properties, and the like. Brief Description of the Drawings

[0122] FIG.1 illustrates a cut-through depiction of a transfer device (1) described herein, as well as a blown-up illustration of a transfer well (5) to show the finer details of these wells. Specifically, the transfer device (1) comprises: a bottom surface (2), a top surface (3), and side walls (4) wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises aAttorney Docket No.009775.00044\WO multiplicity of transfer wells (5), each comprising a floor FL1, an opening diameter OD1, a height of D and a volume defined thereby, V-1, and each of which is configured to receive a defined number of effector moieties or targets; and inlet and outlet ports (7 and 8).

[0123] In addition, protrusions (4B) extend above the top surface (3) of the transfer device (1) as well as over nanowells (5) by a distance (D2). The protrusions depicted are separated from each other by a distance (3A) which defines an area that is populated by transfer wells (5) each of which is sized to contain a defined number of targets or effector moieties that are to be used in the assay. The inlet (7) and outlet (8) ports are placed on the transfer device (1) and create a fluid flow that enters the inlet port (7) and traverses over the top surface (3), which, in combination with protrusions (4B), defines a fluid retention space having a volume (V-4). The fluid flow continues by exiting the transfer device (1) through the outlet port (8).

[0124] In addition, FIG.1 illustrates a blown-up view of a transfer well (5) found on the top surface (3) of said transfer device (1). The top surface of the transfer well has an opening diameter (OD1) which can be elliptical, circular, oblong, or polygonal in shape. However, the OD-1 is preferably circular, and the transfer well (5) is preferably cylindrical in shape. In some embodiments, transfer well (5) is sized to fit a single effector or target.

[0125] FIG.2 illustrates a cross-section of the assay device (9) mated to a transfer device (1), which combination is created by mating indentations (14A – occupied) with protrusions (4B) of the transfer device (1). This requires that the assay device (9) be inverted such that its top surface (13) faces downward against the top surface (3) of the transfer device (1). Because protrusions (4B) are longer in length than indentations (14A), upon mating, a gap (G) is formed between the two surfaces (13 and 3). When protrusion (4B) encompasses the entirety of said gap (G), then said gap (G) is a water-tight volume, but for the inlet and outlet ports (7 and 8). When so constructed, a microfluidic channel is formed with a flow from the inlet port (7) through the 3-dimensional gap (G) and then through the outlet port (8).

[0126] In FIG.2, the transfer wells (5) are positioned vertically upright on the top surface (3) of the transfer device (1), whereas the nanowells (15) of the assay device (9) are positioned downward facing the top surface (3) of the transfer device (1). When so positioned, it is possible to initiate a flow of an aqueous composition comprising, e.g., a target such as a cell through the microfluidic channel, which then allows for depositing aAttorney Docket No.009775.00044\WO single cell into a single transfer well (5) merely by controlling the flow rate through the channel. Note that in FIG.2, the transfer wells (5) are aligned over the center of the nanowells (15) of the assay device.

[0127] Aligning the transfer wells (5) over the center of the nanowells (15) of the assay device is important to ensure that the contents of the transfer wells (5) are transferred only to the nanowell (15) aligned thereto and found thereunder. Alignment can be accomplished in a number of ways, including the use of a latching mechanism which forces alignment, the use of a rigid alignment container as described above, optical techniques such as bright field imaging, and the like.

[0128] In FIG.3, protrusions (4B) can be pillars (4C) and mating of the assay device (9) to transfer device (1) is achieved by use of an adhesive such as a 2-sided pressure- sensitive adhesive, or by a gasket, or by a clamp, or by an O-ring (18), a plug, or an insert. Regardless, a water-tight seal is formed between the assay device (9) and the transfer device (1), but for the inlet port (7), the volume defined by the gap (G) and the outlet port (8). In some embodiments, a 2-sided pressure-sensitive adhesive is employed. The pressure- sensitive adhesive employed allows for a unitary device (11) formed from the two devices (1 and 9) to be disassembled by gentle outward stress applied thereto.

[0129] FIGS.4A and 4B illustrate an alternative to the use of a 2-sided pressure- sensitive adhesive and the use of protrusions (4B). As illustrated, neither the assay device (9) nor the transfer device (1) employs protrusions (4B). Rather, each contains a partial hemispheric indentation (17A and 17B) into which an O-ring (18) can be fitted. After fitting the O-ring, gentle inward pressure can be applied by a clamp (not shown), which compresses the O-ring (18) and possibly squeezes the O-ring (18) such that a water-tight seal is formed, but for the inlet port (7), the volume defined by the gap (G) and the outlet port (8).

[0130] FIGS.4A and 4B illustrate the use of a hemispheric or partial hemispheric indentations (17A and 17B). However, other indentation shapes can be used, such as triangular indentations (17C and 17D), as illustrated in FIGS.4C and 4D.

[0131] FIG. FIG.5A illustrates a top view of a unitary device (11) formed using a pressure-sensitive adhesive (16) or a two-sided pressure-sensitive adhesive. FIGS.5B-5D provide an exploded view of the components of the unitary device (11) with the transfer device (1) on the top and the assay device (9) on the bottom.

[0132] FIG.6 illustrates a top view of a transfer device (1). Internal to side walls (4)Attorney Docket No.009775.00044\WO are four connected protrusion walls (4D) that extend upward from the top surface (3) of the transfer device (1). These connected protrusions form a rectangular confinement means around the transfer wells (5) and are designed to mate with a corresponding set of indentations on the assay device (9) to form a unitary device (11) that encompasses transfer wells (5) thereby forming a water-tight confinement means but for inlet port (7), gap (G) and outlet port (8).

[0133] FIGS.7A and 7B illustrate a slidably engageable transfer device (1) and assay device (9). Specifically, a tongue-in-groove configuration is shown where the assay device (9) contains a groove (29) that mates with a tongue (29A) on the transfer device (1). In this case, sliding tongue (29A) found on the transfer device (1) into the groove (29) of the assay device (9) is achieved by the lateral movement of each relative to the other. As shown, assay device (9) contains a distal wall (30) that terminates the far end of the groove (29), which wall acts as a stopping mechanism. Likewise, transfer device (1) contains a proximal wall (30A) that terminates the near end of the tongue (29A). Both walls (30 and 30A) are configured such that when each contacts the unitary device (11), then the transfer wells (5) are aligned with the nanowells (15). In some embodiments, a clamp or clamps or a latch or latches can be used as a locking mechanism to prevent unintended movement of the assay device during use.

[0134] FIG.7C illustrates the unitary device (11) after mating. Perturbation Beads and Capture Beads

[0135] In some embodiments, assay components such as perturbation beads and / or capture beads can be delivered into the assay device (9) by a transfer device described herein. Such beads are employed when a target, such as a cell, is to be perturbed by a perturbation element (e.g., an effector and / or effector attached to the bead) that is released from the perturbation bead. In some embodiments, the perturbation element is a perturbation compound that is released from a perturbation bead. In some embodiments, the perturbation bead comprises an oligonucleotide that codes for the structure or the synthetic steps used to make the perturbation compound. In some embodiments, the capture bead comprises a capture element that binds to perturbation components generated by a cell in response to the perturbation. Such perturbation elements are typically, but not necessarily, recovered after cell lysis.

[0136] In some embodiments, each perturbation bead comprises multiple copies of aAttorney Docket No.009775.00044\WO same single unique perturbation compound, which are attached to the bead in a releasable manner. In some embodiments, the perturbation bead further comprises multiple copies of the same oligonucleotide releasably attached to the bead, which code for the structure of the compound attached thereto or the reaction steps used to synthesize that compound.

[0137] In some embodiments, each perturbation bead comprises a unique label or a unique set of labels that correspond to the assay well (nanowell) where that particular bead was used during the assay. In some embodiments, multiple copies of an oligonucleotide code are bound to the perturbation bead that codes for the unique label or unique labels attached to the perturbation bead. In some embodiments, multiple copies of the same oligonucleotide code are releasably attached to the perturbation bead, which codes for the structure of the compound attached thereto or the reaction steps used to synthesize that compound. The code also specifies the unique label or set of labels attached to the bead.

[0138] In some embodiments, there is provided a labeled perturbation bead comprising: multiple copies of the same perturbation compound releasably attached to that bead and which is unique to that bead; an optically detectable label or set of labels that is / are unique to and associated with that perturbation bead; and multiple copies of a unique perturbation oligonucleotide releasably attached to said perturbation bead and which code for the perturbation element as well as for the unique label or combination of labels on the perturbation bead.

[0139] In some embodiments, said perturbation oligonucleotide further comprises: a first and optionally a second binding element or precursor thereof wherein said first binding element is complementary to and associates with a complementary capture binding element releasably bound on a capture bead; and said second binding element is complementary to and associates with a complementary binding element present on one or more perturbation components from a lysed cell.

[0140] In some embodiments, there is provided a second binding component or precursor thereof on the perturbation oligonucleotide, which is complementary to and associates with one or more perturbation components released by a lysed cell, provided that if said first binding element binds to the capture bead, then said second binding element is present and binds to a perturbation component.

[0141] In some embodiments, there is provided a population of perturbation beads, each bead comprising:Attorney Docket No.009775.00044\WO a) multiple copies of the same perturbation element releasably attached to that bead, and which is unique to that bead; b) an optically detectable label or set of labels that is / are unique to and associated with each of said perturbation beads in said population; and c) multiple copies of a unique perturbation oligonucleotide releasably attached to said perturbation bead and which codes for the perturbation element as well as for the unique label or combination of labels on the perturbation bead, wherein said perturbation oligonucleotide further comprises a first and optionally a second binding component or precursor thereof wherein said first binding element is complementary to and associates with a complementary functionality releasably bound onto a capture bead or a perturbation component released from a lysed cell, and wherein said optional second binding component or precursor thereof is complementary to and associates with one or more perturbation components released by a lysed cell provided that if said first binding element binds to the capture bead, then said second binding element is present and binds to a perturbation component.

[0142] In some embodiments, said perturbation oligonucleotide further comprises a first and a second binding element or precursor thereof, wherein said first binding element is complementary to and associate with a complementary functionality releasably bound onto a capture bead or a perturbation component released from a lysed cell, and further wherein said second binding component or precursor thereof is complementary to and associates with one or more perturbation components released by a lysed cell.

[0143] In some embodiments, the perturbation oligonucleotide codes for both the perturbation element and the unique label or set of labels associated with the perturbation bead.

[0144] In some embodiments, the perturbation oligonucleotide comprises two distinct components. One component codes for the perturbation element on the bead, and the other component codes for the unique label or set of labels on said perturbation bead. In some embodiments, the two components are attached to each other to form a single perturbation oligonucleotide.

[0145] In some embodiments, there is provided a perturbation bead of formula I:Attorney Docket No.009775.00044\WO where: W is an optically detectable label or a set of optically detectable labels that uniquely identify a perturbation bead; PB is a perturbation bead; PE is a perturbation element; L is a releasable linker; L1is a releasable linker which may be the same or different from L, and when different, L and L1may be cleaved with a same stimulus or with a different stimuli; Q is a perturbation oligonucleotide that codes for the perturbation element and the unique label or set of labels; Q1is a binding element; X is a bond or a binding element wherein, when X is a binding element, it can be the same or different from Q1; m is an integer from 1 to 100 (or more, depending on a size of the bead and / or label, for example); n represents the multiplicity of such (L-X-Q-Q1) groups bound to the perturbation bead; and p represents the multiplicity of such (PE) groups bound to the perturbation bead.

[0146] In some embodiments, L and L1are cleaved using the same stimulus. When both are cleaved, the assay solution will contain a multiplicity of PE and a multiplicity of the group represented by formula II: X-Q-Q1(II) where X, Q, and Q1are as defined above. In some embodiments, provided herein is an oligonucleotide represented by formula II. Capture Bead – First Iteration

[0147] In some embodiments, there is provided a capture bead can be employed in the assay and has a structure such as found in formula III: CB-(L2-CE)s (III) where CB is a capture bead, CE is a capture element on CB, L2is a releasable linker that links a multiplicity of a capture element, CE to CB, and s represents the number of such groups in that multiplicity. In such a case, one of X and Q1of formula II is a complementary binding element to CE, whereas the other of X and Q1can bind perturbation components recovered from the lysed cell. The resulting structures of the capture bead after such bindingAttorney Docket No.009775.00044\WO are represented by formulae IV and V:and CB-(L2-CE-Q1-Q-X)s(V) based on which of X and Q1is complementary to CE.

[0148] In some embodiments, X is a binding element, which can be different from that of Q1.

[0149] In some embodiments, Q1is a complementary functionality to a capture bead binding element, CE, and X is binding element to a perturbation component released from a lysed cell. In other embodiments, X is a complementary functionality to a capture bead binding element, CE, and Q1is binding element to a perturbation component released from a lysed cell.

[0150] In some embodiments, X has a complementary functionality to a perturbation component.

[0151] In some embodiments, n, p, and s range from 1 x 109to 6.02 x 1017.

[0152] In some embodiments, for each of the n L-X-Q-Q1groups, Q1can be a same capture element or different capture elements. In some embodiments, for each of the n L-X- Q-Q1groups, X can be a same capture element or different capture elements. Such same or different capture elements can include, by way of example only, a complementary oligonucleotide strand, avidin, streptavidin, biotin, an antibody, a binding portion of an antibody, an enzyme, an enzyme binding domain, a substrate for an enzyme or enzyme binding domain, and the like.

[0153] In some embodiments, when X is a binding element and X-Q-Q1is released from the perturbation bead in the absence of a capture bead, there is provided an oligonucleotide that is obtained by cleavage of the releasable linker, L, and after lysing of the cell that is represented by formula VI: PC1-X-Q-Q1-PC, (VI) where X, Q, Q1, and PC are as defined above, and PC1is a perturbation component that can be the same or different from that of PC.

[0154] In some embodiments, the perturbation element in each of the above embodiments is a multiplicity of the same compound that is unique to each bead.

[0155] In some embodiments, the perturbation oligonucleotide comprises at its distal and / or proximal end relative to the perturbation bead an X and a Q1group which comprises a chain polynucleotide having repeating units of up to 2,000 guanine units, or cytosine units, orAttorney Docket No.009775.00044\WO adenine units, or thymine units. In some embodiments, X and Q1are different.

[0156] In some embodiments, the perturbation oligonucleotide comprises at its proximal end a chain polynucleotide having up to 2,000 units of cytosine or guanine and at its distal end relative to the bead up to 2,000 units of thymine.

[0157] In some embodiments, the perturbation oligonucleotide comprises poly- thymine (Poly-T) having at either its distal or proximal end relative to the perturbation bead an avidin, streptavidin, or biotin group.

[0158] In some embodiments, there is provided a combination of a perturbation bead and a capture bead, wherein said combination comprises: a perturbation bead as described in formula I above; and a capture bead comprising multiple copies of a same capturing element releasably bound thereto; wherein said combination of said perturbation bead and said capture bead is included in a register that memorializes the combination; provided that: a) if said perturbation bead comprises a unique label or set of labels, then said label or labels are coded by a unique oligonucleotide which is incorporated into or onto the perturbation oligonucleotide; and b) if the perturbation oligonucleotide does not code for said label or labels, then the perturbation bead further comprises a label oligonucleotide which codes for said label or labels, and said label oligonucleotide is releasably attached to said perturbation bead, and said released label oligonucleotide and said perturbation oligonucleotide are attachable to each other.

[0159] In some embodiments, the label oligonucleotide is reversibly bound to the perturbation bead and terminates at either end with a capture element with a poly-G or poly-C oligonucleotide; whereas the perturbation oligonucleotide is reversibly bound to the bead and terminates at one end in a poly-T oligonucleotide capture element and at the other end with a poly-C or poly-G oligonucleotide such that upon release of both oligonucleotides with their binding element(s), the label oligonucleotide will hybridize with the perturbation oligonucleotide thereby providing a continuous oligonucleotide having the label oligonucleotide and the perturbation oligonucleotide in a single strand while retaining the poly-T oligonucleotide for capturing the nucleic acids released from the lysed cells.

[0160] In some embodiments, the captured nucleic acids are mRNA.

[0161] In some embodiments, the captured nucleic acids are mRNA and nucleic-acidAttorney Docket No.009775.00044\WO barcodes attached to antibodies bound to proteins in the fixed or lysed cells.

[0162] In some embodiments, there is provided a combination which comprises a single perturbation bead as described herein and a single capture bead in at least a plurality of nanowells of a mapped assay device, wherein each of said single perturbation beads comprises a label or set of labels that is unique to that bead, wherein the bead is coded by an oligonucleotide releasably bound thereto, and said capture bead comprises multiple copies of a same capturing element releasably bound thereto, and further wherein a register of said specific perturbation bead located in each of the populated nanowells has been recorded to memorialize the specific perturbation bead to that specific nanowell.

[0163] In some embodiments, the optically detectable label or set of optically detectable labels are selected from images, codes, shapes, colors, inducible colors, lettering, numbering, symbols, bar codes, universal product code (UPC) codes, quantum dots (QDs), light emitting diodes (LEDs), and materials introduced onto the perturbation bead, or combinations thereof.

[0164] In some embodiments, the perturbation oligonucleotide codes for each of the reaction steps used in the synthesis of the perturbation compound in a split-pool protocol as described herein. In some embodiments, during each step of the split-pool synthesis, a unique label is attached to each of the beads in each of the reaction vessels such that the fully extended oligonucleotide codes for both the compound that is attached to that bead as well as the unique code associated with the bead.

[0165] In some embodiments, each oligonucleotide strand coding a reaction step in each vessel comprises from about 6 to about 2,000 individual nucleotides; preferably from about 100 to about 2,000 nucleotides; and more preferably from about 800 to about 1,500 nucleotides.

[0166] In some embodiments, the capturing element is an oligonucleotide.

[0167] In some embodiments, the oligonucleotide capturing element on the capture bead attaches a complementary capturing element on the perturbation oligonucleotide by hybridization.

[0168] In some embodiments, the perturbation oligonucleotide comprises one or more capture elements or can be modified to comprise an additional capture element. In some embodiments, one of the capture elements on the perturbation oligonucleotide is complementary to the capture element on the capture bead. In some embodiments, the otherAttorney Docket No.009775.00044\WO capture element on the perturbation oligonucleotide is orthogonal to the capture element on the capture bead.

[0169] In some embodiments, the attachment of the perturbation oligonucleotide including capture element(s) is / are either direct or through a linker.

[0170] In some embodiments, the linker is a releasable linker.

[0171] In some embodiments, the capture element is not an oligonucleotide. In some embodiments, the capture element is a receptor or ligand for the perturbation component to be captured. Such receptors include antibodies or antibody binding fragments for the perturbation compound; an enzyme or enzyme receptor for the perturbation component, a receptor for a given cytokine or chemokine, and the like.

[0172] In some embodiments, the capturing element on the capture bead is avidin / streptavidin that captures perturbation components or the perturbation oligonucleotide that has been modified to contain an avidin group. For example, the distal terminal end of the perturbation oligonucleotide could include biotin, and at its proximal end, a poly-T oligonucleotide capture element. Meanwhile, the capture bead could have avidin or streptavidin as part of its capture element. Upon release of the biotin-perturbation oligonucleotide-poly-T from the perturbation bead, a tight complex is formed between avidin and biotin at one end of the perturbation oligonucleotide, leaving the other end with a poly-T binding element available to bind to nucleic acids.

[0173] In some embodiments, the capturing element is poly-T.

[0174] In some embodiments, there is provided a library or register comprising a recitation of each label or set of detectable labels that code for a specific perturbation bead and the specific nanowell in the assay device for each perturbation bead.

[0175] In some embodiments, there is provided a method for correlating each perturbation bead found in a single nanowell in a mapped assay device having a plurality of nanowells each comprising a single perturbation bead, which method comprises: a) coding each of said perturbation beads with an optically detectable label or a set of optically detectable labels unique to that bead, and wherein each of said perturbation beads comprises a multiplicity of perturbation compounds wherein both the structure of the perturbation compounds and the structure of the unique label or labels is coded onto or into a perturbation oligonucleotide; b) associating each unique labeled perturbation bead to each nanowell in said device; and c) memorializing each position of each perturbation bead to the specific nanowell on theAttorney Docket No.009775.00044\WO mapped assay device.

[0176] In some embodiments, the correlation is performed using an image, aligning each of the optically detectable capture beads with a site on the map.

[0177] In some embodiments, the optically detectable labels can be a micro- component embedded into the perturbation bead, which micro-component can include additional information that is related to the assay to be conducted in that nanowell. The additional information includes, by way of example only, the conditions of the assay, the change in functionality of the cell during the assay, the synthetic reactions used in at least one step of the compound synthesis, the identity of one or more reaction conditions used during synthesis, the technician conducting the assay, the date of the assay, the pH of the assay solution, or combinations thereof. The unique, optically detectable label on each of the perturbation beads in each nanowell can be recorded using photography, videography, barcodes, QR codes, and the like.

[0178] In some embodiments, such additional information can be included on the perturbation bead by a micro-component added to the nanowell. Such micro-components coded with such additional information are described in U.S. patent application Serial No. 18 / 195,049, entitled “High Throughput Single Cell Based Assay for Capturing Genomic Information for Functional and Imaging Analysis and Methods of Use”, filed May 9, 2023, which claims benefit to U.S. provisional patent application Serial No.63 / 339,782, filed May 9, 2024; International patent application Serial No. PCT / US24 / 23594, entitled “Cell Transfer Component for Multi-Well Assay Device,” filed April 8, 2024, and / or U.S. provisional application Serial No.63 / 494,621, U.S. Provisional application Serial No.63 / 494,628, and U.S. provisional application Serial No.63 / 494,636, all filed on April 6, 2023. Each of these applications is incorporated by reference herein in its entirety for all purposes.

[0179] In some embodiments, the micro components can be of different shapes, same shapes but different colors, or different images, such as different descriptions, barcodes, messages, QR codes, and the like placed thereon. In some embodiments, different micro- components are used in combination. In some embodiments, the different images are included on the same micro component such that the combination of such images provides for a unique micro component.

[0180] In some embodiments, the unique micro component or combination of unique micro components provide(s) for a unique image that is uniquely associated with a given nanowell. In some embodiments, each micro component used has a multiplicity of the same unique oligonucleotide strand associated therewith. In some embodiments, the unique strandAttorney Docket No.009775.00044\WO is reversibly attached to the micro component.

[0181] In some embodiments, the linker is used in combination with a unique perturbation bead. In some embodiments, the linker is reversibly attached to the micro component using a linker that is released in an orthogonal manner to the linkers used on the perturbation bead.

[0182] In some embodiments, the perturbation oligonucleotide on the perturbation bead codes only for the structure of the compound synthesized thereon. In some embodiments, the perturbation oligonucleotide comprises a binding element at its distal end. In some embodiments, the oligonucleotide strands on the microcomponents can have a complementary oligonucleotide binding element bound thereto, allowing for hybridization to the binding element on the perturbation oligonucleotide.

[0183] In some embodiments, a single micro component having a unique label or set of labels thereon has a unique oligonucleotide strand that codes for the label and which is attached to the micro component by a cleavable linker that is cleaved in an orthogonal manner to the linker or linkers used on the bead. In a preferred embodiment, the oligonucleotide strand coding for such a micro component comprises a binding element that is complementary to a binding element on the distal end of the perturbation oligonucleotide, such that upon release, the oligonucleotide strand from the micro component will hybridize to the binding element at the distal end of the perturbation oligonucleotide.

[0184] In some embodiments, multiple microcomponents are used such that the combination of these microcomponents is unique to an examination on an assay device. In some embodiments, the oligonucleotide strands on the micro component are capable of being hybridized to the perturbation oligonucleotide by use of unique combinations of binding elements on each component or by use of orthogonally cleaved linkers, such that the oligonucleotide strands can be released and hybridized to the perturbation oligonucleotide in a sequential manner.

[0185] In some embodiments, the microcomponents described above can be used in combination with a labeled or partially labeled perturbation bead that encodes or partially encodes the compound synthesized thereon. In some embodiments, one or more steps of the reaction sequence used to synthesize the compound is / are coded by the micro component or a combination of micro components. In some embodiments, the unique set of micro components provides information regarding the reaction conditions and the like as described above.

[0186] In some embodiments, the unique micro component or combination of microAttorney Docket No.009775.00044\WO components is memorialized by photography or videography to correlate the nanowells where each unique micro component or combination of micro components are found. In some embodiments, the photography or videography is memorialized digitally.

[0187] Still further, the unique label or set of unique labels on said perturbation bead can be further compiled into said library, thereby providing a complete set of information allowing the technician to correlate the perturbation bead to the specific nanowell. Further, the perturbation component captured by the bead will be identified as originating from the specific perturbation bead by the perturbation oligonucleotide that codes for the perturbation element and the unique label or labels. In turn, the correlation between the detectable label(s) and the nanowell through the map will identify the nanowell used with that capture bead. This allows for tracking the captured perturbation component back to the perturbation oligonucleotide and then to the label coded thereby, which identifies the specific nanowell from where it originated, as well as the perturbation element that induced the perturbation.

[0188] In some embodiments, the perturbation oligonucleotide codes only for the perturbation element, and a label oligonucleotide codes only for the label or set of labels that uniquely define the perturbation bead. Each of these two labels is either attached to the other on the bead or is separately attached to the perturbation bead. In some embodiments, the perturbation oligonucleotide can have a capture element at one end designed to capture the label nucleotide having a complementary capture element and, at the other end, a capture element that is complementary to a perturbation component. In such a case, the perturbation bead can be represented by formula IX and / or X:and / orAttorney Docket No.009775.00044\WO where L, L1, PE, Q, Q1, X, m, n, and p are as defined above, LO is a label oligonucleotide, X2is a complementary functionality to either Q1or X, L3is a releasable linker, and k represents a multiplicity of the same L3-LO-X2group and ranges from 1 x 109to 6.02 x 1017.

[0189] In some embodiments, L3is orthogonal to both L and L1such that LO-X2can be released while retaining both PE and L-X-Q-Q1on the bead. In this embodiment, the X2group of LO-X2will attach to Q1, thereby providing for in situ attachment of the label oligonucleotide to the perturbation oligonucleotide, Q, by forming a (L-X-Q-Q1-LO-X2) oligonucleotide.

[0190] In some embodiments, L and L3are released with the same stimulus (and optionally L1), thereby allowing X2to attach to either X or Q1, which forms X-Q-Q1-X2-LO or LO-X2-X-Q-Q1. When X, X2, and Q1are oligonucleotides, and the perturbation component is also an oligonucleotide, then the entirety of either group is an oligonucleotide that can be sequenced.

[0191] In some embodiments of the formulas depicted above, the first binding element can be a poly-C group on the perturbation oligonucleotide, and the binding element on the capture bead can be a poly-G group linked to the capture bead by a cleavable group. The second binding element on the perturbation oligonucleotide can be a poly-T group. So, when released, the poly-C group on the perturbation oligonucleotide will hybridize to the poly-G group on the capture bead, leaving an orthogonal poly-T binding element at the distal end of the perturbation oligonucleotide. This poly-T group will then hybridize with the poly- A functionality found on the nucleic acids released by cell lysis, thereby providing an oligonucleotide that, when sequenced, will provide the relevant information regarding the structure of the perturbation element, the unique label on the perturbation bead, and the identity of the nucleic acids released by cell lysis. In some embodiments, the poly-T binding groups on the capture bead may be replaced by complementary sequences to nucleic acids of interest present within a cell, for instance, complementary sequences for specific regions in the T-cell receptor sequence (TCR) or B cell receptor sequence (BCR).

[0192] In some embodiments, the identity of the unique label or set of labels on the perturbation bead can be ascertained by sequencing the resulting nucleic acid comprising the following formulas XI and XII:where PC, Q, Q1, PO are as defined above and X1and X2are independently binding elements.Attorney Docket No.009775.00044\WO

[0193] In some embodiments, formula XI is the result of the appropriately timed release of cleavable groups L and L1in formula I, followed by hybridization, then lysing of the cell, which releases perturbation components PO (such as mRNA), which are captured by Q1. In some embodiments, formula XII is the result of the appropriately timed release of cleavable groups L, L1, and L3in formula IX or X, followed by hybridization, then lysing of the cell, which releases perturbation components PO (such as mRNA), which are captured by Q1and / or X1 / X2again by hybridization. In formula XI, Q1and X1are interchangeable as to what binds PC and PO. In formula XII, Q1and X2are also similarly interchangeable.

[0194] This then allows the technician to assess the specific perturbation bead corresponding to the given label and, subsequently, using the register recited above, to identify the exact nanowell from which that bead was located. Taken together with visualization of the assay in that nanowell (video or pictures) allows for the dynamic functionalities, features and effects of the cell in the nanowell observed in that visualization before and / or after perturbation to be correlated to the static functionality (e.g., mRNA expressed and captured by the capture elements) determined by sequencing.

[0195] Still further, since each oligonucleotide sequenced will be self-identifying to the particular perturbation bead and the particular nanowell, the technician can merely pool all of the capture beads used in the assay. Once pooled, cleaving the releasable bond between the capture bead and the capture element attached thereto will release capture elements, many of which will contain the oligonucleotide described above (some will be capture element free of such oligonucleotides). Since the oligonucleotides formed by hybridization to the capture bead, followed by hybridization of the perturbation nucleic acids, are self-identifying, pooling of all of the capture beads becomes routine.

[0196] In some embodiments, there is provided a capture bead that self-identifies a unique perturbation bead from a population of perturbation beads which capture bead comprises a multiplicity of capture elements having bound to at least a portion thereof a perturbation oligonucleotide that codes for a perturbation element and a unique label associated with a particular perturbation bead wherein said code self-identifies said unique perturbation bead from a population of uniquely labeled perturbation beads.

[0197] In some embodiments, there is provided a method for identifying a specific nanowell associated with a perturbation component released from a perturbed lysed cell in said nanowell of an assay device comprising a multiplicity of said nanowells, which method comprises: a) conducting an assay in each of a multiplicity of nanowells in a mapped assay deviceAttorney Docket No.009775.00044\WO wherein each of said nanowells comprises a cell in an assay solution; b) including in each of said nanowells: i) a perturbation bead which comprises a multiplicity of perturbation elements releasably bound thereto, an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies that bead wherein said bead comprises a multiplicity of the same perturbation oligonucleotide which is unique to said perturbation bead and is attached to thereto through a releasable linker wherein said perturbation oligonucleotide codes for the structure of the perturbation element on said bead and the unique label associated with that bead further wherein said perturbation oligonucleotide comprises one or more capture elements; and ii) a capture bead comprising a multiplicity of one or more capture elements releasably bound to said bead, wherein at least one of the capture elements is complementary to at least one of the capture elements on the perturbation oligonucleotide, provided that the perturbation oligonucleotide or the capture bead comprises at least two capture elements thereon or can be modified to include a second capture element; c) correlating and memorializing the unique optically detectable label or set of labels in each nanowell by associating the unique optically detectable labels on said capture bead to said mapped assay device, thereby providing for a 1:1 relationship between each capture bead in each of the nanowells; d) inducing a perturbation on said cell by releasing at least a portion of the perturbation element from the perturbation bead, thereby inducing a functional change in said cell which comprises at least a change evidenced by one or more perturbation components expressed by said cell; e) releasing at least a portion of the perturbation oligonucleotide from the perturbation bead for capture by the capture element on the capture bead; f) lysing said cell to release said perturbation components into said assay solution and capturing at least one of said components onto a capture element of said perturbation oligonucleotide or said capture bead; g) sequencing the oligonucleotide comprising the capture element, the perturbation oligonucleotide, and one or more perturbation components; h) identifying the structure of the perturbation element and the unique label or set of labels that identify the unique perturbation bead; and i) correlating said label to a specific nanowell in said mapped assay device by referenceAttorney Docket No.009775.00044\WO to the memorialized position of each uniquely labeled bead, thereby identifying the particular nanowell from which the perturbation component was retrieved.

[0198] In some embodiments, the method provided above further comprises: j) obtaining images of each of the nanowells during the assay; k) correlating the images to the specific nanowell defined in g) above; l) evaluating changes in cellular morphology during the assay as a result of the perturbation of the cell; and m) combining the information obtained from sequencing in h) above with the information in l) above to provide a detailed analysis from l) above.

[0199] In some embodiments, a single unique label is associated with the perturbation bead.

[0200] In some embodiments, a set of labels is used, which set comprises multiple members that, in combination, uniquely identify the perturbation bead to which they were initially bound.

[0201] In some embodiments, there is provided a population of perturbation beads as defined above, wherein each perturbation bead in said population is uniquely correlated by a label or a set of labels attached thereto as well as an oligonucleotide which correlates to the perturbation bead. In some embodiments, the oligonucleotide identifying the label also identifies the structure of the perturbation element on said bead. In some embodiments, the oligonucleotide identifying the unique label is attached to or incorporated into the perturbation oligonucleotide.

[0202] In some embodiments, the perturbation element is selected from compounds, antibodies, immune cells, siRNA, viruses, bacteria, fungi, a change in one or more conditions of the assay, such, but not limited to buffers, salts, pH, temperature, nutrients, oxygen levels, oxidizing agents, physical stress, and the like. In some embodiments, the perturbing element is a compound released from a perturbing bead.

[0203] In some embodiments, the capture bead is positioned in the nanowell before, during, or after completion of the assay.

[0204] In some embodiments, the capture bead described above is isolated from the nanowell prior to removal of the oligonucleotides from the bead.

[0205] In some embodiments, the capture bead is added to the nanowell prior to the assay. In some embodiments, the capture bead is added to the nanowell during the assay.

[0206] In some embodiments, there is provided a register for a given assay device, which register contains a record for each unique set of labels for each perturbation bead, theAttorney Docket No.009775.00044\WO specific nanowell where said perturbation bead is located in the assay device, and the unique perturbation oligonucleotide (including those having label information attached thereto or incorporated therein.

[0207] In some embodiments, the register comprises one or more digital photographs that record the code associated with each nanowell and the optically detectable label on the perturbation bead.

[0208] In some embodiments, the digital photography uses megapixels, wherein the record for each nanowell comprises a different set of pixels from the other nanowells. Capture Bead – Second Iteration

[0209] In some embodiments, a capture bead for use in an assay can be delivered by the transfer device described herein, wherein said capture bead comprises: a) a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead; b) a multiplicity of a capture bead index which codes for the detectable label or set of labels which is unique to said capture bead and is attached thereto, optionally through a linker; and c) a multiplicity of one or more capture elements attached to the capture bead index.

[0210] In some embodiments, there is provided a mapped assay device comprising a multiplicity of examination areas and at least one capture bead in each of the examination areas in use in the device, wherein said mapped assay device correlates a given examination area to a specific location on said map and the capture bead(s) included in each examination area, wherein said capture bead comprises: a) a detectable label or a set of detectable labels, attached to said bead, that uniquely identifies the capture bead; b) a multiplicity of a capture bead index that codes for the detectable label or set of labels, is unique to said capture bead and is attached thereto optionally through a linker; and c) at least one capture element attached to the capture bead index.

[0211] In some embodiments, the detectable label or set of detectable labels used in said capture beads is / are optically detectable.

[0212] In some embodiments, the detectable labels or sets of labels comprise different colors and / or different intensities of one or more colors that are associated with different types of said capture beads.

[0213] In some embodiments, the detectable label or set of labels is selected from theAttorney Docket No.009775.00044\WO group consisting of images, codes, shapes, colors, inducible colors, lettering, numbering, symbols, barcodes, Universal Product Code (UPC), and materials associated with the capture bead.

[0214] In some embodiments, the optional linker on said capture bead is a releasable linker.

[0215] In some embodiments, the capture elements comprise from about 6 to about 2,000 nucleotides.

[0216] In some embodiments, the capture elements attached to the multiplicity of the same capture bead index are selected from a receptor or ligand for the perturbation component to be captured or a complementary oligonucleotide to a corresponding oligonucleotide sequence contained in the perturbation oligonucleotide to be captured.

[0217] In some embodiments, the capture element is an oligonucleotide.

[0218] In some embodiments, a capture element, such as an oligonucleotide capture element, is attached to the proximal and / or distal end of the oligonucleotide index (where the proximal end is adjacent to the releasable linker or the bead).

[0219] In some embodiments, the attachment is either direct or through a linker.

[0220] In some embodiments, the linker is a releasable linker.

[0221] In some embodiments, the capture element is a receptor or ligand for the perturbation component to be captured. Such receptors include antibodies or antibody binding fragments for the perturbation component; an enzyme or enzyme receptor for the perturbation component, a receptor for a given cytokine or chemokine, and the like.

[0222] In some embodiments, the capture element on the capture bead is avidin / streptavidin.

[0223] In some embodiments, the capture element is biotin, which captures perturbation components that have been modified to contain an avidin / streptavidin group.

[0224] In some embodiments, the capture bead index is attached to an oligonucleotide capture element, which is attached either directly or through a releasable linker.

[0225] In some embodiments, the capture element is poly-T, poly-G, or poly-C.

[0226] In some embodiments, there is provide a capture bead as described above and represented by Formula I: (W10)r-CB10-(L10-X10-Q10-Q11)n(I) where: CB10is a capture bead; L10is an optionally releasable linker;Attorney Docket No.009775.00044\WO W10is an optically detectable label or a set of optically detectable labels that uniquely identify the capture bead; X10is a bond or a capture element; Q10is a capture bead index that codes the optically detectable label or set of optically detectable labels on the capture bead; Q11is a capturing element which is independently selected for each of the n L10-X10- Q10-Q11groups; n represents the multiplicity of such (L10-X10-Q10-Q11) groups bound to the bead, and r is an integer from 0 to 100 (or more, depending on the size of the bead and type / size of the optically detectable label.

[0227] In some embodiments, r ranges from 1 to 10.

[0228] In some embodiments, n ranges from about 105to 6.02 x 1017.

[0229] In some embodiments, only multiple copies of the same single oligonucleotide index are bound to the capture bead.

[0230] In some embodiments, X10and Q11are binding elements that capture different targets (e.g., target perturbation components, as may be released by a cell during an assay, and / or perturbation oligonucleotides as discussed herein).

[0231] In some embodiments, each X10and Q11is independently selected for each of the n L10- X10-Q10-Q11groups.

[0232] In some embodiments, each X10and Q11is selected from a functionality that is complementary to a functionality found either on the perturbation oligonucleotide or on nucleic acids or other perturbation components released by the lysed cell.

[0233] In some embodiments, each Q11is an oligonucleotide.

[0234] In some embodiments, one of X10and Q11captures a perturbation oligonucleotide, and the other captures perturbation components.

[0235] In some embodiments, there is provided a capture bead having the Formula I- A: (W10)r -CB10-[L10-X10-Q10-Q11-Y10]n ( I-A) where: Q10is a capture bead index; X10is a bond or a capture element; Y10is a perturbation component PC or a perturbation oligonucleotide PO; Q11is a capture element wherein, when X is not a bond, both X and Q1areAttorney Docket No.009775.00044\WO independently capture elements wherein the capture element on each of Q1and X are complementary to a corresponding functional group on a target; L10is a releasable linker; and n, r, and W10are as defined above. the optically detectable label.

[0236] In some embodiments, r ranges from 1 to 10.

[0237] In some embodiments, there is provided an oligonucleotide represented by the Formula I-B: A10-X11-Q10-Q11-Y10(I-B) where: Q10is the capture index; Q11is a capture element complementary to a corresponding functional group on Y; X11is a capture element complementary to a corresponding functional group on A; Y10is a perturbation component PC or a perturbation oligonucleotide PO; and A10is a perturbation component PC or the perturbation oligonucleotide PO. the optically detectable label.

[0238] In some embodiments, PC is mRNA and Q11is poly-T.

[0239] In some embodiments, A is a perturbation oligonucleotide and X1is a complementary group to a functional group on said perturbation oligonucleotide.

[0240] In some embodiments, A is a nucleic acid other than mRNA.

[0241] In some embodiments, a method is provided for associating sequence data from a solution phase capture index comprising one or more capture elements, wherein at least one of said elements has bound thereto a perturbation component to a specific examination area in a mapped assay device, which method comprises: a) coding each of said capture beads through a releasable bond with an optically detectable label or a set of optically detectable labels unique to that bead, wherein said capture bead comprises an oligonucleotide index that codes for said unique label or set of labels and further comprises a capture element thereon, wherein said capture index is releasably bound to the capture bead; b) adding a coded capture bead from a) to those examination areas in an assay device where an assay is to be conducted; c) generating a registry which records the unique bead in each examination area; d) conducting the assay in the presence of a cell, which is perturbed during the assay;Attorney Docket No.009775.00044\WO e) lysing the cell and capturing at least one nucleic acid perturbation component onto the capture element; f) releasing the capture index from the capture bead; g) sequencing the capture index and the nucleic acid perturbation component to provide a correlation between the capture index and the labels on the capture bead; and h) ascertaining the examination area that correlates to that unique label or set of labels by reference to the registry. the optically detectable label.

[0242] In some embodiments, the correlation is done with an image, aligning each of the optically detectable capture beads to a site on the map.

[0243] In some embodiments, the optically detectable labels on said capture bead can include additional information that is related to the assay to be conducted in that examination area. The additional information includes, by way of example only, the conditions of the assay, the change in functionality of the cell during the assay, the synthetic reactions used in at least one step of the compound synthesis, the identity of one or more reaction conditions used during synthesis, the technician conducting the assay, the date of the assay, the pH of the assay solution, or combinations thereof. The unique optically detectable label on each of said capture beads in each examination area can be recorded by photography, bar codes, QR codes, and the like.

[0244] In some embodiments, only multiple copies of the same single capture bead index oligonucleotide are bound to the capture bead.

[0245] In some embodiments, X10and Q11are the same.

[0246] In some embodiments, X10and Q11are binding elements that capture different targets. In some embodiments, the capture of perturbation components and / or the perturbation oligonucleotide by X10is accomplished after cleaving the releasable linker.

[0247] In some embodiments, the capture element comprises avidin or streptavidin and the perturbation component comprises biotin; or the capture element comprises biotin and the perturbation component comprises avidin or streptavidin. Similarly, the binding pair can be an enzyme and its binding partner, a cytokine or chemokine and its binding partner, an antibody / binding portion thereof, and its target.

[0248] When the capture elements on Q10are oligonucleotides that have bound thereto a perturbation oligonucleotide and / or a nucleic acid perturbation component, such as mRNA, then the combination of X10, Q10, and Q11, along with the components captured byAttorney Docket No.009775.00044\WO X11and Q11, allows for sequencing multiple information components in a single step.

[0249] In some embodiments, the perturbation oligonucleotide further comprises an mRNA capture element such that after the assay is completed, Q11now has attached thereto the capture bead index–Q11-perturbation oligonucleotide-mRNA. For example, attached to the distal terminus of the oligonucleotide index (Q10) would be a poly-C group (Q11), whereas the perturbation oligonucleotide could be constructed to have a poly-T group at one terminus and a poly-G group at the other terminus. When the perturbation oligonucleotide is released from the perturbation bead, the poly-G group can hybridize with the poly-C group of Q11. When the cell is lysed, the poly-T group of the perturbation oligonucleotide will capture the poly-A tail of the mRNA released by the lysed cell.

[0250] In some embodiments, capture can be conducted either on the capture bead or in solution. Perturbation and Capture Bead Definitions

[0251] The following additional terms, used in reference to the perturbation and capture beads discussed above, are provided below. If terms are not defined, they have their generally accepted definitions as used in chemical synthesis, biology, and combinatorial chemistry. a) assay device and mapped assay device; b) associated therewith; c) capture bead; d) capture element or binding element; e) capture bead index, capture index, capture oligonucleotide, or oligonucleotide index; f) cell; g) cellular components; h) cellular morphology; i) change in functionality; j) complementary functionality; k) compound; l) detectable label or set of detectable labels; m) examination area; n) linker and releasable linker; o) multiplicity of perturbation beads; p) multiplicity of perturbation elements; q) multiplicity of oligonucleotides or capture / binding elements; r) nucleic acids; s) oligonucleotide; t) oligonucleotide strand; u) optically detectable; v) or a precursor thereof;Attorney Docket No.009775.00044\WO w) perturbation bead; x) perturbation component; y) perturbation compound; z) perturbation element; aa) perturbation oligonucleotide; ab) releasably attached; and ac) unique.

[0252] As used herein, the term “oligonucleotide strand” refers to oligonucleotides (e.g. RNA, DNA, single and / or double stranded) having from about 6 to about 20,000 nucleotides (and / or base-pairs) or from 16 to 50,000 nucleotides (and / or base-pairs). Such strands can be attached serially to each other to form an oligonucleotide, as defined below. The terms “RNA” and “ribonucleic acid” when used herein encompass transfer RNA (tRNA), messenger RNA (mRNA), hairpin RNA, ribosomal RNA (rRNA), small nucleolar (snoRNAs), small nuclear RNAs (snRNA), microRNAs (mi(RNA), and siRNAs (siRNAs). The term “DNA” and “deoxynucleic acid” when used herein include nuclear DNA and mitochondrial DNA. The genus for RNA and DNA also include their double-stranded and single-stranded forms.

[0253] As used herein, the term “oligonucleotide” refers to an oligonucleotide having from about 6 to about 500,000 nucleotides, provided that if the oligonucleotide comprises two or more oligonucleotide strands attached together in series, then the oligonucleotide comprises from about 12 to about 500,000 individual nucleotides. It is understood that if an oligonucleotide is intended to be attached to another oligonucleotide, it is referred to as an oligonucleotide strand, as above. If, however, the oligonucleotide is to be used and / or discussed as a standalone component, then it is not referred to as an oligonucleotide strand, as above.

[0254] As used herein, the term “perturbation bead” or “PB” refers to the bead that comprises multiple copies of the same “perturbation element”, as defined herein, which is releasably bound to the perturbation bead. In addition, each perturbation bead further comprises a unique detectable label or set of detectable labels that uniquely identifies that bead from a population of other uniquely labeled perturbation beads, as well as a perturbation oligonucleotide that codes for all or part of the perturbation element on the bead. In some embodiments, a “perturbation oligonucleotide” can code for a unique label or set of labels associated with a given bead.

[0255] In some embodiments, the perturbation bead may not carry a “perturbationAttorney Docket No.009775.00044\WO element” as the perturbation is generated by physical changes such as pH, temperature, the presence or absence of nutrients, constitutes one or more conditions of the assay as noted above or, alternatively, the perturbation element can be a virus, an antibody (or fragment thereof), or a cell such as an immune cell. However, to identify the impact of such a perturbation on the nucleic acids released from a lysed cell, a uniquely labeled perturbation bead is required to correlate a particular perturbation bead with a specific nanowell. In such a case, the perturbation bead used is represented by the formula XIII: (W)m -PB-(L-X-Q*-Q1)n (XIII) where each of W, PB, L-X, Q1, m and n are as defined above and Q* is a label oligonucleotide.

[0256] Multiple perturbation beads can be used in a single nanowell. For example, a first perturbation element is released from a first perturbation bead, then a second perturbation element is released from a second perturbation bead, where the two perturbations are intended to perturb a cell, or a first perturbation element separately is intended to perturb the second perturbation element and then the second now perturbed perturbation element is evaluated for its impact on the cell.

[0257] As used herein, the term “label oligonucleotide” or “LO” refers to an oligonucleotide coding for the unique label or set of labels. A label oligonucleotide may be added to the perturbation bead in a situation when the perturbation oligonucleotide codes only for the perturbation element (e.g., not the label) and / or in a situation when the perturbation bead does not comprise a discrete perturbation element (e.g., Q* in formula XIII). In some embodiments, the label oligonucleotide can become bound to the perturbation oligonucleotide either on the perturbation bead, on the capture bead, or in the solution phase.

[0258] As used herein, the term “capture bead” or “CB” refers to a bead used in an assay to capture the perturbation oligonucleotide, which, in turn, directly or indirectly captures one or more cellular components after perturbing the cell and then lysing the cell to release such cellular components that are generated due to the perturbation (“perturbation components”). In some cases, the perturbation components include “nucleic acids” such as mRNA, tRNA, DNA, and the like. In some cases, the perturbation components can be chemokines, cytokines, enzymes, proteins, glycoproteins, peptides, hormones, metabolites, protein complexes, protein-nucleic acid complexes, endogenous cellular ligands, and the like. One or more capture beads can be included in a nanowell. Multiple capture beads can be used when different perturbation components are to be captured. Alternatively, a singleAttorney Docket No.009775.00044\WO capture bead is used such that different perturbation components are to be captured by that bead.

[0259] As used herein, the terms “capture bead index,” “capture index,” “capture oligonucleotide index,” and “an oligonucleotide index” are used interchangeably and refer to an oligonucleotide that codes for the unique label or set of labels on the capture bead thereby correlating the oligonucleotide index to the unique label or set of labels and then to the unique specific capture bead with that unique labeling. In some embodiments, the oligonucleotide index is attached to the capture bead, optionally in a releasable manner. In some embodiments, the oligonucleotide index can be attached to a “capture element” that can capture a perturbation oligonucleotide and / or a perturbation component, both as defined above.

[0260] In some embodiments, when the capture beads include a unique detectable label or a set of detectable labels associated therewith, the capture bead does not include any oligonucleotide indices that code for the unique label or set of labels on the capture bead.

[0261] In some embodiments, a single capture bead and a single perturbation bead are employed in each nanowell. In some embodiments, multiple capture beads and a single perturbation bead are employed in each nanowell. In some embodiments, a single capture bead and multiple perturbation beads are employed in each nanowell.

[0262] As used herein, the term “detectable labels” refers to any source of information that can be detected by visual, chemical, biological, audio, or other means to evidence the presence of the information generated by the label. In some cases, the detectable label can be an “optically detectable label” which contains, generates or can be stimulated to generate a visual or optically detectable image / signal such as a color, visible light, lettering, numbers, symbols, barcodes, fluorescence, particles, micro-components such as microchips, Universal Product Codes (UPC), tags, light emitting diodes (LEDs), or combinations thereof that can be seen with an unaided eye or by use of instrumentation such as a microscope or a fluorometer and the like. In some cases, the optically detectable label generates chemically or biologically induced and optically detectable signals, optionally requiring suitable instrumentation to visualize, such as bioluminescence. Such detectable labels include quantum dots, fluorescence (e.g., fluorescent particles, mass spectra, nuclear magnetic spectra (e.g., H1or C13spectrum), and the like. In some cases, the detectable label is an oligonucleotide such as siRNA, and the like. In some cases, the detectable signal generatesAttorney Docket No.009775.00044\WO an audio or electromagnetic signal, such as those generated by a radio frequency identification device (RFID), WiFi, or a Bluetooth device. The particular detectable label or labels used in the embodiments disclosed herein are within the skill of the art, being dependent on the constraints of the assays to be conducted and the need to uniquely identify a component or components in that assay. Combinations of different labels can be used.

[0263] As used herein, the term “examination area” refers to assay wells in which individual assays can be performed. An examination area may be, for example, a microwell, picowell, and / or nanowell, defined by its dimensions and / or volumetric capacity.

[0264] A “nanowell,” as used herein, refers to assay wells having a maximum linear dimension of no greater than 1 mm and a volume of no more than 1 µL (1 microliter), and the term “maximum linear dimension” refers to the distance along the longest axis for that shape. For example, the diameter of an ellipse would be the semi-major diameter, and the diameter of a square would be from the top corner of the square to the opposite lower corner of the square.

[0265] Multiple nanowells are employed in one or more assay devices. Each nanowell is configured to contain at least one cell, at least one capture bead, and at least one perturbation element in isolation from other nanowells. In some embodiments, nanowells do not include those wells within a device that do not participate in an assay. That is to say that only those wells where an assay is conducted are deemed to be “nanowells” as discussed herein.

[0266] In some embodiments, a single capture bead and a single perturbation bead is employed in each nanowell. In some embodiments, multiple capture beads and a single perturbation bead are employed in each nanowell. In some embodiments, a single capture bead and multiple perturbation beads are employed in each nanowell. In some embodiments, multiple capture beads and multiple perturbation beads are employed in each nanowell.

[0267] As used herein, the term “perturbation oligonucleotide” or “PO” refers to an oligonucleotide that uniquely codes for all or part of the perturbation element on the perturbation bead. In some embodiments, the perturbation oligonucleotide also codes for the unique label or set of labels on the perturbation bead, thereby uniquely identifying the perturbation bead. The perturbation oligonucleotide further comprises a complementary binding domain to the binding element on the capture bead. The complementary binding domain preferably is either the proximal or distal portion of the perturbation oligonucleotide.Attorney Docket No.009775.00044\WO In some embodiments, the perturbation oligonucleotide still further comprises or can be modified to comprise a complementary binding domain to the perturbation components released from the lysed cell. In some embodiments, the perturbation oligonucleotide is attached to the perturbation bead in a releasable manner.

[0268] In some embodiments, the perturbation oligonucleotide simultaneously codes for the perturbation element as well as the unique label or set of labels on the perturbation bead. In some embodiments, the number of reaction steps required to form the “perturbation compound” exceeds the number of labels that are required to provide for a unique set of labels. In such cases, the perturbation oligonucleotide still simultaneously codes for both the perturbation compound and the unique set of labels, as the skilled artisan would know that labeling stopped at a given step in the synthesis.

[0269] As used herein, the term “capture element” refers to a complementary functional group that recognizes and binds to a corresponding complementary functional group for which it will capture. Such complementary functional groups include, by way of example only, complementary strands of nucleic acids (e.g., poly-T and poly-A; poly-G and poly-C, etc.), avidin or streptavidin and biotin, an enzyme and its binding partner, a cytokine or chemokine and its binding partner, an antibody or the binding portion thereof, and its target. The complementary group that is captured by the capture element on the capture bead is sometimes referred to as the “target.”

[0270] As used herein, the term “unique” refers to the likelihood of duplication, e.g., of a single bead having the given label, being less than 1 in 1,000; or, in some embodiments, less than 1 in 10,000; or, in some embodiments, less than 1 in 100,000.

[0271] For example, in a split pool reaction scheme illustrated herein, a population of 100,000 beads can be split into 10 groups of approximately 10,000 beads each and then placed into separate reaction vessels. In a 6-step split-pool reaction scheme, step 1 will provide for 10 sets of beads, each set with different labels. When pooled together, mixed to a homogenous mixture, and split again into 10 reaction vessels in step 2, the probability that a bead will be found in the same reaction vessel in step 2 as in step 1 is 10%. So, after step 2, the probability that beads will have the same two labels is now down to 1,000 for each reaction vessel or an aggregate of 10,000 duplications. Steps 3, 4, and 5 will likewise have a 10% probability of beads having been in the same reaction vessel throughout the steps 1 to 5. Hence, for step 3, the probability is 10% of step 2 (10,000) or 1,000 out of 100,000. ForAttorney Docket No.009775.00044\WO step 4, that becomes 100 beads with the same likelihood of having duplicate labels, and for step 5, that number becomes 10 out of 100,000; and step 6, the probability is 1 out of 100,000. If desired, a step 7 and step 8 would reduce the number to less than 0.01 out of 100,000. At any point, after step 5, the limited probability of duplication has been reduced to the point that the overwhelming number of such beads are unique.

[0272] In some embodiments, the presence of duplicates can act as a control for the assay as such duplicates should provide the same results.

[0273] As used herein, the term “perturbation element” or “PE” refers to any source that perturbs a cell. A perturbation may be an effector and / or effector described herein. Such sources include, by way of example only, compounds, antibodies, immune cells, siRNA, viruses, bacteria, fungi, a change in one or more of the conditions of the assay, such as but not limited to buffers, salts, pH, temperature, nutrients, oxygen levels, oxidizing agents, physical stress, and the like. In some embodiments, the perturbing element is a compound released from a perturbation bead. Such a compound is sometimes referred to as a “perturbation compound” herein.

[0274] As used herein, the term “compound” or “perturbation compound” or “PC” is an example of a perturbation element and refers to small molecules (having a molecular weight of less than about 1500 Dalton) that are synthesized bound to a perturbation bead through a releasable (cleavable) bond that is located between the compound and the bead. When the perturbation element is a compound on a perturbation bead that comprises multiple copies of the same compound releasable from the bead and multiple copies of a perturbation oligonucleotide which identifies the structure of or the identity of the compound, or the synthetic steps used to make the compound, the perturbation element is sometimes combined with the term “perturbation bead”.

[0275] As used herein, the term “assay device” refers to a device having a multiplicity of nanowells for conducting assays using the perturbation beads as described herein.

[0276] As used herein, the terms “mapped assay device” and “assay device” refer to any assay mechanism where the nanowells can be identified by the geometry of the device (e.g., X and Y coordinates or any orientation that allows the individual nanowells to be identified, such as triangular shapes, trapezoidal shapes, etc.) or by images, lettering, numbering, etc. associated with each nanowell, such that the nanowell can be uniquely identified. In some cases, a mark or label, or other indicia on the assay device allows theAttorney Docket No.009775.00044\WO technician to properly orient the mapped assay device.

[0277] As used herein, the term “multiplicity of perturbation beads” means more than one bead, such as at least 90 beads, or at least 1,000 beads, or at least 10,000 beads, or at least 100,000 beads. An upper limit of beads in all cases can be as many as 5,000,000 beads or more, but generally is about 2,000,000.

[0278] As to the term “multiplicity” when used in conjunction with perturbation elements, oligonucleotides, binding elements, and the like means that there is a sufficient number of each to perturb a cell, to capture perturbation components from a lysed cell, and to assess the identity of the perturbation components as well as the perturbation element that generated the perturbation components, and the nanowell from which the perturbation components were generated. In some embodiments, such a multiplicity ranges from about 1 x 109to about 6.02 x 1017.

[0279] As used herein, the terms “releasably attached,” “releasably bound thereto,” and “releasably attached thereto” means that the specific component so described is attached to another component such as a bead in a releasable manner such that release / cleavage of at least a portion (meaning, e.g., 50 out 100 are released instead of half of an element is released) of such compounds, oligonucleotides or nucleic acids from the bead can be initiated (e.g., by the technician) at an appropriate (e.g., desired, scheduled or controlled) time in the assay. Such controlled releasability is achieved, in one instance, by using a cleavable covalent bond – a bond that is cleaved under appropriate stimulation, such as light (e.g., UV light), heat, pH change, electromagnetic stimulation, sound, salt, oxidation change, enzymatic activity, and the like. Such cleavable bonds are well known in the art.

[0280] As used herein, the term “cellular morphology” refers to any one or a combination of two or more properties of a cell or its components, such as size, shape, structure, and / or form. Cellular components are well known in the art and include the nucleus, the mitochondria, and ribosomes.

[0281] As used herein, the term “change in functionality” of a cell refers to one or more changes in cellular morphology as well as changes in the cellular expression of nucleic acids, chemokines, cytokines, enzymes, peptides, hormones, and the like as compared to the cell prior to exposure to the perturbation element. Other changes in functionality include the generation of apoptotic or other biological markers by the cellular perturbation. Such apoptotic markers include chromatin condensation, blebbing, DNA fragmentation, andAttorney Docket No.009775.00044\WO others, some of which can be readily visualized in the cell.

[0282] As used herein, the term “cell” refers to a bacterial, viral, fungal, eukaryotic cell (which includes yeast and fungi), protists, as well as animals and plants. Exemplary animal cells include human or primate cells. In some embodiments, the cell is a mammalian cell, which is preferably a cell from a primate, and more preferably, a human cell.

[0283] As used herein, the term “linker” refers to any structure that attaches one entity to another. Such a linker can be releasable or non-releasable. In some embodiments, the linker is a nucleotide or an oligonucleotide having from 1 to 2,000 nucleotide units. In some embodiments, the oligonucleotide incorporates a uracil (U) group, which is cleaved by the User enzyme. In some embodiments, the oligonucleotide can be cleaved by a restriction enzyme. In some embodiments, the oligonucleotide is cleaved by a nicking enzyme. In some embodiments, CRISPR®technology is used to cleave the oligonucleotide. Chemical linkers such as carbonate, an ester, or carbamate groups can be cleaved by pH, esterases, lipases, and the like. Photocleavable linkers are well known in the art and include coumarin groups, ortho-nitrobenzyl groups, and others found in FIG.2 of Johan, et al., Pharmaceuticals (Basel), 15(6):655 et seq. (June 2022), which is incorporated herein by reference in its entirety. The selection of a suitable linker is not critical and is well known to the skilled artisan.

[0284] In some embodiments, when the linker is a non-nucleotide or oligonucleotide, then it comprises at least one and up to about 40 non-hydrogen atoms, including carbon, nitrogen, oxygen, sulfur, and phosphorus. Where appropriate, these atoms include hydrogen (including all isotopes), hydroxy, oxo (=O), amino, or halo to satisfy the valence of the atoms. When the term “linker” is employed, unless stated otherwise or implicit from its use, it is intended to include direct attachment of the two groups without the introduction of any additional atoms.

[0285] The term “releasable linker” or “cleavable linker” means that the linker comprises a cleavable functionality – that is to say that the functionality or a covalent bond is readily cleaved into a first component and a second component by stimulation that breaks the bond. Such stimuli that can cleave a releasable bond include enzymes, UV light, heat, pH change, salt, and other components well known in the art.

[0286] The term “associated therewith” refers to any and all interactions whereby a first component becomes associated with a second component by, for example, its physicalAttorney Docket No.009775.00044\WO location, covalent bonding, ligand and receptor interactions, electrostatic interactions, magnetic interactions, hybridization, chelation, molecular sieve capturing, micelle or liposome formation, and the like. In a preferred embodiment, the term “associated therewith” utilizes either physical location, bonding, or ligand receptor interactions. In some instances, the term “associated therewith” encompasses complementary functionalities where two functional groups, when present, are capable of becoming associated with each other.

[0287] The term “unique perturbation oligonucleotide,” “perturbation oligonucleotide” or “PO” refers to an oligonucleotide that codes for the perturbation element on the perturbation bead, wherein said PO is unique to other oligonucleotides used in the assay. In some embodiments, the unique perturbation oligonucleotide also codes for the unique label or set of labels on the perturbation bead.

[0288] In some embodiments, a label oligonucleotide (LO) can be attached to the PO to provide for a POL. That is to say that after forming the PO, a unique label oligonucleotide is then added at any stage of the assay to generate a POL group on the perturbation bead to identify the unique label or set of labels found on that bead.

[0289] Alternatively, during the synthesis of the perturbation element and the perturbation oligonucleotide in the split-pool reaction scheme, one or more steps of that reaction scheme can include the addition of a label to the perturbation bead, and a corresponding oligonucleotide strand is added to the precursor oligonucleotide being formed on the bead. In such a case, the resulting PO will again code for both the perturbation compound and the unique label or set of labels.

[0290] Still further, only a sufficient number of labels and their corresponding oligonucleotide strands need to be added during the synthesis of the perturbation element to uniquely define the label. So, assuming that in a 7-step split-pool reaction scheme using 10 reaction vessels in each step and that each perturbation bead can be uniquely labeled with six different labels, then, at a minimum, only six of the seven steps need to add a label and a corresponding reaction step label oligonucleotide. That is to say that it is not necessary to label a perturbation bead at each reaction step when the number of reaction steps in the synthesis of the perturbation element exceeds the number of labels required to uniquely identify that bead. However, in practice, one can continue with labeling the perturbation bead at each reaction step even if such becomes unnecessary.

[0291] The term “static information” refers to changes in one or more cellular functionsAttorney Docket No.009775.00044\WO that have changed during the assay but collected after the assay is completed such as the expression of mRNA, upregulating or down regulating one or more cellular components that are captured by one or more capture beads such as chemokines, cytokines, hormones, enzymes, peptides, peptide fragments, and the like. Static information also includes perturbation components that are captured by the capture elements on the capture beads, as well as perturbation components that can be analyzed directly in the lysed cellular milieu. For example, protein degradation components can be observed at the end of the assay but do not need to be captured to assess the perturbation components. In some embodiments, protein degradation components can be evaluated by immunofluorescent staining after the assay is completed, provided that the perturbed and lysed cell is not from a stable cell line that contains innate fluorescence.

[0292] The term “dynamic information” refers to information regarding the cell as it occurs during the assay. Such dynamic information can include photo images of the cell undergoing perturbation, including picture(s) or a video of the perturbed cell during a portion or all of the assay. Such images evidence changes in cellular functionality such as changes in cellular morphology, movement, size, shape, adhesion, division, induction of apoptosis, gene expression (e.g., expression of fluorescent / fluorescently labeled proteins), intracellular organization / structure (e.g., cytoskeletal organization / structure, organelle structure), extracellular expression, evidence of other cellular structural / behavioral changes, and the like. For example, such dynamic information may include optically detectable evidence of the presence of apoptotic markers or other markers, protein upregulation or downregulation that can be monitored live and / or in real time (e.g., fluorescent proteins, etc.), activation of T- cells, thereby observing proliferation and change in their sizes, T-cells killing target cells, and the like. Such changes are deemed “live” or “real time” as they are observed as occurring during the assay and can be recorded via pictures, video recordation, three-dimensional analysis of the cell, and the like.

[0293] As used herein, the term “or a precursor thereof” means that the distal end of the perturbation oligonucleotide is either still bound to the bead or released from the bead and has a perturbation component bound to a first end, then the second end can be modified to provide for a second binding element. For example, if the distal end of the perturbation oligonucleotide has a poly-T binding element attached thereto, then, if the proximal end contains a known oligonucleotide leader sequence (not part of the code for the perturbation compound or label(s)), then that sequence can be used to attach a binding element thereto byAttorney Docket No.009775.00044\WO hybridization. As such, the leader sequence would constitute “or a precursor thereof”.

[0294] In all cases described above, at the completion of the split pool process, each bead will have a unique perturbation compound and a unique perturbation label compared to the other beads within a population of beads. Embodiments

[0295] The following is a listing of various embodiments to which variants as taught in the specification would be understood by one of skill. Embodiment [1]. A transfer device (1) comprising: a) a bottom surface (2), a top surface (3), and side walls (4), wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, a height of D, and a volume defined thereby, V-1, and each of which is configured to receive a defined number of effector moieties or targets; and b) inlet and outlet ports (7 and 8). Embodiment [2]. The transfer device of Embodiment [1], wherein said device further comprises a mating means to attach said transfer device (1) to a second device. Embodiment [3]. The transfer device of Embodiment [2], wherein said mating means is reversible. Embodiment [4]. The transfer device of Embodiment [2], wherein said mating means comprises one or more of the following mating means: an adhesive (16); or a clamp; or a latch; or a gasket; or a partial or complete hemispheric groove (17) on the transfer device (1) which aligns with a partial or complete hemispheric groove (17A) on the second device which forms an open tubular opening (17) wherein said tubular opening is adapted to fit an O-ring (18) or other compatible material after combining the transfer device (1) with the second device; or one or more protrusions (4B) on the transfer device (1) that mate with one or more compatible indentations (14A) on a second device; orAttorney Docket No.009775.00044\WO one or more indentations on the transfer device (1) that mate with one or more compatible protrusions, which align with indentations. Embodiment [5]. The transfer device of Embodiment [2], wherein said second device is an assay device (9) comprising a multiplicity of nanowells. Embodiment [6]. The transfer device of Embodiment [5], wherein said mating means comprises an adhesive (16) of a two-sided pressure-sensitive adhesive having a thickness which defines a gap (G) between the transfer device (1) and the assay device (9). Embodiment [7]. The transfer device of Embodiment [5], wherein said the mating means between the transfer device (1) and the assay device (9) comprise indentations on both the transfer device (1) and the assay device (9) that are alignable with each other such that, when aligned, form a tubular opening (19) suitable to fit an O-ring (18). Embodiment [8]. The transfer device of Embodiment [7], wherein said indentations are hemispheric or partially hemispheric indentations (17A and 17B) on said transfer device (1) and on said assay device that alignable and when aligned are able to fit an O-ring (18) within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined. Embodiment [9]. The transfer device of Embodiment [7], wherein said indentations are a polygonal open sided indentations found on both said transfer device (1) and said assay device (9) which, when aligned, fit an O-ring (18) is sized to fit within the tubular opening (19) formed thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined. Embodiment

[0010] . The transfer device of either Embodiment [8] or Embodiment [9], wherein said O-ring provides a distance between the top surface (3) of said transfer device (1) and the top surface (13) of said assay device (9), which defines gap (G). Embodiment

[0011] . The transfer device of Embodiment [4], wherein said mating means comprises at least one indentation or at least one protrusion (4B) on the transfer device (1) which mates with a compatible protrusion or compatible indentation (14A) respectively on said second device to form a unitary assay device (11) when combined. Embodiment

[0012] . The transfer device of Embodiment

[0011] , wherein said the transfer wells (5) of the transfer device (1) are enclosed in an area bound by said protrusion or protrusions (4B) or said indentation or indentations.Attorney Docket No.009775.00044\WO Embodiment

[0013] . The transfer device of Embodiment

[0011] , wherein said protrusion (4B) or said indentation extends in a continuous manner to encompass said transfer wells (5), wherein said continuous manner is a closed loop. Embodiment

[0014] . The transfer device of Embodiment

[0011] , wherein said closed loop comprises a circular, oval, or oblong configuration. Embodiment

[0015] . The transfer device of Embodiment

[0011] , wherein said protrusions (4B) or said indentations comprise a connected set of closed protrusion walls (4D). Embodiment

[0016] . The transfer device of Embodiment

[0015] , wherein said closed walls form a polygon. Embodiment

[0017] . The transfer device of Embodiment

[0016] , wherein said polygon is selected from a triangle, a rectangle, a trapezoid, a square, a pentagon, a hexagon, an octagon, and the like, as well as any other configuration that is continuous. Embodiment

[0018] . The transfer device of Embodiment [5], wherein said transfer device (1) and said assay device (9) are reversibly slidable with each other in any one of an X, Y, or Z axis. Embodiment

[0019] . A unitary device comprising a transfer device (1) reversibly attached to an assay device (9), wherein: a) said transfer device (1) comprises a bottom surface (2), a top surface (3), and side walls (4) wherein: said top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor FL1, an opening diameter OD1, and a volume, V-1, each of which is configured to receive only a defined number of effector moieties or targets; inlet and outlet ports (7 and 8) that are associated with the transfer device; b) an assay device (9) comprising a multiplicity of nanowells (15) each having a floor FL12, an opening diameter OD12, and a volume V-3 which, in combination, allow for conducting an assay in said nanowells (15); and c) a mating means for connecting the top surface (3) of said transfer device (1) to said top surface (13) of said assay device (9); wherein, upon mating, a gap (G) is formed between said top surface (3) of said transfer device (1) and said top surface (13) of said assay device (9), wherein said gap (G)Attorney Docket No.009775.00044\WO defines a volume (V-4), and further wherein said inlet and outlet ports (7 and 8) of said transfer device (1) are in fluid communication with said gap (G) thereby providing for microfluidic communication into said inlet port (7), through said gap (G) and out of said outlet port (8). Embodiment

[0020] . The unitary device of Embodiment

[0019] , wherein said transfer wells (5) of said transfer device (1) have an opening diameter (OD1) that ranges from about 5% to about 60% of the opening diameter (OD12) of the nanowells (15) of the assay device (9) and further provided that, upon mating, the transfer wells (5) align to said nanowells (15) on a one-to-one basis such that a projection of an opening of each transfer well (5) onto a corresponding nanowell (15) is encompassed by an area of said nanowell (15). Embodiment

[0021] . The unitary device of Embodiment

[0019] , wherein said mating means comprises one or more of the following: an adhesive (16); or a clamp; or a latch; or a gasket; or a partial or complete hemispheric groove (17B) on the transfer device (1) which aligns with a partial or complete hemispheric groove (17A) on the assay device (9) which forms a tubular channel (17) wherein said tubular opening is adapted to fit an O-ring (18) or other compatible material after combining the transfer device (1) with the assay device (9); or one or more protrusions (4B) on the transfer device (1) that mate with one or more compatible indentations (14A) on the assay device (9); or one or more indentations on the transfer device (1) that mate with one or more compatible protrusions, which align with indentations. Embodiment

[0022] . The unitary device of Embodiment

[0021] , wherein said mating means comprises an adhesive (16) of a two-sided pressure-sensitive adhesive having a thickness which defines a gap (G) between the transfer device (1) and the assay device (9). Embodiment

[0023] . The unitary device of Embodiment

[0021] , wherein said the mating means between the transfer device (1) and the assay device (9) comprises hemispheric or partial hemispheric indentations (17A and 17B) that are alignable with each other such that, when aligned, form a tubular opening (19) suitable to fit an O-ring (18). Embodiment

[0024] . The unitary device of Embodiment

[0023] , wherein saidAttorney Docket No.009775.00044\WO hemispheric indentations (17A and 17B) of said transfer device (1) and said assay device are aligned and said O-ring (18) is sized to fit within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined. Embodiment

[0025] . The unitary device of Embodiment

[0023] , wherein said partial hemispheric indentations (17A and 17B) of said transfer device (1) and said assay device are aligned and said O-ring (18) is sized to fit within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined. Embodiment

[0026] . The unitary device of either Embodiment

[0024] or Embodiment

[0025] , wherein said O-ring (18) provides a distance between the top surface (3) of said transfer device (1) and the top surface (13) of said assay device (9), which defines gap (G). Embodiment

[0027] . The unitary device of Embodiment

[0021] , wherein said mating means comprises at least one indentation or at least one protrusion (4B) on the transfer device (1) which mates with a compatible protrusion or compatible indentation (14A) respectively on said assay device (9) to form a unitary assay device (11) when combined. Embodiment

[0028] . The unitary device of Embodiment

[0027] , wherein said the transfer wells (5) of the transfer device (1) are enclosed in an area bound by said protrusion or protrusions (4B) or said indentation or indentations. Embodiment

[0029] . The unitary device of Embodiment

[0028] , wherein said protrusion (4B) or said indentation extends in a continuous manner to encompass said transfer wells (5), wherein said continuous manner is a closed loop. Embodiment

[0030] . The unitary device of Embodiment

[0029] , wherein said closed loop comprises a circular, oval, or oblong configuration. Embodiment

[0031] . The unitary device of Embodiment

[0029] , wherein said protrusions (4B) or said indentations comprise a connected set of closed protrusion walls (4D). Embodiment

[0032] . The unitary device of Embodiment

[0031] , wherein said closed protrusion walls (4D) form a polygon. Embodiment

[0033] . The unitary device of Embodiment

[0032] , wherein saidAttorney Docket No.009775.00044\WO polygon is selected from a triangle, a rectangle, a trapezoid, a square, a pentagon, a hexagon, an octagon, and the like, as well as any other configuration that is continuous. Embodiment

[0034] . The unitary device of Embodiment

[0019] , wherein said transfer device (1) and said assay device (9) are reversibly slidable with each other in any one of an X, Y or Z axis. Embodiment

[0035] . The unitary device of Embodiment

[0019] , wherein said inlet port (7) and an outlet port (8) on the same surface of the transfer device (9). Embodiment

[0036] . The unitary device of Embodiment

[0019] , wherein the same surface is the top surface (3) of said transfer device (1). Embodiment

[0037] . The unitary device of Embodiment

[0019] , wherein said target in said nanowell is a cell. Embodiment

[0038] . The unitary device of Embodiment

[0037] , wherein said effector in said nanowell is a perturbation compound. Embodiment

[0039] . The unitary device of Embodiment

[0038] , wherein multiple copies of the same perturbation compound are reversibly attached to a perturbation bead in said nanowell. Embodiment

[0040] . The unitary device of Embodiment

[0019] , wherein each nanowell comprises an aqueous assay composition comprising a single cell, a perturbation bead, a capture bead, and spatial indices. Embodiment

[0041] . A sequential method for transferring a cell (55) and an additional assay component (24) into a nanowell (15) of an assay device (9), which method comprises: a) selecting a transfer device (1) comprising a bottom surface (2), a top surface (3), and side walls (4) wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor FL1, an opening diameter OD1, a height of D and a volume defined thereby, V-1, and each of which is configured to receive a single cell wherein said transfer device (1) further comprises inlet and outlet ports (7 and 8); b) selecting an assay device (9) wherein said assay device comprises a multiplicity of nanowells (15), each of which comprises an opening diameter (OD12) and a volume (V-4) wherein said nanowells (15) comprise a cell-adhering protein or peptideAttorney Docket No.009775.00044\WO adhering to an interior surface of said nanowell (15); c) reversibly attaching said assay device (9) to said transfer device (1) to form a unitary device (11) wherein the top surface (3) of said transfer device (1) comprising transfer wells (5) is faced upward while the top surface of said assay device (9) is facing downward; d) aligning the nanowells (15) of said assay device (9) over the transfer wells (5) such that the opening diameter (OD12) of the nanowells (15) encompasses an entirety of the opening diameter (OD1) of the transfer wells, wherein said attachment further defines a gap (G) having a volume V-4 which is connected by said inlet port (7) and said outlet port (8), thereby defining a microfluidic channel over said transfer wells (5); e) creating a flow of an aqueous solution comprising a heterogeneous population of effector moieties into the inlet port (7), through gap (G), and out of outlet port (8), wherein a rate of said flow is controlled to allow at least a portion of said moieties to deposit in said transfer wells (5); f) continuing the flow of said aqueous solution until at least a portion of said transfer wells (5) are populated with a single effector; g) terminating the flow of said aqueous solution; h) inverting the unitary device (11) whereby a single cell in a transfer well is transferred into the nanowell (15) aligned thereto, wherein said cell adheres to said protein or peptide; i) removing the transfer device (1) from the unitary device (11); j) attaching a second transfer device (1) to said assay device (9) wherein said transfer wells (5) of said second transfer device (1) are sized to accept an effector and further wherein said attachment further defines a gap (G) having a volume V-4 which is connected by said inlet port (7) and said outlet port (8) thereby defining a microfluidic channel over said transfer wells (5); k) repeating steps d) through i) to provide for an assay device comprising a multiplicity of nanowells (15) each comprising a single cell (25) and a single effector. l) continuing the flow of said aqueous solution until at least a portion of said transfer wells (5) are populated with an effector; m) terminating the flow of said aqueous solution and reversing the positions of the assay device and the second transfer device, thereby transferring the effector into the nanowell (15) aligned with the transfer well (5).Attorney Docket No.009775.00044\WO Embodiment

[0042] . The method of Embodiment

[0041] , which method further comprises replacing the second transfer device (1) with a third transfer device (1) having one or more additional effectors, targets, perturbation beads, capture beads, spatial indices, and assay reagents, which are to be delivered to the nanowells (15). Embodiment

[0043] . The method of Embodiment

[0042] , wherein said second transfer device delivered a perturbation bead or a capture bead to each nanowell (15). Embodiment

[0044] . The method of Embodiment

[0043] , wherein said bead is a magnetic bead that is retained in the nanowell when the assay device is inverted by magnetic forces. Embodiment

[0045] . A unitary device (11) comprising an assay device (9) and a transfer device (1) wherein the assay device (9) comprises: a) a top surface (13) having a multiplicity of nanowells (15) facing upward, wherein the nanowells (13) comprise an aqueous assay composition (21); and b) a transfer device having a top surface (3) positioned above said assay device (9) and facing downward; c) confinement means extending above said top surface (13) of said assay device (9) and below the top surface (3) of said transfer device (1) that maintains a 3- dimensional space suitable to hold a water-insoluble cover therebetween; and d) a water-insoluble cover retained in said confinement means wherein said cover remains on the top surface (13) of the assay device (9), including the top surface of the aqueous assay composition in the nanowells (15), resulting in a protective covering over the nanowells (15). Embodiment

[0046] . The unitary device of Embodiment

[0045] , wherein said water-insoluble cover positioned in said confinement means comprises an oil layer, a wax, or an epoxy sheet which covers the top surface (13) of said assay device (9). Embodiment

[0047] . The unitary device of Embodiment

[0045] , wherein said aqueous assay composition in said nanowells (15) comprises an effector and a target. Embodiment

[0048] . The unitary device of Embodiment

[0047] , wherein the target is a mammalian cell, and the effector is a perturbation compound reversibly bound to a perturbation bead which comprises a plurality of substantially the same perturbation compound and a DNA barcode configured to identify a structure and / or a reaction step used to synthesize said compound.Attorney Docket No.009775.00044\WO Embodiment

[0049] . The unitary device of Embodiment

[0048] , wherein the perturbation bead is coded with optically visible codes that identify the nanowell where the perturbation bead is located. Embodiment

[0050] . The unitary device of Embodiment

[0049] , wherein the optically visible code or codes for each nanowell are memorialized by an oligonucleotide or a set of oligonucleotides that are ligated together. Embodiment

[0051] . The unitary device of Embodiment

[0045] , wherein the nanowells (15) in said assay device (9) are spatially indexed, which identifies each of said nanowells (15) by its X and Y axis coordinates. Embodiment

[0052] . A method for reversibly applying and removing a cover positioned on a top surface (13) of an assay device (9) including over nanowells (15) found on said surface, wherein said nanowells (15) comprise an aqueous assay composition including one or more detergent-sensitive components, said method comprises: a) selecting an assay device comprising a top surface (13) and a multiplicity of nanowells (15) on said surface wherein said nanowells (15) comprise an aqueous assay solution including one or more detergent sensitive components and further wherein said top surface (13) of said assay device (9) comprises confinement means to maintain a volume of fluid over the top surface of the assay device (9) including over the nanowells (15); and b) adding a cover to said confinement means, which overlays the surface (13) of the assay device (9) and the aqueous assay solution in said nanowells (15), wherein said cover protects individual nanowell contents; c) attaching a transfer device (1) or a second device to said assay device (9) to form a unitary device (11); d) at one or more intervals during an assay performed in the assay device (9), remove the transfer device (1) or the second device to expose the cover and remove said cover (20), thereby exposing the aqueous assay composition in said nanowells (15); e) optionally i) modifying said aqueous assay composition by addition of one or more assay components to said aqueous assay composition; or ii) removing components from said aqueous assay composition; or iii) removing an aliquot from said aqueous assay composition; and f) adding a cover over the top surface (13) of said assay device (9), including the top surface of said nanowells (15) as per b) above, and then mating said assay device (9)Attorney Docket No.009775.00044\WO to a transfer device (1) or a second device. Embodiment

[0053] . The method of Embodiment

[0052] , which method further comprises analyzing said assay components or the aliquot of aqueous assay composition removed from the nanowell. Embodiment

[0054] . An assay method to screen a library of perturbation compounds to determine an effect of a perturbation compound from said library of perturbation compounds on a cell which allows for reversible access to components of the assay in an assay device (9) comprising a multiplicity of nanowells (15) during the assay without disrupting the assay or the cell, said method comprising: a) selecting an assay device (9) having a top surface (13) comprising a multiplicity of nanowells (15) comprising an aqueous assay composition and further wherein said top surface (13) of said assay device (9) comprises confinement means to maintain a volume of fluid over the top surface (13) of the assay device (9) including over the nanowells (15), wherein each nanowell (15) further comprises a perturbation bead which itself comprises a plurality of substantially the same, unique compound reversibly bound thereto, wherein said bead and said cell are maintained in an aqueous assay composition in said nanowells; b) adding a layer of a volatile oil to said confinement means, which layer overlays the top surface (13) of the assay device (9) and the aqueous assay composition in said nanowells (15), and optionally placing a lid, a transfer device (1), or a second device over the assay device (9) to form a unitary device (11); c) initiating the assay by releasing at least a portion of said perturbation compounds from their respective perturbation beads, thereby allowing the released portion of the perturbation compounds to interact with the cell in each nanowell; d) at one or more intervals during an assay performed in the assay device (9), removing the lid or replacement device from the top surface (13) of said assay device (9), thereby exposing an oil layer, and optionally removing a portion of said exposed oil layer to provide for an oil film on said surface (13); e) maintain said assay device under assay conditions, thereby allowing said volatile oil to dissipate due to evaporation, thereby exposing the aqueous assay composition of each nanowell (15); f) identifying and accessing one or more nanowells (15) of interest without disrupting the cell and / or the nanowell (15) contents and removing an aqueous aliquot or one or more assay components from the nanowells (15) to assess functional changes in the cell;Attorney Docket No.009775.00044\WO g) adding an oil layer over the top surface (13) of said assay device (9), including the top surface of said nanowells (15) and continuing the assay. Embodiment [1A]. A transfer device (1) comprising: a) a bottom surface (2), a top surface (3), and side walls (4), wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, a height of D, and a volume defined thereby, V-1, and each of which is configured to receive a defined number of effector moieties or targets; and b) inlet and outlet ports (7 and 8); wherein the transfer device optionally comprises a mating means to attach said transfer device (1) to a second device, said mating means is optionally reversible, optionally the second device is an assay device (9) comprising a multiplicity of nanowells. Embodiment [2A]. The transfer device of Embodiment [1A] wherein said mating means comprises one or more of the following mating means: an adhesive (16); or a clamp; or a latch; or a gasket; or a partial or complete hemispheric groove (17) on the transfer device (1) which aligns with a partial or complete hemispheric groove (17A) on the second device which forms an open tubular opening (19) wherein said tubular opening is adapted to fit an O-ring (18) or other compatible material after combining the transfer device (1) with the second device; or one or more protrusions (4B) on the transfer device (1) that mate with one or more compatible indentations (14A) on a second device; or one or more indentations on the transfer device (1) that mate with one or more compatible protrusions, which align with indentations; and optionally wherein said mating means comprises at least one indentation or at least one protrusion (4B) on the transfer device (1) which mates with at least one compatible protrusion or compatible indentation (14A) on said second device to form a unitary assay device (11) when combined. Embodiment [3A]. The transfer device of Embodiment [1A], wherein said mating means comprises an adhesive (16) of a two-sided pressure-sensitive adhesive having a thickness which defines a gap (G) between the transfer device (1) and the assay device (9), orAttorney Docket No.009775.00044\WO said mating means between the transfer device (1) and the assay device (9) comprises indentations on both the transfer device (1) and the assay device (9) that are alignable with each other such that, when aligned, form a tubular opening (19) suitable to fit an O-ring (18). Embodiment [4A]. The transfer device of Embodiment [3A], wherein said indentations are hemispheric or partially hemispheric indentations (17A and 17B) on said transfer device (1) and on said assay device that alignable and when aligned are able to fit an O-ring (18) within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined, and / or wherein said indentations are a polygonal open sided indentations found on both said transfer device (1) and said assay device (9) which, when aligned, fit an O-ring (18) is sized to fit within the tubular opening (19) formed thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined, wherein optionally said O-ring provides a distance between the top surface (3) of said transfer device (1) and the top surface (13) of said assay device (9) which defines gap (G). Embodiment [5A]. The transfer device of Embodiment [2A], wherein (i) said the transfer wells (5) of the transfer device (1) are enclosed in an area bound by said protrusion or protrusions (4B) or said indentation or indentations; (ii) said protrusion (4B) or said indentation extends in a continuous manner to encompass said transfer wells (5), wherein said continuous manner is a closed loop, and wherein said closed loop comprises a circular, oval, or oblong configuration, and / or said protrusions (4B) or said indentations comprise a connected set of closed protrusion walls (4D); and / or said closed walls form a polygon. Embodiment [6A]. The transfer device of Embodiment [5A], wherein said polygon is selected from a triangle, a rectangle, a trapezoid, a square, a pentagon, a hexagon, an octagon, and the like, as well as any other configuration that is continuous. Embodiment [7A]. The transfer device of Embodiment [1A], wherein said transfer device (1) and said assay device (9) are reversibly slidable with each other in any one of an X, Y, or Z axis. Embodiment [8A]. A unitary device comprising a transfer device (1) reversibly attached to an assay device (9), wherein: a) said transfer device (1) comprises a bottom surface (2), a top surface (3), and sideAttorney Docket No.009775.00044\WO walls (4) wherein: said top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, and a volume, V-1, each of which is configured to receive only a defined number of effector moieties or targets; inlet and outlet ports (7 and 8) that are associated with the transfer device; b) an assay device (9) comprising a multiplicity of nanowells (15) each having a floor, an opening diameter OD12, and a volume V-3 which, in combination, allow for conducting an assay in said nanowells (15); and c) a mating means for connecting the top surface (3) of said transfer device (1) to said top surface (13) of said assay device (9); wherein, upon mating, a gap (G) is formed between said top surface (3) of said transfer device (1) and said top surface (13) of said assay device (9), wherein said gap (G) defines a volume (V-4) and further wherein said inlet and outlet ports (7 and 8) of said transfer device (1) are in fluid communication with said gap (G) thereby providing for microfluidic communication into said inlet port (7), through said gap (G) and out of said outlet port (8), optionally wherein said transfer wells (5) of said transfer device (1) have an opening diameter (OD1) that ranges from about 5% to about 60% of the opening diameter (OD12) of the nanowells (15) of the assay device (9), and further provided that, upon mating, the transfer wells (5) align to said nanowells (15) on a one-to-one basis such that a projection of an opening of each transfer well (5) onto a corresponding nanowell (15) is encompassed by an area of said nanowell (15). Embodiment [9A]. The unitary device of Embodiment [8A], wherein said mating means comprises one or more of the following: an adhesive (16); or a clamp; or a latch; or a gasket; or a partial or complete hemispheric groove (17B) on the transfer device (1) which aligns with a partial or complete hemispheric groove (17A) on the assay device (9) which forms a tubular channel (17) wherein said tubular opening is adapted to fit an O-ring (18) or other compatible material after combining the transfer device (1) with the assay device (9); orAttorney Docket No.009775.00044\WO one or more protrusions (4B) on the transfer device (1) that mate with one or more compatible indentations (14A) on the assay device (9); or one or more indentations on the transfer device (1) that mate with one or more compatible protrusions, which align with said indentations; optionally wherein said mating means comprises an adhesive (16) of a two-sided pressure- sensitive adhesive having a thickness which defines a gap (G) between the transfer device (1) and the assay device (9); and / or said mating means between the transfer device (1) and the assay device (9) comprises hemispheric or partial hemispheric indentations (17A and 17B) that are alignable with each other such that, when aligned, form a tubular opening (19) suitable to fit an O-ring (18); and / or said hemispheric indentations (17A and 17B) of said transfer device (1) and said assay device are aligned, and said O-ring (18) is sized to fit within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined; and / or said partial hemispheric indentations (17A and 17B) of said transfer device (1) and said assay device are aligned, and said O-ring (18) is sized to fit within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined. Embodiment [10A]. The unitary device of Embodiment [9A], wherein said O- ring provides a distance between the top surface (3) of said transfer device (1) and the top surface (13) of said assay device (9), which defines gap (G). Embodiment [11A]. The unitary device of Embodiment [9A], wherein said mating means comprises at least one indentation or at least one protrusion (4B) on the transfer device (1) which mates with a compatible protrusion or compatible indentation (14A) respectively on said assay device (9) to form a unitary assay device (11) when combined, optionally wherein said the transfer wells (5) of the transfer device (1) are enclosed in an area bound by said protrusion or protrusions (4B) or said indentation or indentations, and optionally wherein said protrusion (4B) or said indentation extends in a continuous manner to encompass said transfer wells (5), wherein said continuous manner is a closed loop, and said closed loop comprises a circular, oval, or oblong configuration.Attorney Docket No.009775.00044\WO Embodiment [12A]. The unitary device of Embodiment [10A], wherein said protrusions (4B) or said indentations comprise a connected set of closed protrusion walls (4D), wherein said closed protrusion walls (4D) form a polygon, and optionally wherein said polygon is selected from a triangle, a rectangle, a trapezoid, a square, a pentagon, a hexagon, an octagon, and the like, as well as any other configuration that is continuous. Embodiment [13A]. The unitary device of Embodiment [8A], wherein (i) said transfer device (1) and said assay device (9) are reversibly slidable with each other in any one of an X, Y, or Z axis; (ii) said inlet port (7) and an outlet port (8) are on the same surface of the transfer device (9); the same surface is the top surface (3) of said transfer device (1); said target in said nanowell is a cell; or each nanowell comprises an aqueous assay composition comprising a single cell, a perturbation bead, a capture bead, and spatial indices. Embodiment [14A]. The unitary device of Embodiment [12A], wherein said effector in said nanowell is a perturbation compound. Embodiment [15A]. The unitary device of Embodiment [8A], wherein each nanowell comprises an aqueous assay composition comprising a single cell, a perturbation bead, a capture bead, and spatial indices. Embodiment [16A]. A sequential method for transferring a cell and an additional assay component into a nanowell (15) of an assay device (9), which method comprises: a) selecting a transfer device (1) comprising a bottom surface (2), a top surface (3), and side walls (4) wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, a height of D and a volume defined thereby, V-1, and each of which is configured to receive a single cell wherein said transfer device (1) further comprises inlet and outlet ports (7 and 8); b) selecting an assay device (9) wherein said assay device comprises a multiplicity of nanowells (15), each of which comprises an opening diameter (OD12) and a volume (V-4), wherein said nanowells (15) comprise a cell adhering protein or peptide adhering to an interior surface of said nanowell (15); c) reversibly attaching said assay device (9) to said transfer device (1) to form a unitary device (11) wherein the top surface (3) of said transfer device (1) comprising transfer wells (5) is faced upward while the top surface of said assay device (9) is facing downward;Attorney Docket No.009775.00044\WO d) aligning the nanowells (15) of said assay device (9) over the transfer wells (5) such that the opening diameter (OD12) of the nanowells (15) encompasses an entirety of the opening diameter (OD1) of the transfer wells, wherein said attachment further defines a gap (G) having a volume V-4 which is connected by said inlet port (7) and said outlet port (8), thereby defining a microfluidic channel over said transfer wells (5); e) creating a flow of an aqueous solution comprising a heterogeneous population of effector moieties into the inlet port (7), through gap (G), and out of outlet port (8), wherein a rate of said flow is controlled to allow at least a portion of said moieties to deposit in said transfer wells (5); f) continuing the flow of said aqueous solution until at least a portion of said transfer wells (5) are populated with a single effector; g) terminating the flow of said aqueous solution; h) inverting the unitary device (11) whereby a single cell in a transfer well is transferred into the nanowell (15) aligned thereto, wherein said cell adheres to said protein or peptide; i) removing the transfer device (1) from the unitary device (11); j) attaching a second transfer device (1) to said assay device (9) wherein said transfer wells (5) of said second transfer device (1) are sized to accept an effector and further wherein said attachment further defines a gap (G) having a volume V-4 which is connected by said inlet port (7) and said outlet port (8) thereby defining a microfluidic channel over said transfer wells (5); k) repeating steps d) through i) to provide for an assay device comprising a multiplicity of nanowells (15) each comprising a single cell and a single effector. l) continuing the flow of said aqueous solution until at least a portion of said transfer wells (5) are populated with an effector; m) terminating the flow of said aqueous solution and reversing the positions of the assay device and the second transfer device, thereby transferring the effector into the nanowell (15) aligned with the transfer well (5); wherein said method optionally comprises replacing the second transfer device (1) with a third transfer device (1) having one or more additional effectors, targets, perturbation beads, capture beads, spatial indices, and assay reagents which are to be delivered to the nanowells (15); wherein optionally said second transfer device delivered a perturbation bead or aAttorney Docket No.009775.00044\WO capture bead to each nanowell (15); and / or said bead is a magnetic bead which is retained in the nanowell when the assay device is inverted by magnetic forces. Embodiment [17A]. A unitary device (11) comprising an assay device (9) and a transfer device (1) wherein the assay device (9) comprises: a) a top surface (13) having a multiplicity of nanowells (15) facing upward, wherein the nanowells (13) comprise an aqueous assay composition; and b) a transfer device having a top surface (3) positioned above said assay device (9) and facing downward; c) confinement means extending above said top surface (13) of said assay device (9) and below the top surface (3) of said transfer device (1) that maintains a 3-dimensional space suitable to hold a water-insoluble cover therebetween; and d) a water-insoluble cover retained in said confinement means wherein said cover remains on the top surface (13) of the assay device (9), including the top surface of the aqueous assay composition in the nanowells (15), resulting in a protective covering over the nanowells (15); wherein (i) said water-insoluble cover positioned in said confinement means comprises an oil layer, a wax, or an epoxy sheet which covers the top surface (13) of said assay device (9); (ii) said aqueous assay composition in said nanowells (15) comprises an effector and a target, optionally wherein the target is a mammalian cell, and the effector is a perturbation compound reversibly bound to a perturbation bead which comprises a plurality of substantially the same perturbation compound and a DNA barcode configured to identify a structure and / or a reaction step used to synthesize said compound, and / or optionally wherein the perturbation bead is coded with optically visible codes that identify the nanowell where the perturbation bead is located. Embodiment [18A]. The unitary device of Embodiment [17A], wherein the optically visible code or codes for each nanowell are memorialized by an oligonucleotide or a set of oligonucleotides that are ligated together; and / or the nanowells (15) in said assay device (9) are spatially indexed, which identifies each of said nanowells (15) by its X and Y axis coordinates. Embodiment [19A]. A method for reversibly applying and removing a cover positioned on a top surface (13) of an assay device (9) including over nanowells (15) found on said surface, wherein said nanowells (15) comprise an aqueous assay compositionAttorney Docket No.009775.00044\WO including one or more detergent-sensitive components, said method comprises: a) selecting an assay device comprising a top surface (13) and a multiplicity of nanowells (15) on said surface wherein said nanowells (15) comprise an aqueous assay solution including one or more detergent sensitive components and further wherein said top surface (13) of said assay device (9) comprises confinement means to maintain a volume of fluid over the top surface of the assay device (9) including over the nanowells (15); and b) adding a cover to said confinement means, which overlays the surface (13) of the assay device (9) and the aqueous assay solution in said nanowells (15), wherein said cover protects individual nanowell contents; c) attaching a transfer device (1) or a second device to said assay device (9) to form a unitary device (11); d) at one or more intervals during an assay performed in the assay device (9), remove the transfer device (1) or the second device to expose the cover and remove said cover, thereby exposing the aqueous assay composition in said nanowells (15); e) optionally i) modifying said aqueous assay composition by adding one or more assay components to said aqueous assay composition; or ii) removing components from said aqueous assay composition; or iii) removing an aliquot from said aqueous assay composition; and f) adding a cover over the top surface (13) of said assay device (9), including the top surface of said nanowells (15) as per b) above, and then mating said assay device (9) to a transfer device (1) or a second device, and optionally said method further comprises analyzing said assay components or the aliquot of aqueous assay composition removed from the nanowell. Embodiment [20A]. An assay method to screen a library of perturbation compounds to determine an effect of a perturbation compound from said library of perturbation compounds on a cell, which allows for reversible access to components of the assay in an assay device (9) comprising a multiplicity of nanowells (15) during the assay without disrupting the assay or the cell, said method comprising: a) selecting an assay device (9) having a top surface (13) comprising a multiplicity of nanowells (15) comprising an aqueous assay composition and further wherein said top surface (13) of said assay device (9) comprises confinement means to maintain a volume of fluid over the top surface (13) of the assay device (9) including over the nanowells (15), wherein each nanowell (15) further comprises a perturbation bead which itself comprises aAttorney Docket No.009775.00044\WO plurality of substantially the same, unique compound reversibly bound thereto, wherein said bead and said cell are maintained in an aqueous assay composition in said nanowells; b) adding a layer of a volatile oil to said confinement means, which layer overlays the top surface (13) of the assay device (9) and the aqueous assay composition in said nanowells (15), and optionally placing a lid, a transfer device (1), or a second device over the assay device (9) to form a unitary device (11); c) initiating the assay by releasing at least a portion of said perturbation compounds from their respective perturbation beads, thereby allowing the released portion of the perturbation compounds to interact with the cell in each nanowell; d) at one or more intervals during an assay performed in the assay device (9), removing the lid or replacement device from the top surface (13) of said assay device (9), thereby exposing an oil layer, and optionally removing a portion of said exposed oil layer to provide for an oil film on said surface (13); e) maintain said assay device under assay conditions, thereby allowing said volatile oil to dissipate due to evaporation, thereby exposing the aqueous assay composition of each nanowell (15); f) identifying and accessing one or more nanowells (15) of interest without disrupting the cell and / or the nanowell (15) contents and removing an aqueous aliquot or one or more assay components from the nanowells (15) to assess functional changes in the cell; g) adding an oil layer over the top surface (13) of said assay device (9), including the top surface of said nanowells (15) and continuing the assay.

Claims

Attorney Docket No.009775.00044\WO WHAT IS CLAIMED IS:

1. A transfer device (1) comprising: a) a bottom surface (2), a top surface (3), and side walls (4), wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, a height of D, and a volume defined thereby, V-1, and each of which is configured to receive a defined number of effector moieties or targets; and b) inlet and outlet ports (7 and 8); wherein the transfer device optionally comprises a mating means to attach said transfer device (1) to a second device, said mating means is optionally reversible, optionally the second device is an assay device (9) comprising a multiplicity of nanowells.

2. The transfer device of claim 1, wherein said mating means comprises one or more of the following mating means: an adhesive (16); or a clamp; or a latch; or a gasket; or a partial or complete hemispheric groove (17) on the transfer device (1) which aligns with a partial or complete hemispheric groove (17A) on the second device which forms an open tubular opening (19) wherein said tubular opening is adapted to fit an O-ring (18) or other compatible material after combining the transfer device (1) with the second device; or one or more protrusions (4B) on the transfer device (1) that mate with one or more compatible indentations (14A) on a second device; or one or more indentations on the transfer device (1) that mate with one or more compatible protrusions, which align with indentations; and optionally wherein said mating means comprises at least one indentation or at least one protrusion (4B) on the transfer device (1) which mates with at least one compatible protrusion or compatible indentation (14A) on said second device to form a unitary assay device (11) when combined.Attorney Docket No.009775.00044\WO 3. The transfer device of claim 1, wherein said mating means comprises an adhesive (16) of a two-sided pressure-sensitive adhesive having a thickness which defines a gap (G) between the transfer device (1) and the assay device (9), or said mating means between the transfer device (1) and the assay device (9) comprises indentations on both the transfer device (1) and the assay device (9) that are alignable with each other such that, when aligned, form a tubular opening (19) suitable to fit an O-ring (18).

4. The transfer device of claim 3, wherein said indentations are hemispheric or partially hemispheric indentations (17A and 17B) on said transfer device (1) and on said assay device that alignable and when aligned are able to fit an O-ring (18) within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined, and / or wherein said indentations are a polygonal open sided indentations found on both said transfer device (1) and said assay device (9) which, when aligned, fit an O-ring (18) is sized to fit within the tubular opening (19) formed thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined, wherein optionally said O-ring provides a distance between the top surface (3) of said transfer device (1) and the top surface (13) of said assay device (9) which defines gap (G).

5. The transfer device of claim 2, wherein (i) said transfer wells (5) of the transfer device (1) are enclosed in an area bound by said protrusion or protrusions (4B) or said indentation or indentations; (ii) said protrusion (4B) or said indentation extends in a continuous manner to encompass said transfer wells (5), wherein said continuous manner is a closed loop, and wherein said closed loop comprises a circular, oval, or oblong configuration, and / or said protrusions (4B) or said indentations comprise a connected set of closed protrusion walls (4D); and / or said closed walls form a polygon.

6. The transfer device of claim 5, wherein said polygon is selected from a triangle, a rectangle, a trapezoid, a square, a pentagon, a hexagon, an octagon, and the like, as well as any other configuration that is continuous.

7. The transfer device of claim 1, wherein said transfer device (1) and said assay device (9) are reversibly slidable with each other in any one of an X, Y, or Z axis.Attorney Docket No.009775.00044\WO 8. A unitary device comprising a transfer device (1) reversibly attached to an assay device (9), wherein: a) said transfer device (1) comprises a bottom surface (2), a top surface (3), and side walls (4) wherein: said top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, and a volume, V-1, each of which is configured to receive only a defined number of effector moieties or targets; inlet and outlet ports (7 and 8) that are associated with the transfer device; b) an assay device (9) comprising a multiplicity of nanowells (15) each having a floor, an opening diameter OD12, and a volume V-3 which, in combination, allow for conducting an assay in said nanowells (15); and c) a mating means for connecting the top surface (3) of said transfer device (1) to said top surface (13) of said assay device (9); wherein, upon mating, a gap (G) is formed between said top surface (3) of said transfer device (1) and said top surface (13) of said assay device (9), wherein said gap (G) defines a volume (V-4) and further wherein said inlet and outlet ports (7 and 8) of said transfer device (1) are in fluid communication with said gap (G) thereby providing for microfluidic communication into said inlet port (7), through said gap (G) and out of said outlet port (8), optionally wherein said transfer wells (5) of said transfer device (1) have an opening diameter (OD1) that ranges from about 5% to about 60% of the opening diameter (OD12) of the nanowells (15) of the assay device (9), and further provided that, upon mating, the transfer wells (5) align to said nanowells (15) on a one-to-one basis such that a projection of an opening of each transfer well (5) onto a corresponding nanowell (15) is encompassed by an area of said nanowell (15).

9. The unitary device of claim 8, wherein said mating means comprises one or more of the following: an adhesive (16); or a clamp; or a latch; or a gasket; or a partial or complete hemispheric groove (17B) on the transfer device (1) whichAttorney Docket No.009775.00044\WO aligns with a partial or complete hemispheric groove (17A) on the assay device (9) which forms a tubular channel (17) wherein said tubular opening is adapted to fit an O-ring (18) or other compatible material after combining the transfer device (1) with the assay device (9); or one or more protrusions (4B) on the transfer device (1) that mate with one or more compatible indentations (14A) on the assay device (9); or one or more indentations on the transfer device (1) that mate with one or more compatible protrusions, which align with said indentations; optionally wherein said mating means comprises an adhesive (16) of a two-sided pressure-sensitive adhesive having a thickness which defines a gap (G) between the transfer device (1) and the assay device (9); and / or said mating means between the transfer device (1) and the assay device (9) comprises hemispheric or partial hemispheric indentations (17A and 17B) that are alignable with each other such that, when aligned, form a tubular opening (19) suitable to fit an O-ring (18); and / or said hemispheric indentations (17A and 17B) of said transfer device (1) and said assay device are aligned, and said O-ring (18) is sized to fit within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined; and / or said partial hemispheric indentations (17A and 17B) of said transfer device (1) and said assay device are aligned, and said O-ring (18) is sized to fit within the tubular opening (19) thereby providing for a watertight fitting between said transfer device (1) and said assay device (9) when combined.

10. The unitary device of claim 9, wherein said O-ring provides a distance between the top surface (3) of said transfer device (1) and the top surface (13) of said assay device (9), which defines gap (G).

11. The unitary device of claim 9, wherein said mating means comprises at least one indentation or at least one protrusion (4B) on the transfer device (1) which mates with a compatible protrusion or compatible indentation (14A) respectively on said assay device (9) to form a unitary assay device (11) when combined, optionally wherein said the transfer wells (5) of the transfer device (1) are enclosed in an area bound by said protrusion or protrusions (4B) or said indentation or indentations, and optionally wherein said protrusion (4B) or said indentation extends in a continuous mannerAttorney Docket No.009775.00044\WO to encompass said transfer wells (5), wherein said continuous manner is a closed loop, and said closed loop comprises a circular, oval, or oblong configuration.

12. The unitary device of claim 10, wherein said protrusions (4B) or said indentations comprise a connected set of closed protrusion walls (4D), wherein said closed protrusion walls (4D) form a polygon, and optionally wherein said polygon is selected from a triangle, a rectangle, a trapezoid, a square, a pentagon, a hexagon, an octagon, and the like, as well as any other configuration that is continuous.

13. The unitary device of claim 8, wherein (i) said transfer device (1) and said assay device (9) are reversibly slidable with each other in any one of an X, Y, or Z axis; (ii) said inlet port (7) and an outlet port (8) are on the same surface of the transfer device (9); the same surface is the top surface (3) of said transfer device (1); said target in said nanowell is a cell; or each nanowell comprises an aqueous assay composition comprising a single cell, a perturbation bead, a capture bead, and spatial indices.

14. The unitary device of claim 12, wherein said effector in said nanowell is a perturbation compound.

15. The unitary device of claim 8, wherein each nanowell comprises an aqueous assay composition comprising a single cell, a perturbation bead, a capture bead, and spatial indices.

16. A sequential method for transferring a cell and an additional assay component into a nanowell (15) of an assay device (9), which method comprises: a) selecting a transfer device (1) comprising a bottom surface (2), a top surface (3), and side walls (4) wherein the top surface (3) extends upward from said bottom surface (2) by a distance D1 and comprises a multiplicity of transfer wells (5), each comprising a floor, an opening diameter OD1, a height of D and a volume defined thereby, V-1, and each of which is configured to receive a single cell wherein said transfer device (1) further comprises inlet and outlet ports (7 and 8); b) selecting an assay device (9) wherein said assay device comprises a multiplicity of nanowells (15), each of which comprises an opening diameter (OD12) and a volume (V-4), wherein said nanowells (15) comprise a cell adhering protein or peptide adhering to an interior surface of said nanowell (15); c) reversibly attaching said assay device (9) to said transfer device (1) to form aAttorney Docket No.009775.00044\WO unitary device (11) wherein the top surface (3) of said transfer device (1) comprising transfer wells (5) is faced upward while the top surface of said assay device (9) is facing downward; d) aligning the nanowells (15) of said assay device (9) over the transfer wells (5) such that the opening diameter (OD12) of the nanowells (15) encompasses an entirety of the opening diameter (OD1) of the transfer wells, wherein said attachment further defines a gap (G) having a volume V-4 which is connected by said inlet port (7) and said outlet port (8), thereby defining a microfluidic channel over said transfer wells (5); e) creating a flow of an aqueous solution comprising a heterogeneous population of effector moieties into the inlet port (7), through gap (G), and out of outlet port (8), wherein a rate of said flow is controlled to allow at least a portion of said moieties to deposit in said transfer wells (5); f) continuing the flow of said aqueous solution until at least a portion of said transfer wells (5) are populated with a single effector; g) terminating the flow of said aqueous solution; h) inverting the unitary device (11) whereby a single cell in a transfer well is transferred into the nanowell (15) aligned thereto, wherein said cell adheres to said protein or peptide; i) removing the transfer device (1) from the unitary device (11); j) attaching a second transfer device (1) to said assay device (9) wherein said transfer wells (5) of said second transfer device (1) are sized to accept an effector and further wherein said attachment further defines a gap (G) having a volume V-4 which is connected by said inlet port (7) and said outlet port (8) thereby defining a microfluidic channel over said transfer wells (5); k) repeating steps d) through i) to provide for an assay device comprising a multiplicity of nanowells (15) each comprising a single cell and a single effector; l) continuing the flow of said aqueous solution until at least a portion of said transfer wells (5) are populated with an effector; m) terminating the flow of said aqueous solution and reversing the positions of the assay device and the second transfer device, thereby transferring the effector into the nanowell (15) aligned with the transfer well (5); wherein said method optionally comprises replacing the second transfer device (1) with a third transfer device (1) having one or more additional effectors, targets, perturbation beads, capture beads, spatial indices, and assay reagents which are to beAttorney Docket No.009775.00044\WO delivered to the nanowells (15); wherein optionally said second transfer device delivered a perturbation bead or a capture bead to each nanowell (15); and / or said bead is a magnetic bead which is retained in the nanowell when the assay device is inverted by magnetic forces.

17. A unitary device (11) comprising an assay device (9) and a transfer device (1) wherein the assay device (9) comprises: a) a top surface (13) having a multiplicity of nanowells (15) facing upward, wherein the nanowells (13) comprise an aqueous assay composition; and b) a transfer device having a top surface (3) positioned above said assay device (9) and facing downward; c) confinement means extending above said top surface (13) of said assay device (9) and below the top surface (3) of said transfer device (1) that maintains a 3- dimensional space suitable to hold a water-insoluble cover therebetween; and d) a water-insoluble cover retained in said confinement means wherein said cover remains on the top surface (13) of the assay device (9), including the top surface of the aqueous assay composition in the nanowells (15), resulting in a protective covering over the nanowells (15); wherein (i) said water-insoluble cover positioned in said confinement means comprises an oil layer, a wax, or an epoxy sheet which covers the top surface (13) of said assay device (9); (ii) said aqueous assay composition in said nanowells (15) comprises an effector and a target, optionally wherein the target is a mammalian cell, and the effector is a perturbation compound reversibly bound to a perturbation bead which comprises a plurality of substantially the same perturbation compound and a DNA barcode configured to identify a structure and / or a reaction step used to synthesize said compound, and / or optionally wherein the perturbation bead is coded with optically visible codes that identify the nanowell where the perturbation bead is located.

18. The unitary device of claim 17, wherein the optically visible code or codes for each nanowell are memorialized by an oligonucleotide or a set of oligonucleotides that are ligated together; and / or the nanowells (15) in said assay device (9) are spatially indexed, which identifies each of said nanowells (15) by its X and Y axis coordinates.Attorney Docket No.009775.00044\WO 19. A method for reversibly applying and removing a cover positioned on a top surface (13) of an assay device (9) including over nanowells (15) found on said surface, wherein said nanowells (15) comprise an aqueous assay composition including one or more detergent-sensitive components, said method comprises: a) selecting an assay device comprising a top surface (13) and a multiplicity of nanowells (15) on said surface wherein said nanowells (15) comprise an aqueous assay solution including one or more detergent sensitive components and further wherein said top surface (13) of said assay device (9) comprises confinement means to maintain a volume of fluid over the top surface of the assay device (9) including over the nanowells (15); and b) adding a cover to said confinement means, which overlays the surface (13) of the assay device (9) and the aqueous assay solution in said nanowells (15), wherein said cover protects individual nanowell contents; c) attaching a transfer device (1) or a second device to said assay device (9) to form a unitary device (11); d) at one or more intervals during an assay performed in the assay device (9), remove the transfer device (1) or the second device to expose the cover and remove said cover, thereby exposing the aqueous assay composition in said nanowells (15); e) optionally i) modifying said aqueous assay composition by adding one or more assay components to said aqueous assay composition; or ii) removing components from said aqueous assay composition; or iii) removing an aliquot from said aqueous assay composition; and f) adding a cover over the top surface (13) of said assay device (9), including the top surface of said nanowells (15) as per b) above, and then mating said assay device (9) to a transfer device (1) or a second device, and optionally said method further comprises analyzing said assay components or the aliquot of aqueous assay composition removed from the nanowell.

20. An assay method to screen a library of perturbation compounds to determine an effect of a perturbation compound from said library of perturbation compounds on a cell, which allows for reversible access to components of the assay in an assay device (9) comprising a multiplicity of nanowells (15) during the assay without disrupting the assay or the cell, said method comprising: a) selecting an assay device (9) having a top surface (13) comprising a multiplicity of nanowells (15) comprising an aqueous assay composition and further wherein said topAttorney Docket No.009775.00044\WO surface (13) of said assay device (9) comprises confinement means to maintain a volume of fluid over the top surface (13) of the assay device (9) including over the nanowells (15), wherein each nanowell (15) further comprises a perturbation bead which itself comprises a plurality of substantially the same, unique compound reversibly bound thereto, wherein said bead and said cell are maintained in an aqueous assay composition in said nanowells; b) adding a layer of a volatile oil to said confinement means, which layer overlays the top surface (13) of the assay device (9) and the aqueous assay composition in said nanowells (15), and optionally placing a lid, a transfer device (1), or a second device over the assay device (9) to form a unitary device (11); c) initiating the assay by releasing at least a portion of said perturbation compounds from their respective perturbation beads, thereby allowing the released portion of the perturbation compounds to interact with the cell in each nanowell; d) at one or more intervals during an assay performed in the assay device (9), removing the lid or replacement device from the top surface (13) of said assay device (9), thereby exposing an oil layer, and optionally removing a portion of said exposed oil layer to provide for an oil film on said surface (13); e) maintain said assay device under assay conditions, thereby allowing said volatile oil to dissipate due to evaporation, thereby exposing the aqueous assay composition of each nanowell (15); f) identifying and accessing one or more nanowells (15) of interest without disrupting the cell and / or the nanowell (15) contents and removing an aqueous aliquot or one or more assay components from the nanowells (15) to assess functional changes in the cell; g) adding an oil layer over the top surface (13) of said assay device (9), including the top surface of said nanowells (15) and continuing the assay.

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