Capture beads for use in assays

Abstract

Disclosed are capture beads comprising one or more index or indices bound thereto wherein said indices identify a unique set of micro components from an assay conducted in a given examination area in an assay device comprising multiple examination areas.

Description

CAPTURE BEADS FOR USE IN ASSAYSRelated Applications

[0001] This Application claims the benefit of U.S. Provisional Application No. 63 / 624,181, filed January 23, 2024, which is incorporated by reference herein in its entirety.Field

[0002] This disclosure provides for a capture bead that is suitable for use in an assay device which is configured to correlate the capture bead to a specific examination area in the device as well as correlating any captured nucleic acid or other cellular components generated to during the assay to that bead. This disclosure further provides for methods, devices and systems that include said capture bead.Background

[0003] Capture beads can be used in assays to isolate certain components generated by an assay which are desired for analysis after assay completion. In general, assay devices have a multiplicity of examination areas that allow for thousands to millions of assays to be conducted simultaneously in one device. Examination areas in the device can be populated with one or more cells of interest and a separate assay can be conducted in each examination area. The assay typically includes a perturbation element to perturb the cell or cells and then determines the consequences that the perturbation had on the cell or cells. A capture bead is included in the examination area and, when the assay is complete and the cell lysed, nucleic acids released from the cell are then captured by the bead. Typically, but not necessarily, a single perturbation is combined with a single cell which is to be perturbed. After capture of the nucleic acid(s), the capture bead is isolated, and the nucleic acid is sequenced.

[0004] In one conventional system, the assay employs a perturbation bead that comprises multiple copies of the same perturbation compound which are releasably attached to the bead. In addition, the perturbation bead includes multiple copies of an oligonucleotide releasably bound to the bead. This oligonucleotide is generated during synthesis of the compound on the bead, and which records the reaction steps used to synthesis the compound and is sometimes referred to herein as the “compound oligonucleotide”. In some embodiments, the compound oligonucleotide includes a nucleic acid capturing element such as a mRNA capturing element. The assay is conducted by releasing the perturbation compound from the perturbation bead thereby perturbing the cell. Aftercompletion of the assay, the cell is lysed, and the compound oligonucleotide having nucleic acid capturing elements thereon can capture those nucleic acids whether the compound oligonucleotide is released from the bead into the examination area or retained on the bead. In one embodiment, the nucleic acid is mRNA and the nucleic acid capturing element is poly-T.

[0005] When attaching poly-T to the compound oligonucleotide, mRNA released from the lysed cell is captured by the compound oligonucleotide. In some embodiments, the compound oligonucleotide comprising poly-T and mRNA bound thereto can be sequenced in a single sequencing step on the bead. The resulting information provides an insight into the changes in functionality of the cell induced by the perturbation as well as the structure of the perturbation compound based on the reactions conducted to make this compound.

[0006] To obtain more meaningful information from these assays such as additional changes in functionality of the cell induced by the perturbation both on a static and in a real time basis, it is necessary to identify the perturbation elements generated by cellular perturbation and then correlate these informational components back to the capture bead and then back to the specific examination area where the assay was run. That is to say that limiting the knowledge of the cellular perturbation only to the compound that induced the perturbation and the mRNA generated by the perturbation also limits the information that is otherwise available from the assay.

[0007] Limiting the amount of information regarding the changes in cellular functionality induced by the perturbation to just the mRNA and perturbation compound used also limits the value of that information. Other information regarding changes in cellular functionality left unaddressed include, by way of example only, the specific well from which the capture bead was retrieved, changes in cellular morphology, how the assay conditions affect the changes in cellular functionality, as well as other information such as the technician conducting the assay, the date of the assay, and other perturbation elements that might be captured.

[0008] However, all or some of this additional information is often not collected during an assay for a variety of reasons including the inability to correlate the mRNA generated by the perturbation compound back to the specific examination area where the assay was conducted. Information regarding the specific examination area which generated the nucleic acid is lost as is any information regarding other functional changes in the cell itself. This prevents association a more complete description of the changes infunctionality of the cell and a more complete understanding of the impact of the perturbation element on the cell. Such would include changes in cellular functionality during the assay and when those changes occurred after initiating cellular perturbation. Adding such this information provides a more complete picture of how the perturbation altered the functionality of the cell and, whether such changes were deemed to be positive or negative.

[0009] Accordingly, there is an ongoing need to expand the amount of information retrieved from one or more of the examination areas found in an assay of a library of diverse compounds. Such additional information would expand the knowledge obtained from each assay and would facilitates drug discovery. / . Definitions

[0010] In order to provide clarity to the reader, the following terms are defined. Terms that are not defined have their scientifically accepted definition.

[0011] 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.

[0012] 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 a sub value therebetween. In one embodiment, the term “about” when used with regard to a dose amount means that the dose may vary by + / - 10%.

[0013] 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.

[0014] 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 disclosure.

[0015] 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 disclosure.

[0016] As used herein, the term “oligonucleotide strand” refers to oligonucleotides having from about 6 to about 10,000 nucleotides. Such strands can be attached to each other to form an oligonucleotide.

[0017] As used herein, the term “oligonucleotide” refers to an oligonucleotide having from about 6 to about 10,000 nucleotides provided that if the oligonucleotide comprises 2 or more oligonucleotide strands attached together, then the oligonucleotide comprises from about 12 to about 30,000 individual nucleotides.

[0018] As used herein, the term “capture bead” refers to any and all beads used in an assay to capture one or more cellular components after perturbing the cell and then lysing the cell to release such cellular components that are uniquely generated due to the perturbation (“perturbation components”). In some cases, the perturbation components include nucleic acids such as mRNA, 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 an examination area. Multiple capture beads can be used when different perturbation components are to be captured. Alternatively, a single capture bead is used such that different perturbation components are to be captured by that bead.

[0019] 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 generates or can be stimulated to generate a visual image such as exhibit a color, visible light, lettering, numbers, symbols, barcodes, fluorescent particles, micro-components such as microchips, UPC codes, tags or combinations thereof that can be seen with an unaided eye or by use of a microscope. In some cases, the detectable label generates chemical signals optionally requiring suitable instrumentation such as quantum dots, fluorescence (e.g., fluorescent particles, mass spectra, nuclear magnetic spectra (e.g., H1or C13spectrum), oligonucleotides, and the like. In some cases, the detectable signal generates an audio or electromagnetic signal such as those generated by a radio frequency identification (RFID), WiFi or a Bluetooth device. In some cases, the detectable label generates a biological signal or a biologically induced signal such nucleic acids, bioluminescence, and the like. The particular detectable label or labels used in the embodiments disclosed herein is 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.

[0020] As used herein, the term an “examination area” or “area” is a defined point (e.g., region, position, space, etc.), in an assay device or portion of an assay device, where asingle assay is performed at a given time. Multiple examination areas are employed in one or more assay device. Examination areas may include droplets, wells, channels, and the like. Each area is capable of containing at least one cell, at least one capture bead, and at least one perturbation element in isolation from other locations. In some embodiments, examination areas do not include points within a device that do not participate in an assay.

[0021] As used herein, the term “an oligonucleotide index” refers to an oligonucleotide which uniquely correlates to a specific capture bead. 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 “capturing element” that is able to capture a perturbation component as defined above. In some embodiments, the oligonucleotide index can be attached to the capture bead. In some embodiments, the index is attached to the perturbation component (e.g., mRNA) which is then captured on the capture bead. In some embodiments, this occurs during the assay and, in other embodiments, this occurs after the bead is removed from the examination area. In some embodiments, a single capture bead is employed in each examination area. In some embodiments, multiple capture beads are employed in each examination area. In some embodiments, each of the capture beads in a single examination area is indexed with the same unique oligonucleotide specific for that capture bead. In some embodiments, some or all of the capture beads in a single examination area are indexed with a unique oligonucleotide index all of which correlate to the same capture bead. For example, one or more of said additional capture beads are indexed with a unique capturing element and a unique oligonucleotide index in order to capture different perturbation components generated by the perturbation.

[0022] As used herein, the term “unique” as used relative to its use in the phrase “unique to a capture bead” refers to both the unique label or set of labels and the oligonucleotide index where both the label or set of labels and the index are unique relative to other labels or set of labels and indices used with other capture beads as found in other examination areas of the same assay device during a particular assay. Such does not infer that the label or set of label or the specific oligonucleotide index or indices is strictly unique but rather means that they are unique in the assay in question. Accordingly, such a unique label or set of labels and a unique oligonucleotide index need only be unique relative to labels or sets of labels and unique oligonucleotide indices used on other capture beads in other examination areas in the same assay in question.

[0023] As used herein, the term “perturbation element” refers to any source that perturbs a cell. Such sources include, by way of example only, compounds, antibodies, immune cells, siRNA, viruses, bacteria, fungi, change in one or more the conditions of the assay such but not limited to buffers, salts, pH, temperature, nutrients, oxygen levels, oxidizing agents, physical stress, and the like. In one embodiment, the perturbing element is a compound released from a perturbing bead.

[0024] As used herein, the term “perturbation bead” refers to a bead that carries the perturbation element in a releasable form. During an assay in an examination area, the perturbation element carried by the perturbation bead is released and allowed to interface with the cell found in the examination. Typically, the perturbation bead carries multiple copies of the same compound in releasable form which compound constitutes the perturbation element. In some embodiments, the assay does not include a perturbation bead as the perturbation element constitutes one or more conditions of the assay as noted above or, alternatively, the perturbation element can be a virus, an antibody, or a cell such as an immune cell.

[0025] As used herein, the term “compound” or “perturbation compound” 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.

[0026] As used herein, the term “assay device” refers to a device having multiple examination areas for conducting assays using the capture beads as described herein. When the perturbation element is a bead comprising multiple copies of the same compound releasable from the bead and multiple copies of a compound oligonucleotide identifying the structure of the compound or the synthetic steps used to make the compound, the perturbation element is sometimes referred to herein as a “perturbation bead”.

[0027] As used herein, the term “mapped assay device” refers to any assay device where the examination areas can be identified by the geometry of the device (e.g., X and Y coordinates or any orientation that allows the individual examination areas to be identified such as triangular shapes, trapezoidal shapes, etc.) or by images, lettering, numbering, etc. associated with each examination area such that the examination area can be uniquely identified. In some cases, a mark or label or other indicia on the assay device to allow the technician to properly orient the mapped assay device.

[0028] As used herein, the term “plurality of perturbation beads” means more than one bead, such as at least 100 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 of 5,000,000 beads or more but generally is about 2,000,000.

[0029] As used herein, the term “releasably bound thereto” means that the specific component so described is attached to a capture bead in a releasable manner such that release / cleavage of at least a portion 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 bonds - bonds that are cleaved under appropriate stimulation from light (e.g., UV light), heat, pH change, electromagnetic stimulation, sound, salt, change in oxidation, and the like. Such cleavable bonds are well known in the art.

[0030] As used herein, the term “cellular morphology” refers to any one or a combination of two or more properties of a cell or components thereof such as the size, shape, structure, and / or form of a cell or components thereof. Cellular components are well known in the art and include the nucleus, the mitochondria, and ribosomes.

[0031] 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, and the like in the cell some of which can be readily visualized.

[0032] As used herein, the term “cell” refers to a mammalian cell, preferably, a cell from a primate, and more preferably, a human cell.

[0033] As used herein, the term “linker” refers to group which attaches one entity to another. Such a linker releasable or non-releasable. In some embodiments the linker is nucleotide or an oligonucleotide having from 2 to 40 nucleotide units. In some embodiments, the linker is a non-nucleotide or oligonucleotide but 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) or halo to satisfy the valence of the atoms.

[0034] The term “releasable” 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 UV light, heat, pH change, salt, and other components well known in the art. . Summary

[0035] This disclosure provides for capture beads that are configured to collect information regarding changes in cellular functionality generated during an assay induced by exposing a cell to a perturbation element. In particular, the capture beads described herein are capable of associating the change in functionality directly to the perturbation element and the examination area used. This allows collection of information both statically (at the end of the assay) as well as in real time or dynamic changes in functionality that occur during the assay. Static information includes assessing changes in one or more cellular functions that have changed during the assay 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 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 need not be captured to assess the perturbation components. In one embodiment, protein degradation components can be evaluated by immunofluorescent staining after the assay is completed if the perturbed and lysed cell is not from a stable cell line which contains innate fluorescence.

[0036] In addition, one can assess changes in cellular functionality due to the use of a other perturbation elements that are not compounds such as a different pH, and / or a different temperature, or using different reaction conditions such as a different buffer, and / or a different salt, and / or a different concentration of buffer and / or salt, and / or a different length of time for the assay to be conducted, and the like. Still further, the perturbation element can be an antibody or a cell such an immune cell.

[0037] Real time information can include a picture(s) or a video of the perturbed cell during a portion or all of the assay to assess the change in functionality evidenced by a change in morphology of that cell, evidence of the presence of apoptotic markers or other markers,protein upregulation or downregulation that can be monitored live and / or at the end of the assay, activation of T-cells thereby observing proliferation and change in their sizes, T- cells killing target cells, and the like. Coupling the static information with the real time (or “dynamic”) information provides for a more complete analysis of the impact of exposing the perturbation element(s) to the functionality of the cell.

[0038] The ability to correlate static and dynamic information necessitates that one can correlate the static functionality captured by the capture bead(s) to the examination area that where the assay was conducted which then correlates the dynamic information to the static information generated during the assay. Such allows coupling of multiple changes in cellular functionality into a more global analysis. As described herein, such is accomplished by including on the capture bead: a label or a set of detectable labels that are unique to that capture bead; a binding element, and an oligonucleotide index on the capture bead that can correlate the captured perturbation component(s) to that bead. Still further, the assay must employ a mapped assay device that allows correlation of the capture bead to the examination area where the capture bead is located.

[0039] By coupling each of these informational components, the technician can correlate the captured perturbation components generated to the capture bead using the index or indices. The technician can then correlate the capture bead to a specific examination area using the map on the assay device. This then allows one to observe the dynamic changes in cellular functionality during the assay such as the changes in morphology and assign this to a particular capture bead. Combining all or some of this information using knowledge as to what examination area was used, what perturbation element was used in that examination area, what capture bead was in that examination area, and what perturbation component was retrieved from that examination area by the capture bead is substantial static evidence of the changes in functionality. Still further, if pictures or film of the assay device are taken during the assay, dynamic changes in cellular functionality occuring during the assay for a given examination area of interest can be observed.

[0040] When the perturbation element is a compound, the perturbation bead has bound thereto via a linker, which is optionally releasable, a compound oligonucleotide that identifies at least a portion of the compound bound to the bead. The one or more capture beads included in an examination area will also capture the compound oligonucleotide thereby allowing the technician to ascertain the structure of the perturbation compound that was responsible for the perturbation of the cell captured both in a static and in real time.

[0041] Specifically, an assay device comprising a multiplicity of examination areas is mapped such that each examination area is readily defined. Such mapping can include the X and Y coordinates for each examination area. Alternatively, each examination area in the device can be marked with a unique image or code. However, such is typically unnecessary when mapping the X and Y coordinates suffice. In those assay devices where the X and Y coordinates are ambiguous due to lack of uniformity of the examination areas to each other, marking each area will resolve the ambiguity. Still further, an indicia or mark can be applied to the assay device to orient the device along its proper X and Y axis. See, for example, FIGS. 1 A and IB.

[0042] In addition, the methods described herein permit the use of multiple perturbations in an examination area. For example, multiple perturbations can include a first perturbation designed to generate a second perturbation and the assay measures the impact of the second perturbation on a cell of interest. Such can be illustrated by using a perturbation compound (first perturbation) to contact and perturb a T-cell which then contacts a tumor cell to determine the impact of the perturbed T-cell on the tumor cell.

[0043] In some embodiments, when the capture element attached to the capture bead is an oligonucleotide (“capture oligonucleotide”) configured to capture a nucleic acid from the perturbed and lysed cell, an oligonucleotide index can be attached to the capture oligonucleotide optionally through a linker to ensure that the captured nucleic acid is combined with the index oligonucleotide thereby allowing for the technician to correlate the captured nucleic acid to the index oligonucleotide by sequencing the combination. This allows for correlation back to the specific capture bead that captured that nucleic acid. Then, using the map from the mapped assay device, a final correlation provides the examination area from which the capture bead was used.

[0044] The combination of these otherwise separate informational components allows for real time information generated during the assay to be associated to the cell used in that examination area. This allows the technician to combine the real time and static information in a manner that provides significant information as to the change in cellular functionality that occurred during the assay.

[0045] In some embodiments, there is provide a capture bead for use in an assay said capture bead comprising: a) a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead;b) a multiplicity of an oligonucleotide index which is unique to said capture bead and is attached to thereto optionally through a linker; and c) a multiplicity of a capturing element.

[0046] 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 said examination areas which capture beads comprise: a) a detectable label or a set of detectable labels attached to said bead which uniquely identifies each capture bead; b) a multiplicity of an oligonucleotide index which is unique to each of said capture beads and is attached thereto; and c) a multiplicity of a capturing element, 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.

[0047] In some embodiments, the detectable label or set of detectable labels is / are optically detectable.

[0048] In some embodiments, the set of detectable labels have from 2 to 10 members.

[0049] In some embodiments, the label employed is a visible label selected from images, codes, shapes, colors, inducible colors, lettering, numbering, symbols, bar codes, UPC codes, and materials introduced onto the capture bead or electromagnetic signals such as radio waves selected from BlueTooth, WiFi, and RFID, or combinations thereof.

[0050] In some embodiments, the oligonucleotide index is attached to the bead through a linker.

[0051] In some embodiments, the oligonucleotide index is attached to the capture bead by a releasable linker.

[0052] In some embodiments, the oligonucleotide index comprises from about 6 to about 10,000 individual nucleotides; preferably from about 8 to about 200 nucleotides; and more preferably from about 8 to about 100 nucleotides.

[0053] In some embodiments, the capturing element is an oligonucleotide.

[0054] In some embodiments, the oligonucleotide capturing element is attached to the oligonucleotide index.

[0055] In some embodiments, the attachment is either direct or through a linker.

[0056] In some embodiments, the linker is a releasable linker.

[0057] In some embodiment, the capturing element is a receptor or ligand for the perturbation component to be captured. Such receptors include antibodies or antibody bindingfragments for the perturbation compound; an enzyme or enzyme receptor for the perturbation component, a receptor for a given cytokine or chemokine, and the like.

[0058] In some embodiments, the capturing element is avi din / streptavidin that captures perturbation components that has been modified to contain a biotin group.

[0059] In some embodiments, the capturing element is biotin that captures perturbation components that has been modified to contain an avi din / streptavidin group.

[0060] In some embodiments, the oligonucleotide index and an oligonucleotide capturing element are attached to each other either directly or through a releasable linker.

[0061] In some embodiments, the capturing element and the capture index are not attached to each other either directly or through a linker.

[0062] In some embodiments, the capturing element is poly-T.

[0063] In some embodiments, there is provided a library or registry comprising a recitation of each label or set of detectable labels that code for a specific capture bead and the specific examination area in the assay device for each capture bead.

[0064] In some embodiments, there is provide a method for associating a capture bead to a specific examination area in a mapped assay device which method comprises: a) coding each of said capture beads with an optically detectable label or a set of optically detectable labels unique to that bead; b) adding a capture bead to at least a portion of the examination areas in said device; and c) associating each unique labeled capture bead to each examination area in said device.

[0065] In some embodiments, this method further comprises: d) memorializing each position of each capture bead to the specific examination area on the mapped assay device.

[0066] In some embodiment, the capture bead further comprises with a unique oligonucleotide index attached thereto and a capturing element.

[0067] In some embodiments, the correlation is done with an image aligning each of the optically detectible capture beads to a site on the map.

[0068] 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 opticallydetectable label on each of said capture beads in each examination area can be recorded by photography, bar codes, QR codes, and the like.

[0069] In some embodiments, such additional information can be included on the capture bead or by a micro device added to the examination area. Such micro devices coded with such additional information are described in US Provisional patent application Serial No. 63 / 494,628, entitled “Systems for Employing an Encoded Micro-Component to Identify a Cell”, filed April 6, 2023, which application is incorporated by reference herein in its entirety.

[0070] Still further, the label or set of unique labels on said capture bead can be further compiled into said library thereby providing a complete set of information allowing the technician to correlate the capture bead to the specific examination area. Further, the perturbation component captured by the bead will be identified as originating from the specific capture bead by the oligonucleotide index. In turn, the correlation between the detectable label and the examination area through the map will identify the examination area used with that capture bead. This allows for tracking the captured perturbation component back to the specific examination area from where it originated as well as to identify the perturbation element that induced the perturbation.

[0071] In one embodiment, the capture bead can be presented by formula I: (WyCB- L-Q-Q^nI where CB is a capture bead, L is a linker, n represents the multiplicity of such (linker-Q- Q1) groups bound to the bead, and one of Q and Q1is a capturing element and the other is an oligonucleotide index that uniquely associates the capture bead to the perturbation component captured by the bead, W is a set a optically detectable label or a set of optically detectable labels that uniquely identify the capture bead, and r is an integer from 1 to 10.

[0072] In Formula I, the attachment of Q to Q1can be direct or through a linker. When the capturing element is an oligonucleotide having bound thereto a nucleic acid perturbation component such as mRNA, then the combination of Q and Q1allows for sequencing the captured perturbation component together with the oligonucleotide index. This allows one to assess the impact of the perturbation on the cell as well as correlating that perturbation to a specific capture bead and then to the specific examination area from which the capture bead was used.

[0073] In one embodiment, the nucleic acid perturbation component is mRNA and the mRNA capturing element is poly-T which is attached to the oligonucleotide index such that formula I is then reduced to formula IA and IB as follows:(W)r-CB-[L-(poly-T)-Q1]n(W)r-CB-[L-Q1-(poly-T)]nI-A I-B where, in each case, Q1is the index oligonucleotide and n, r, L, and W are as defined above. In some embodiments, the linker is a releasable linker. When that linker is cleaved, the resulting structure is Q1-(poly-T). Note that release results in the oligonucleotide index being coupled to the mRNA attached to the poly-T such that the index now identifies the capture bead from where it came.

[0074] In an alternative embodiment, the index oligonucleotide can comprise individual oligonucleotide strands that are used in combination to identify the capture bead. In such an embodiment, the individual strands taken together would generate a code that identifies the capture bead. So, assuming that ATGCTA is defined as “1”, TACGTA is defined as “2”, AAGFTAis defined as “3” and so forth, the strands create a code that uniquely identify the capture bead.

[0075] In some embodiments, there is provided a method for identifying a specific examination area associated to a perturbation component released from a perturbed lysed cell in said area of an assay device comprising a multiplicity of said examination areas, which method comprises: a) conducting an assay in each of a multiplicity of examination areas in a mapped assay device wherein each of said examination areas comprise a cell, a perturbating agent for said cell; an assay solution b) including in each of said examination areas a capture bead which comprises: an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead; a multiplicity of the same oligonucleotide index which is unique to said capture bead and is attached to thereto optionally through a linker; and a multiplicity of the same capturing element; c) correlating and memorializing the optically detectable label or set of labels in each examination area by associating the unique optically detectible 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 examination areas;d) inducing a perturbation on said cell 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) lysing said cell to release said perturbation components into said assay solution and capturing at least one of said component onto the capture bead; f) identifying the perturbation component(s) captured on said bead; g) sequencing the oligonucleotide index to identify the specific index which correlates to a specific capture bead and then correlating the capture bead to the specific examination area by reference to the memorialized correlation in c) above thereby identifying the particular examination area from which the perturbation component was retrieved.

[0076] In one embodiment, the method provided above further comprises: h) obtaining images of each of the examination areas during the assay; i) correlating the images to the specific examination area defined in g) above; and j) evaluating changes in cellular morphology during the assay as a result of the perturbation of the cell.

[0077] In one embodiment, the set of labels on said capture bead comprises a single member.

[0078] In one embodiment, the set of labels comprises multiple members which, in combination, identifies the capture bead to which they were initially bound.

[0079] In one embodiment, there is provided a population of capture beads as defined above wherein each capture bead in said population is uniquely correlated by a label or a set of labels attached thereto as well as an oligonucleotide index which correlates to the capture bead.

[0080] In one embodiment, the perturbation element is an compounds, antibodies, immune cells, siRNA, viruses, bacteria, fungi, change in one or more the conditions of the assay such but not limited to buffers, salts, pH, temperature, nutrients, oxygen levels, oxidizing agents, physical stress, and the like. In one embodiment, the perturbing element is a compound released from a perturbing bead.

[0081] In one embodiment, the capture bead is positioned in the examination area before, during, or after completion of the assay.

[0082] In one embodiment, the capture bead described above is isolated from the examination area prior to removal of the oligonucleotides from the bead.

[0083] In one embodiment, the capture bead is added to the examination area prior to the assay. In another embodiment, the capture bead is added to the examination area during the assay.

[0084] In one embodiment, the capture bead is added to the examination area after completion of the assay.

[0085] In one embodiment, there is provided a register for a given assay device which register contains a record for each unique set labels for each capture beads, the specific examination area where said capture bead is located in the assay device, and the unique oligonucleotide found on said capture bead.

[0086] In one embodiment, the register comprises one or more digital photographs that record the code associated with each examination area and the optically detectable label on the capture bead.

[0087] In one embodiment, the digital photography uses megapixels wherein the record for each examination area comprises a different set of pixels from the other examination areas. . Brief Description of the Drawings

[0088] FIGS. 1 A and IB illustrate two different mapped assay devices having an orientation component.

[0089] FIGS. 2A-2D illustrates capture beads (circle) comprising a set of optical labels attached thereto that uniquely identify each capture bead from other capture beads.

[0090] FIG. 3 illustrates the capture beads of FIGS. 2A-2D in a subset of examination areas depicted in FIG. IB.

[0091] FIG. 4 illustrates a mixed bead population having beads of different colors and different color intensity for the same colors.4. Detailed Description

[0092] This disclosure provides, in part, for a capture bead that is suitable for use in an assay device which is configured to correlate the capture bead to a specific examination area in the device as well as correlating any captured nucleic acid or other cellular components generated to during the assay to that bead.5. Perturbation elements comprising a perturbation compound

[0093] In some embodiments, the perturbation element is a compound releasably bound to a perturbation bead. Such perturbation compounds are synthesized on the beads using conventional split pool synthetic protocols well known in the art to generate a library of perturbation compounds. As per reaction scheme A below, which is provided for illustrative purposes only, a three-step synthesis for preparing a 1,000 member combinatorial library is depicted. Note that a four-step synthesis would provide for 10,000 possible compounds in the combinatorial library and a five-step synthesis would generate up to 100,000 compounds.Reaction Scheme A

[0094] In step 1 of reaction scheme A, a plurality of beads (such as 1,000) are split into 10 vessels numbered 1-10 respectively (e.g., such that each vessel has approximately 100 beads). The beads comprise a cleavable linker for reacting with a component of interest to form the compounds that are to be made. In addition, there is an oligonucleotide site on the beads to which oligonucleotide strands can be added which define at least a portion of the structure of the perturbation compound to be generated thereon or the reaction steps used to make that compound (perturbation oligonucleotide - as per above). In the casewhere the capture bead captures the perturbation oligonucleotide defining the structure or the reaction steps of the compound, the oligonucleotide site is bound to the bead by a releasable linker. In some embodiments, the releasable linker for the perturbation compound and the perturbation oligonucleotide are the same such that when the perturbation compound is released from the bead, so too is the compound oligonucleotide. The releasable linker may be cleaved under any means (e.g., UV light exposure, pH, etc.). In some embodiments, the releasable linker used at the perturbation oligonucleotide site is orthogonal to the releasable linker used for the perturbation compound made on the bead. In some embodiments, the linker used for the oligonucleotide binding site is non- releasable under normal conditions including that used to cleave the perturbation compound from the bead.

[0095] Further in Scheme A, a first component of the perturbation compound to be synthesized can be added to the bead using chemistry that provides for very high yields (e.g., Click chemistry). The reactions can be run under conventional conditions that are maintained until the reactions are deemed complete. At this point, the beads comprise 10 different building blocks (e.g., beads in vessel 1 comprise a first building block in vessel 1, beads in vessel 2 comprise a second building block in vessel 2, and so on until the beads in vessel 10 comprise a tenth building block in vessel 10). An oligonucleotide strand can be introduced into each vessel either before, during or after the reaction to memorialize the reaction performed in step 1 (e.g., oligonucleotide strands are coded by a sequence unique to each vessel and each strand is connected to the preceding strand). Uniquely sequenced oligonucleotide strands in each reaction vessel can then be added to the bead, thereby memorializing and allowing for identifying the reaction conducted in that vessel.

[0096] In step 2, all of the beads from each of the reaction vessels are typically washed and then pooled and mixed (e.g., shaken, stirred, etc.) to insure homogeneity. The beads are then split into a second set of reaction vessels in the manner described above (e.g., resulting in an approximately equal number of beads in each vessel). Each reaction vessel will receive a portion of the beads previously subjected to reactions in vessels 1- 10. As such, each reaction vessel will receive beads comprising each of the 10 building blocks (e.g., beads comprising the first building block, beads comprising the second building block, etc.) The reaction vessels for step 1 can be used for step 2 (e.g., after washing vessels and / or otherwise removing any reagents remaining from the prior step). The reactions conducted on the beads in each vessel are memorialized by addition ofanother strand of an oligonucleotide unique to each reaction vessel. The oligonucleotide strand is attached to the prior oligonucleotide strand to from a single strand. The beads are then pooled and mixed as described above. At this point, the beads comprise up to 100 different building blocks (e.g., all combinations of the first 10 building blocks and the second 10 building blocks, in a case that the first 10 building blocks and the second 10 building blocks are mutually exclusive). Step 2 may be repeated as many times as desired to form the combinatorial library.

[0097] In step 3 (e.g., the final synthesis step), the beads are once again split and added to different reaction vessels in the manner described above. As above, the reaction in step 3 results in the generation of 1,000 compounds on the beads (e.g., if different components / reactions are performed in each vessel in each step). The generated library of compounds on the beads can be tested for activity with a cell, for example, in an assay where a single bead and a single cell are combined.

[0098] In one embodiment, the final step can utilize an oligonucleotide strand to identify the component added to the compound to be synthesized thereby identifying the compound or the reaction steps used. In another embodiment, and unlike steps 1-2, described above, no oligonucleotide is added to memorialize the reaction in step 3. Rather, the beads from each vessel are washed and optionally dried and then are placed into an isolated location on the assay device (i.e., beads from each vessel are transferred to separate locations on a single assay device or into separate assay devices, wherein the separate locations or separate assay devices each contain examination areas, such as wells or droplets, for conducting the assay). Alternatively, the beads from each vessel are placed in separate assay devices marked with the reaction vessel from which they were retrieved. This allows the technician to immediately identify the last step of the reaction used for those examination areas that evidence interesting results.6. Other Perturbation elements

[0099] Other perturbing elements that are useful in the assays described herein include agents such as antibodies, immune cells, synthetically engineered cells, siRNA, viruses, bacteria, fungi, a change in one or more the conditions of the assay such but not limited to buffers, salts, pH, temperature, nutrients, oxygen levels, oxidizing agents, physical stress, and the like. Each of these can be used alone or in combination with one or more of such agents including a perturbation compound.

[0100] As to immune cells, their addition to an examination area allows for the evaluation of whether such a cell will perturb a cell such a tumor cell in a therapeutic manner. In addition, the examination area also can contain a perturbation compound to evaluate whether that compound will perturb an immune cell which previously was either non- responsive marginally responsive in a manner rendering the immune cell therapeutic to the tumor cell. Still further, the immune cell can be used alone and a perturbation compound added to assess whether such attenuates or enhances the activity of that cell.

[0101] As to antibodies, the addition of a weakly reactive antibody into an examination area containing a tumor cell allows for the addition of a perturbation compound that perturbs the tumor cell. One can then evaluate whether the perturbation induced in the tumor cell enhances the therapeutic activity of the antibody.

[0102] As to engineered cells, these may comprise cells engineered to produce secreted or membrane associated agents capable of interacting with the test cells in the examination area. In some embodiments, the engineered cells may be engineered to secrete synthetic proteins, cytokines or antibodies. In some embodiments, the engineered cells may by engineered to produce unique small molecules or metabolites. Engineered cells are the foundation of biotechnology and the various methods are well known to technicians engaged in the art of biotechnology and / or synthetic biology.

[0103] As to other perturbation agents, the use of conditions to perturb a cell provides valuable information as to whether the perturbation is beneficial or detrimental to the cell. Where conditions are detrimental, therapeutic agents which induce a similar perturbation to a diseased cell are appropriate candidates for inducing similar changes in vivo.

[0104] As per the above, combinations of perturbing agents can be used to ascertain the perturbation impact of combination as compared to the separate perturbating agents when used alone.7. Capture Beads

[0105] Capture beads, as described herein, are used to directly capture perturbation components generated by a perturbed cell during the assay. This is accomplished by the addition of attaching capture elements onto capture bead which capture perturbation components released by the lysed cell including by way of example, mRNA , DNA, nucleic acids, cytokines, chemokines, enzymes, proteins, hormones, and the like. Such diverse selection of perturbation components not only assesses the changes generated by the perturbation as compared to an unperturbed cell but also to assess whether suchchanges are beneficial or not. In total, a technician is able to provide a more thorough evaluation of the changes in functionality of the cell due to the perturbation by assessing as many components as possible.

[0106] In addition, if compound oligonucleotide is released form the perturbation bead into the examination area during an assay, then such can also be captured by the capture bead. In such cases, the capture bead will contain one or more of the perturbation components (e.g., perturbation nucleic acids) generated during the assay as well as compound oligonucleotide which corresponds to the perturbation compound responsible for the cellular perturbations. The combination of both the perturbation components generated and the perturbation compound responsible for components is further information available to the technician.

[0107] When the perturbation component is mRNA, such can be captured by poly-T. As to cytokines and chemokines, a receptor specific for these can be attached to the capture bead. Likewise, enzymes can be captured by a substrate to which they act upon, and the like. Suffice it to note that the extent of targets to be captured by the capture bead is limited only by the recognition of a binding element specific for the target. In some embodiments, a binding element can be created for a particular perturbation component. For example, monoclonal antibody can be generated by conventional means to bind to a specific perturbation component generated during the assay.

[0108] In some embodiments, multiple capture beads can be used in a single examination area. Each capture bead is directed to capturing different perturbation elements from the lysed cell. For illustrative purposes only, a first capture bead has bound thereto multiple copies of the same capturing element for mRNA. A second capture bead has bound thereto multiple copies of the same capturing element for a specific cytokine. Additional capture beads can be used to bind to other perturbation components. Each capture bead has bound thereto an oligonucleotide index unique to that capture bead. In some embodiments, the same oligonucleotide index can be used for each capture bead used in the same examination area. In some embodiments, a different oligonucleotide index is used for each capture bead albeit that such different oligonucleotide indices all uniquely code for the same capture bead.

[0109] In some embodiments, the capture bead comprises the following structure III:Ill where n, r, L, Q, Q1, and W are as defined above and CB inside the shaded circle represents the capture bead.

[0110] Regarding L, it is a linker. In some embodiments, it can be a releasable linker that can be cleaved at the appropriate time during or after the assay including after the capture bead is retrieved from the examination area. Suitable linkers and releasable linkers are well known in the art.[OHl] As to W, the optically detectable label(s) include one or more colors, induced colors, lettering, numbering, images, insignia, barcodes, quantum dots, QR codes and the like. A sufficient number of labels, r, are used, either alone or in combination, to uniquely identify that capture bead as compared to other capture beads used in the assay. This means that each capture bead to be used in an assay will be uniquely identified. In one embodiment, a single optically detectable label can be used such as a barcode or a QR code. In another embodiment, the capture bead can include multiple labels that in combination provide for a unique code or image. Such embodiments include a mixture of colors, lettering and / or numbering, and the like. In a preferred embodiment, r is an integer from 1 to 10.

[0112] As to Q and Q1, Q is an oligonucleotide index that is unique to that capture bead and Q1is a capturing element as described above. As depicted Q and Q1are directly attached to each other, However, in some embodiments, a linker (including a releasable linker) can be used to facilitate attachment. The captured element, Q1, is coupled to Q optionally through a linker. The oligonucleotide index uniquely identifies the specific capture bead from which the captured element is bound. In one preferred embodiment, Q1is an mRNA capturing element such as poly-T which is coupled to Q thereby rendering sequencing of both Q and the captured mRNA in a manner such that both the identity of the capture bead coded by Q and the identity of the captured mRNA can be determined in a single sequencing step.

[0113] In another embodiment, Q1can also capture the compound oligonucleotide that identifies the compound on the perturbation bead or the synthetic steps used to make thatcompound. So, in practice where a perturbation bead is used containing multiple copies of the same compound releasably bound thereto and where the perturbation bead contains compound oligonucleosides that code for the compound or the reaction steps used, then Q1can be used to capture such compound oligonucleotides.

[0114] In some embodiments, the capture bead comprises the following structure IV:IV where m, n, r, L, L1, Q, Q1, CB and W are as defined above. In this optional embodiment, the capture element is separated from the oligonucleotide index.

[0115] As to n and m (as described above,) these represent multiple copies of the linker bound components on the capture bead. As used herein, the term “multiple copies” or “a multiplicity” as applied to bead bound components means that the population of such bead bound components ranges up to about 1 x 10'8moles or 6.02 x 1015individual molecules. The exact number is immaterial as long as there are sufficient amounts of the bead bound materials to allow the assay to be conducted and to retrieve sufficient amounts of perturbation components in order to assess the cellular perturbations induced during the assay. In some embodiments, the number of attachments ranges from about 1 x 109to about 6.02 x 1015.

[0116] Regarding L and L1, each of these are linkers and optionally releasable linkers. In one embodiment, L and L1are identical, and both will be cleaved in the same step if they are releasable linkers. In another embodiment, L and L1are different. When different and when both are releasable linkers, L and L1can be released using the same procedure but the release rate may be different for each. This means that more of Q or Q1will be released in a given amount of time from that linker which is more efficiently cleaved. In still another embodiment, when L and L1are releasable linkers, such can be selected to be released in an orthogonal manner such that one of the linkers can be cleaved while the other remains intact. The specific combination of L and L1is within the skill of the technician and depends on factors as to whether release of Q and Q1is more efficient being done together or separately.

[0117] The specifics of capture beads suitable for use herein is not critical as long as the beads can be labeled and modified to include -L-Q-Q1or -L-Q and -L-Q1groups as appropriate. In one embodiment, the capture beads can be polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination of the above. The particular material used for the capture bead is conditioned only on it being made of material that is inert to the assay to be conduct including the solvent and reagents used.

[0118] In one embodiment, the size of the capture bead corresponds to a percentage of the size of the examination area where larger examination areas tolerate larger capture beads. In general, the size of the capture bead ranges from about 5 percent to about 50 percent of the size of the examination area. In absolute size, the capture bead is preferably from about 10 microns to about 250 micron along its longest axis and, more preferably from about 20 microns to about 200 microns along its longest axis, and even more preferably from about 30 to about 180 microns along it longest axis.

[0119] In one embodiment, the capture bead is added after assay completion where each of the capture elements as well as is releasable bound to the capture bead. By adding the capture bead after assay completion, any cleavable bonds used with the capture components can be the same as that used during the assay. Once the capture bead is recovered, the captured components bound to the bead can be released and then determined by conventional means.

[0120] Alternatively, the capture bead can have bound thereto, optionally through a linker including a cleavable linker, a multiplicity of biotin or avidin / streptavidin groups. For example, the compound oligonucleotide can be terminated with an oligonucleotide strand having a biotin or avidin / streptavidin group thereon optionally through a linker. The choice of each group is such that if a biotin group is either on the compound oligonucleotide or on the capture bead , then the avi din / streptavidin group is on capture bead or on the compound oligonucleotide such that they are functionally complementary to each other. In such a case, the biotin - avidin binding mechanism can be used to bind the compound oligonucleotide and Q or Q1can be a perturbation component binding element thereby providing different sites on the capture bead for these different targets. Such a capture bead will then have the formula:V-A V-B where each of n, m, r, CB, L, Q, Q1, and W are as defined above, X is Q- avi din / streptavidin or Q-biotin where Q is the oligonucleotide unique to that capture bead, t represents a multiplicity of avidin / streptavidin or biotin groups releasably bound to the capture bead through L2which is a linker and optionally a cleavable linker. In some embodiments, L2is the same of L and / or L1. In some embodiments, when L, L1and L2are each cleavable linkers, either one or two of these linkers can be selected to be released using an orthogonal cleaving mechanism as compared to one or to both\ When X is avidin / streptavidin, then the compound oligonucleotide comprises a biotin group; and when X is biotin, then the compound oligonucleotide comprises avidin / streptavidin. One advantage of this method is that the compound oligonucleotide can be captured independently from the, e.g., mRNA recovered from the lysed cell.

[0121] In one alternative embodiment, one need not use an oligonucleotide in the compound oligonucleotide or in the index oligonucleotide. Rather, a compound having a known molecular weight can be used as the index on the capture bead. Such a compound would have a molecular weight that identifies the capture bead and is unique to other compounds used as an index on other capture beads. In some embodiments, the compound index is attached to the capture bead through a releasable linker. The index compound, once released, can be assessed by mass spectroscopy. In some embodiments, each of the index compounds on the different capture beads can be bound to the same material that increases its molecular weight. For example, the index compound can have a biotin group attached. Upon release, a compound having an avidin group can be bound to each of the compound indices so released. The resulting conjugate has an increased mass as compared to the index compound so that a mass spectrum of the conjugate is more readily done.

[0122] In one embodiment, the capture bead and the perturbation bead are the same and, in such cases, the resulting bead is termed a “hybrid capture bead” or “HCB”)). In such an embodiment, the elements of the capture bead are included on the perturbation beadresulting in the HCB. In an assay, the perturbation compound is released from the HCB while the compound oligonucleotide is retained on the bead. Perturbation components generated by perturbing a cell with the perturbation compound are captured by capturing elements on the HCB. In addition, the HCB has an index oligonucleotide thereon.8. Real Time Analysis

[0123] As above, this disclosure provides the technician with the ability to associate the perturbation component(s) generated by the perturbed cell to a specific capture bead that captured that perturbation component and then back to the examination area where the perturbed cell was located by reference to the map on the mapped assay device. In turn, generating a record of each cell in each examination area that encompasses the assay period, one can access that record and assess the cellular changes that can be visibly observed as a result of the perturbation. Such cellular changes include changes in morphology, induction of apoptosis, evidence of cellular changes, and the like. Such changes are deemed “real time” as they are taken during the assay and can include pictures, video recordation, three-dimensional analysis of the cell and the like.

[0124] The technician can then combine the real time changes in functionality with the static changes and provide a substantially improved overview of the cellular changes in functionality.9. Systems

[0125] In one embodiment, there is provided a system for providing information regarding an assay said system comprises: a) a mapped assay device comprising multiple examination areas that are individually identifiable; b) a perturbation bead comprising multiple copies of a perturbation compound releasably bound thereto which compound is unique to other perturbation beads to be used in the assay device; c) a capture bead as described herein; and d) a registry that correlates each capture bead to a specific examination area.

[0126] The systems described herein can further comprise one or more additional capture beads, one or more additional cells, a combination of perturbation elements that actcooperatively (a first perturbation element that perturbs a second perturbation element that then perturbs a cell of interest). In one embodiment, sequencing of oligonucleotides bound to the capture bead, or the hybrid capture bead can be done on the bead itself. In another embodiment, sequencing is done after releasing the oligonucleotides by cleaving the releasable bonds.

[0127] The assays described herein are conducted in a mapped assay device having a multiplicity of examination areas. For illustrative purposes only, the assays described herein employ a perturbation bead having multiple copies of the same perturbation compound releasably bound thereto and a compound oligonucleoside that memorializes all or part of the reaction steps used to make such compounds on a plurality of beads. Again, for illustrative purposes only, the compound oligonucleoside has attached thereto a poly-T component that permits its capturing by a poly-A capturing element on the capture bead.

[0128] Each examination area in the assay device includes a perturbation bead as described above, a single cell to be perturbed, a capture bead as described herein including multiple copies of a capture element and multiple copies of an oligonucleotide index, and an aqueous solution suitable for conducting the assay. For illustrative purposes only, in this embodiment, the oligonucleotide index and the capture element are attached to each other and to the capture bead in a manner where each attachment is optionally through a linker.

[0129] The position of each capture bead in each examination area as well as the capture bead itself is memorialized or recorded by identifying the label on each capture bead and associating that label to an examination area that is mapped by the assay device. This provides a 1 : 1 correlation between each examination area and each capture bead maintained therein merely by referencing the unique label or set of labels found on the capture bead to the mapped examination area.

[0130] The assay is initiated by releasing at least a portion of the perturbation compound bound to the perturbation bead into the aqueous solution in a sufficient amount to perturb the cell. In some embodiments, the releasing mechanism (e.g., UV light) will also release the compound oligonucleotide from the perturbation bead as well as any releasable bonds on the capture bead that are responsive to release using the same releasing mechanism. In addition, in this embodiment, the released compound oligonucleotide will be captured by the poly-A capturing element which associates with the poly-T component of thecompound oligonucleotide. The assay is then conducted for a sufficient period of time to induce one or more cellular perturbation(s). In some embodiments, pictures of the cellular response to the perturbation or a video of the same can be made. Upon completion of the assay, the cell is lysed to release cellular contents.

[0131] Upon release, the mRNA released is captured by the poly-T capturing elements on capture element. The capture element, being attached to the oligonucleotide index now contains a population of either the compound oligonucleotide and / or the mRNA associated therewith. In some embodiments, the capture bead is isolated from the examination area, washed, and prepared for sequencing. In some embodiments, the captured compound oligonucleotide, the captured mRNA and the oligonucleotide attached thereto are sequenced to provide the identity of the compound generating the perturbation, the extent of mRNA expression generated or altered by the perturbation, and the identity of the capture bead associated with this information. In the absence of the oligonucleotide index, one could not determine the examination area from where this information was generated.

[0132] Next, the capture bead identity is then correlated to the examination area that generated this information by referencing the memorialized information and finding the specific examination area associated therewith. Such is simply identifying the examination area “address” in the assay device from the memorialized information. Assuming that either pictures and / or a video of the examination areas were generated during the assay, one is now able to correlate changes in cellular morphology to the perturbations generated. Further, if additional capture beads are employed to capture other perturbation components generated during the assay, a substantially more sophistic analysis of the change in cellular functionality can be made

[0133] In some embodiments, record or memorialization is conducted using digital photograph or digital video recording. The digital aspects of such records allows for rapid assessment of colored labels used to uniquely identify a particular capture bead.

[0134] As noted previously, the capture bead can be added to the examination area prior to, during, or after the assay completion. However, addition of the capture bead into the examination area before the start of the assay is preferred as it makes recordation of the examination area address to the particular capture bead easier if for no other reason that assay debris can be avoided when recordation is done.11. Example

[0135] The following examples are provided to illustrate the use of a capture bead as described herein. In this example, the following terms used therein have the following meanings: bp = base pairs poly -A = poly adenine poly-T = poly thymine mRNA = messenger RNAUV = ultravioletExample 1 - Assay Device

[0136] A mapped device is employed which comprises a multiplicity of examination areas (e.g., picowells). Each picowell is aligned in rows and columns such that the number of columns is longer than the number of rows. To orient the assay device, a mark or emblem can be added to one corner of the device to mark the up position. FIG. 1 A illustrates the arrangement of examination areas in a portion of a mapped assay device. Specifically, mapped assay device, 10, has a mark, 12, in the comer of one side of the device that directs the user to reference that comer as the upper left corner of device, 10. Any mark, indentation or other feature can be used provided that it orients the device. Such is shown in FIG. IB where the corner of the assay device, 10, has been cut to create an indentation, 22. In each case, a mapped assay device is provided where each examination area is identifiable by a column and row number.Example 2 - A library of Perturbation Beads

[0137] Step 1. A population of 100,000 beads having multiple copies of an oligonucleotide binding site and multiple copies of a compound synthesis site bound thereto are commercially available from Rapp Polymere GmbH, Ernst-Simon-Strasse 9, 72072 Tuebingen, Germany. The beads are divided in approximately equal number into 10 different reaction vessels. A unique first step of compound synthesis is conducted in each vessel such that each vessel generates a distinct building block different from the other building blocks generated in the other vessels. Either before, during or after reaction completion, a first oligonucleotide strand that is unique to each reaction vessel is added tothe oligonucleotide binding site such that all of the beads in the same reaction vessel will have the same first oligonucleotide strand which is different from the oligonucleotide strand found in the other vessels. After completion of the first step of the compound synthesis the beads are washed and then combined and homogenized.

[0138] Step 2. The procedure of Step 1 is repeated but using a unique second building block for compound synthesis in each of the 10 reaction vessels as well a unique second oligonucleotide strand for each reaction vessel. After completion of this step of the compound synthesis the beads are washed and then combined and homogenized.

[0139] Steps 3-5. The procedure of Step 2 is repeated but using a unique third, fourth, and fifth building block for compound synthesis as well as a unique third, fourth and fifth oligonucleotide strand. After step 5, there are 100,000 different compounds made on the 100,000 different beads. A single assay bead is then added to a single examination area in the assay device. Such a single assay bead can be illustrated as shown in Scheme 1 :Scheme 1 where the circle containing PB represents a perturbation bead, PC is a perturbation compound, L3is a releasable (cleavable bond), X is a bond or a linker which is optionally a releasable linker, Y is the compound oligonucleotide, and s and w are independent integers representing the multiplicity of bound groups to the perturbation bead.Example 3 - Capture Bead

[0140] Each capture bead is made from the same starting material used for the perturbation bead. Each of the capture beads have bound thereto a set of labels comprising oligonucleotides, fluorescent particles or semiconductor microchips etc., or combinations thereof. In addition, a binding element is attached to the capture bead using, e.g., for example a poly-T group for binding to the mRNA poly-A group. Other binding elements can be used as described herein. In addition, an oligonucleotide index is attached to the capture bead using methods well known in the art. As above, each oligonucleotide index is unique to the capture bead in a given examination area. In this embodiment, theresulting capture bead can be represented by the following structure as provided in Scheme 2:Scheme 2 where the circle labeled as CB represents the capture bead having a multiplicity of a capture element, the stars represent the combination of detectable labels that uniquely identify the capture bead from other capture beads to be used in an assay device, L4and L5, which may be the same or different, represent linkers which may be optionally releasable, and y and z represent the multiplicity of an oligonucleotide index and the capture element bound to the capture bead.

[0141] In another embodiment, the oligonucleotide index and the capture element can be attached to each other such that the resulting structure can be illustrated as follows in Scheme 3 :Scheme 3 where the circle with CB inside and the stars are as defined above, L3is an optionally releasable linker, one of Q and Q1is the oligonucleotide index and the other is the capture element and a represents the multiplicity of Q-Q1on the capture bead where, optionally, a linker (not shown) can attach Q to Q1.

[0142] FIGS. 2A- 2D illustrate different optically detectible labels placed on a capture bead that uniquely distinguish that bead from other capture beads. Specifically, a unique combination of fluorescent dyes and / or their intensity of signal can be covalently attachedto a capture bead such that the fluorescent signals generated can be associated specifically with that bead. In FIG. 2A, the first Y can be at one-third of the intensity of the second Y thereby distinguish a first Y from a second Y. A large number of quantum dots are commercially available from ThermoFisher can be used for this purpose. The number of such dots required per capture bead is a function of the number of examination areas to be used in the assay device. For example, ten uniquely identifiable quantum dots can provide for over 3,000,000 unique combinations. In FIGS. 2A-2D, B stands for blue; G stands for green, O stands for orange, R stands for red, and Y stands for yellow. In each case, the combination of colors using only these 5 colors provides for 120 unique combinations which is greatly expanded by adding just another 5 colors as above.

[0143] Alternatively, capture beads can be impregnated with or covalently bound to dyes such that each capture bead has a distinguishable color or fluorescent generated color that acts as a label and differentiates it from other capture beads. FIG. 4 illustrates a mixture of beads with different colors and different intensities of the same color which greatly expands the scope of possible combinations.

[0144] FIG. 3 illustrates placement of the labeled beads of FIGS. 2A-2D into separate examination areas as found in FIG. IB.

[0145] A human cell such as a HeLa is then selected as the cell to be perturbed in the assay. The cell, the perturbation bead, and the capture bead are combined into a single examination area of a mapped assay device which is depicted as in FIG. 1 A containing a suitable aqueous composition for conducting the assay. The combination of components provides for the following in a single examination well as shown in Scheme 4:where L3, L4, L5, PB, CB and the stars on CB are as defined above, L6is a releasable linker, and w, x, y and z represent the multiplicity of each of the components on their respective beads. In addition, the ellipse with the HeLa cell inside is meant to show a uniform elliptical shape of the cell which is provided merely for illustrative purposes.

[0146] After combining these components into an examination area of a mapped assay device also having other so filled examination areas, then at any time during the assay, a registry is created by taking a digital picture of the assay device including each of the examination areas which registry records the uniquely labeled capture bead in each examination area preferably in electronic form.

[0147] The assay is initiated by releasing at least a portion of the perturbation compound from the perturbation bead into the assay solution to allow the perturbation compound to interface with the cell. When the compound oligonucleotide is attached to the perturbation bead via a releasable bond which, for the sake of Scheme, is the same releasable bond attaching the perturbation compound to the perturbation bead, then application of an appropriate stimulus such as UV light will result in at least a portion of both the perturbation compound and the compound oligonucleotide being released into the assay solution. In such a case, the resulting examination area will take the form shown in Scheme 5:Scheme 5 where the circle with HeLa Cell represent a human HeLa cell (sometimes referred to “Cell”) and each of the circles with PB and CB are as defined above as are the terms Perturbation Compounds, Compound Oligonucleotide, Capture Element and Oligonucleotide Index. Scheme 5 depicts the lack of compound oligonucleotide and perturbation compound on the perturbation bead. This is done for illustrative purposesonly as the release rate is not 100% and, as such, the perturbation bead will contain a portion of perturbation compounds bound thereto after release.Assay

[0148] Each of the assays conducted in the mapped assay device are incubated at standard conditions allowing the released compounds to perturb the cell. During the assay, pictures encompassing each assay well are taken at periodic intervals. Such pictures can evidence a change in cellular morphology as evidence by a changed cell shape and other possible attributes. Possible changes in cellular morphology are provided in Scheme 6 below:Scheme 6Here the original elliptical shape of the cell has been distorted into a shape that somewhat resembles an elliptical shape, but which also evidences areas of undulations and bulging due to the perturbation caused by the perturbation compounds. These changes captured during the assay are deemed to reflect dynamic functional changes in the perturbed cell.

[0149] Upon completion of the assay, the cell is lysed in the presence of the capture bead which captures perturbation elements generated by the cell during the assay. Using the capture bead as described above, the perturbation elements of interest in the cellular milieu are captured on the capture bead as are the compound oligonucleosides as shown in Scheme 7 below:Here, a portion of the capture element, represented by b, is dedicated to capturing mRNA from the lysed cell whereas another portion of the capture element, designated as y minus c (y-c), is dedicated to capturing compound oligonucleotide. In this case, c is the sum of b + any capture elements not bound by the mRNA or the compound oligonucleoside.

[0150] In one embodiment, each of the mRNA, the compound oligonucleotide, and the oligonucleotide index are sequence either on the capture bead or separated from the capture bead by cleaving L4and L5using an appropriate cleavage stimulation. The changes in the mRNA evidenced by the perturbation can be combined with any dynamic changes in cellular functionality as noted above to enhance the more of the changes in cellular functionality generated by the perturbation.Embodiments

[0151] Each of the following embodiments are representative of a portion of the possible combinations that can be achieved herein many of which are explicitly provided above.

[0152] Embodiment 1: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device.

[0153] Embodiment 2: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device.

[0154] Embodiment 3: A capture bead for use in an assay conducted in an examination area in a mapped assay device comprising multiple examination areas, said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein the capture bead in said mapped assay device is correlated to a specific examination area thereby identifying that examination area where said capture bead is located.

[0155] Embodiment 4: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead wherein said correlation is memorialized.

[0156] Embodiment 5: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set ofdetectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead and said correlation is memorialized into a registry recording the position of each capture bead in each examination area in the mapped assay device.

[0157] Embodiment 6: A capture bead for use in an assay conducted in an assay device said capture bead comprising an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device.

[0158] Embodiment 7: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device.

[0159] Embodiment 8: A capture bead for use in an assay conducted in an examination area in a mapped assay device comprising multiple examination areas, said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein the capture bead in said mapped assay device is correlated to a specific examination area thereby identifying that examination area where said capture bead is located.

[0160] Embodiment 9: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead wherein said correlation is memorialized.

[0161] Embodiment 10: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead and said correlation is memorialized into a registry recording the position of each capture bead in each examination area in the mapped assay device.

[0162] Embodiment 11: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity ofthe same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0163] Embodiment 12: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0164] Embodiment 13: A capture bead for use in an assay conducted in an examination area in a mapped assay device comprising multiple examination areas, said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein the capture bead in said mapped assay device is correlated to a specific examination area thereby identifying that examination area where said capture bead is located and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0165] Embodiment 14: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead wherein said correlation is memorialized and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0166] Embodiment 15: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead and said correlation is memorialized into a registry recording the position of each capture bead in each examination area in the mapped assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0167] Embodiment 16: A capture bead for use in an assay conducted in an assay device said capture bead comprising an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0168] Embodiment 17: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0169] Embodiment 18: A capture bead for use in an assay conducted in an examination area in a mapped assay device comprising multiple examination areas, said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein the capture bead in said mapped assay device is correlated to a specific examination area thereby identifying that examination area where said capture bead is located and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0170] Embodiment 19: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead wherein said correlation is memorialized and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0171] Embodiment 20: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead and said correlation is memorialized into a registry recording the position of each capture bead in each examination area in the mapped assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0172] Embodiment 21: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same capturing element on said bead.

[0173] Embodiment 22: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set ofdetectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same capturing element on said bead.

[0174] Embodiment 23: A capture bead for use in an assay conducted in an examination area in a mapped assay device comprising multiple examination areas, said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein the capture bead in said mapped assay device is correlated to a specific examination area thereby identifying that examination area where said capture bead is located device and a multiplicity of the same capturing element on said bead.

[0175] Embodiment 24: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead wherein said correlation is memorialized device and a multiplicity of the same capturing element on said bead.

[0176] Embodiment 25: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead and said correlation is memorialized into a registry recording the position of each capture bead in each examination area in the mapped assay device and a multiplicity of the same capturing element on said bead.

[0177] Embodiment 26: A capture bead for use in an assay conducted in an assay device said capture bead comprising an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same capturing element on said bead.

[0178] Embodiment 27: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device .and a multiplicity of the same capturing element on said bead.

[0179] Embodiment 28: A capture bead for use in an assay conducted in an examination area in a mapped assay device comprising multiple examination areas, said capture bead comprises an optically detectable label or a set of optically detectable labels attached tosaid bead which uniquely identifies the capture bead in said assay device wherein the capture bead in said mapped assay device is correlated to a specific examination area thereby identifying that examination area where said capture bead is located and a multiplicity of the same capturing element on said bead.

[0180] Embodiment 29: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead wherein said correlation is memorialized and a multiplicity of the same capturing element on said bead.

[0181] Embodiment 30: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead and said correlation is memorialized into a registry recording the position of each capture bead in each examination area in the mapped assay device and a multiplicity of the same capturing element on said bead.

[0182] Embodiment 31: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0183] Embodiment 32: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0184] Embodiment 33: A capture bead for use in an assay conducted in an examination area in a mapped assay device comprising multiple examination areas, said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein the capture bead in said mapped assay device is correlated to a specific examination area thereby identifying that examination area where said capture bead is located and a multiplicity of the sameoligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0185] Embodiment 34: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead wherein said correlation is memorialized and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0186] Embodiment 35: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead and said correlation is memorialized into a registry recording the position of each capture bead in each examination area in the mapped assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0187] Embodiment 36: A capture bead for use in an assay conducted in an assay device said capture bead comprising an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0188] Embodiment 37: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0189] Embodiment 38: A capture bead for use in an assay conducted in an examination area in a mapped assay device comprising multiple examination areas, said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein the capture bead in said mapped assay device is correlated to a specific examination areathereby identifying that examination area where said capture bead is located and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0190] Embodiment 39: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead wherein said correlation is memorialized and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0191] Embodiment 40: A capture bead for use in an assay conducted in an examination area in a mapped assay device said capture bead comprises an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead in said assay device and said examination area in said mapped assay device is correlated to said capture bead and said correlation is memorialized into a registry recording the position of each capture bead in each examination area in the mapped assay device and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capturing element on said bead.

[0192] Embodiment 41: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label is selected from visual, chemical, biological, audio or other means to evidence the presence of the information generated by the label.

[0193] Embodiment 42 A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label generates or can be stimulated to generate a color.

[0194] Embodiment 43. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label generates or can be stimulated to emit light.

[0195] Embodiment 44. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to saidbead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises lettering.

[0196] Embodiment 45: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises numbers.

[0197] Embodiment 46: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises symbols.

[0198] Embodiment 47: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises one or more barcodes.

[0199] Embodiment 48: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises UPC codes.

[0200] Embodiment 49: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises one or more tags.

[0201] Embodiment 50: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises chemical signals.

[0202] Embodiment 51 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises optionally requiring suitable instrumentation quantum dots.

[0203] Embodiment 52: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises quantum dots,

[0204] Embodiment 53. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises fluorescence.

[0205] Embodiment 54. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises mass spectroscopy.

[0206] Embodiment 55: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises nuclear magnetic spectra (e.g., a proton or C13spectrum).

[0207] Embodiment 56: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises a biological signal or a biologically induced signal.

[0208] Embodiment 57: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises nucleic acids.

[0209] Embodiment 58: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises bioluminescence.

[0210] Embodiment 59: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises radio waves or electromagnetic signals.

[0211] Embodiment 60: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises Bluetooth.

[0212] Embodiment 61 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to saidbead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises RFID.

[0213] Embodiment 62. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises Bluetooth.

[0214] Embodiment 63. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises WiFi.

[0215] Embodiment 64. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises two or more labels selected from colors, codes, shapes, colors, inducible colors, lettering, numbering, symbols, bar codes, UPC codes, Bluetooth, WiFi, RFID, bioluminescence, and nucleic acids.

[0216] Embodiment 65: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label is selected from visual, chemical, biological, audio or other means to evidence the presence of the information generated by the label and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0217] Embodiment 66. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label generates or can be stimulated to generate a color and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0218] Embodiment 67. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label generates or can be stimulated to emit light and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0219] Embodiment 68. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises lettering and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0220] Embodiment 69 A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises numbers and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0221] Embodiment 70: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises symbols and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0222] Embodiment 71 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises one or more barcodes and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0223] Embodiment 72: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises UPC codes and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0224] Embodiment 73: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises one or more tags and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0225] Embodiment 74: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein saiddetectable label comprises chemical signals and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0226] Embodiment 75: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises optionally requiring suitable instrumentation quantum dots and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0227] Embodiment 76 A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises quantum dots and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0228] Embodiment 77: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises fluorescence and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0229] Embodiment 78: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises mass spectroscopy and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0230] Embodiment 79: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises nuclear magnetic spectra (e.g., a proton or C13spectrum) and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0231] Embodiment 80: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises a biological signal or a biologically induced signal and amultiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0232] Embodiment 81 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises nucleic acids and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0233] Embodiment 82. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises bioluminescence and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0234] Embodiment 83. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises radio waves or electromagnetic signals and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0235] Embodiment 84. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises Bluetooth and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0236] Embodiment 85. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises RFID and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0237] Embodiment 86 A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises Bluetooth and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0238] Embodiment 87: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises WiFi and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0239] Embodiment 88 A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises two or more labels selected from colors, codes, shapes, colors, inducible colors, lettering, numbering, symbols, bar codes, UPC codes, Bluetooth, WiFi, RFID, bioluminescence, and nucleic acids, and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay.

[0240] Embodiment 89: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label is selected from visual, chemical, biological, audio or other means to evidence the presence of the information generated by the label, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay and a multiplicity of the same capture element.

[0241] Embodiment 90: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label generates or can be stimulated to generate a color, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element

[0242] Embodiment 91 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label generates or can be stimulated to emit light, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0243] Embodiment 92: A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein saiddetectable label comprises lettering and a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0244] Embodiment 93. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises numbers, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0245] Embodiment 94. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises symbols, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0246] Embodiment 95. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises one or more barcodes, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0247] Embodiment 96. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises UPC codes, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0248] Embodiment 97. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises one or more tags, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0249] Embodiment 98. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises chemical signals, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0250] Embodiment 99. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises optionally requiring suitable instrumentation quantum dots, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0251] Embodiment 100. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises quantum dots, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0252] Embodiment 101 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises fluorescence, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0253] Embodiment 102. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises mass spectroscopy, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0254] Embodiment 103. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises nuclear magnetic spectra (e.g., a proton or C13spectrum), amultiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0255] Embodiment 104 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises a biological signal or a biologically induced signal, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0256] Embodiment 105. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises nucleic acids, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0257] Embodiment 106. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises bioluminescence, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0258] Embodiment 107 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises radio waves or electromagnetic signals, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0259] Embodiment 108. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises Bluetooth, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0260] Embodiment 109. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached tosaid bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises RFID, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0261] Embodiment 110. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises Bluetooth, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0262] Embodiment 111 : A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises WiFi, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0263] Embodiment 112. A capture bead for use in an assay conducted in an assay device said capture bead comprising a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device wherein said detectable label comprises two or more labels selected from colors, codes, shapes, colors, inducible colors, lettering, numbering, symbols, bar codes, UPC codes, Bluetooth, WiFi, RFID, bioluminescence, and nucleic acids, a multiplicity of the same oligonucleotide index which is unique to other oligonucleotide indices used in said assay, and a multiplicity of the same capture element.

[0264] Embodiment 113. A method for associating a capture bead to a specific examination area in a mapped assay device which method comprises coding each of said capture beads with an optically detectable label or a set of optically detectable labels unique to that bead; adding a capture bead to at least a portion of the examination areas in said device; and associating each unique labeled bead to each examination area in said device.

[0265] Embodiment 114: A method for associating a capture bead to a specific examination area in a mapped assay device which method comprises coding each of said capture beads with an optically detectable label or a set of optically detectable labels unique to that bead; adding a capture bead to at least a portion of the examination areas in said device; associating each unique labeled bead to each examination area in said device; andmemorializing each position of each capture bead to the specific examination area on the mapped assay device.

[0266] Embodiment 115: The method of Embodiments 113 and 114 wherein the label is as defined in any one of Embodiments 41 to 66.

[0267] Embodiment 116: The method of Embodiment 115 wherein the capture bead further comprises an oligonucleotide index and a capture element as defined in any one of Embodiments 89 through 113.

[0268] Embodiment 117: A method for identifying a specific examination area associated to a perturbation component released from a perturbed lysed cell in said area of an assay device comprising a multiplicity of said examination areas, which method comprises: a) conducting an assay in each of a multiplicity of examination areas in a mapped assay device wherein each of said examination areas comprise a cell, a perturbating agent for said cell; an assay solution b) including in each of said examination areas a capture bead which comprises: an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead; a multiplicity of the same oligonucleotide index which is unique to said capture bead and is attached to thereto optionally through a linker; and a multiplicity of the same capturing element; c) correlating and memorializing the optically detectable label or set of labels in each examination area by associating the unique optically detectible 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 examination areas; d) inducing a perturbation on said cell 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) lysing said cell to release said perturbation components into said assay solution and capturing at least one of said component onto the capture bead; f) identifying the perturbation component(s) captured on said bead; g) sequencing the oligonucleotide index to identify the specific index which correlates to a specific capture bead and then correlating the capture bead to the specific examination area by reference to the memorialized correlation in c) above thereby identifying the particular examination area from which the perturbation component was retrieved.

[0269] Embodiment 118: The method of Embodiment 117 which further comprises: h) obtaining images of each of the examination areas during the assay; i) correlating the images to the specific examination area defined in g) above; and j) evaluating changes in cellular morphology during the assay as a result of the perturbation of the cell.

[0270] Embodiment 119: The multiplicity of the same capture element on the capture bead is a receptor or ligand for the perturbation component to be captured.

[0271] Embodiment 120: The multiplicity of the same capture element on the capture bead is an antibody or an antibody binding fragment for the perturbation compound.

[0272] Embodiment 121: The multiplicity of the same capture element on the capture bead is an enzyme or enzyme receptor for the perturbation component.

[0273] Embodiment 122: The multiplicity of the same capture element on the capture bead is a receptor for a given cytokine or chemokine.

[0274] Embodiment 123: The multiplicity of the same capture element on the capture bead is comprises avi din / streptavidin that captures perturbation components that have been modified to contain a biotin group.

[0275] Embodiment 124: The multiplicity of the same capture element on the capture bead comprises biotin that captures perturbation components that have been modified to contain an avi din / streptavidin group.

[0276] Embodiment 125: The multiplicity of the same oligonucleotide index and the multiplicity of the same oligonucleotide capturing element are attached to each other either directly or through a releasable linker to the capture bead.

[0277] Embodiment 126: The multiplicity of the same oligonucleotide index the multiplicity of the same capturing element are not attached to each other either directly or through a linker.

[0278] Embodiment 127: The multiplicity of the same oligonucleotide capturing element on said capture bead is poly-T.

[0279] Embodiment 128: A library or registry comprising a recitation of each of the label or set of detectable labels that code for a unique capture bead and the specific examination area in the assay device where each of said capture beads are located.

[0280] Embodiment 129. A capture bead comprising a detectable label or a set of detectable labels that uniquely associates the capture bead to a specific examination area wherein said capture bead comprises additional information that is related to the assay to be conducted in that examination area including the conditions of the assay, the change infunctionality 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.

[0281] Embodiment 130. The unique optically detectable label on each of said capture beads in each examination area recorded by photography, bar codes, or QR codes.

[0282] Embodiment 131 : A capture bead presented by formula I:(WyCB- L-Q-Q^nI where CB is a capture bead, L is a linker, n represents the multiplicity of such (linker-Q- Q1) groups bound to the bead, and one of Q and Q1is a capturing element and the other is an oligonucleotide index that uniquely associates the capture bead to the perturbation component captured by the bead, W is an optically detectable label or a set of optically detectable labels that uniquely identify the capture bead, and r is an integer from 1 to 10.

[0283] Embodiment 132. The capture bead of Embodiment 131 where W is a label as defined in any one of Embodiments 41 to 66.

[0284] Embodiment 133. The capture bead of Embodiment 132 where the capture bead further comprises an oligonucleotide index and a capture element as defined in any one of Embodiments 89 through 113.

[0285] Embodiment 134. The capture bead of Embodiment 131 where the attachment of Q to Q1is either a direct linkage or through a linker.

[0286] Embodiment 135. The capture bead of formula I which is described as in formula IA and IB as follows:(W)r-CB-[L-(poly-T)-Q1]n(W)r-CB-[L-Q1-(poly-T)]nI-A I-B where, in each case, Q1is the index oligonucleotide and n, r, L, and W are as defined above. In some embodiments, the linker is a releasable linker. When that linker is cleaved, the resulting structure is Q1-(poly-T). Note that release results in the oligonucleotide index being coupled to the mRNA attached to the poly-T such that the index now identifies the capture bead from where it came.

[0287] Embodiment 136. The capture bead of Embodiment 135 where W is a label as defined in any one of Embodiments 41 to 66.

[0288] Embodiment 137. The capture bead of Embodiment 136 where the capture bead further comprises an oligonucleotide index and a capture element as defined in any one of Embodiments 89 through 113.

[0289] Embodiment 138. The capture bead of Embodiment 135 where the attachment of Q to Q1is either a direct linkage or through a linker.

[0290] Embodiment 139. The capture bead of Embodiment 135 where the attachment of Q to Q1is through a releasable linker.

[0291] Embodiment 140: A perturbation bead comprising all of the capture bead elements as defined above attached thereto wherein said resulting bead is a “hybrid capture bead” or “HCB”.

[0292] Embodiment 141: A hybrid capture bead comprising a perturbation compound releasably bound thereto, a label or set of labels that uniquely identify said bead, a compound oligonucleotide non-releasably bound thereto, an oligonucleotide index and capture elements for perturbation components generated by perturbing a cell with the perturbation compound.

[0293] Embodiment 142: A hybrid capture bead comprising a label or set of labels that uniquely identify said bead, multiple copies of the same compound oligonucleotide bound thereto, multiple copies of an oligonucleotide index unique to said bead and capture elements for capturing perturbation components generated by perturbing a cell with the perturbation compound.

[0294] Embodiment 141: The hybrid capture bead of any one of Embodiments 140 through 142 wherein said label or set of labels that uniquely identify said bead are as defined in any one of Embodiments 41 to 66, and said oligonucleotide index and said capture element are as defined in any one of Embodiments 89 through 113.

Claims

What is Claimed is:

1. A capture bead for use in an assay conducted in an assay device said capture bead comprising: a) a detectable label or a set of detectable labels attached to said bead which uniquely identifies the capture bead in said assay device; b) a multiplicity of an oligonucleotide index which is unique to said capture bead and is attached thereto optionally through a linker; and c) a multiplicity of a capturing element.

2. A mapped assay device comprising a multiplicity of examination areas and at least one capture bead in each of said examination areas which capture beads comprise: a) a detectable label or a set of detectable labels attached to said bead which uniquely identifies each capture bead; b) a multiplicity of an oligonucleotide index which is unique to each of said capture beads and is attached thereto optionally through a linker; and c) a multiplicity of a capturing element, wherein said mapped assay device correlates a given examination area to a specific location on said map and the capture bead(s) are included in each examination area.

3. The capture bead of claim 1, wherein the detectable label or set of detectable labels is optically detectable.

4. The mapped assay device of claim 2, wherein the detectable label or set of detectable labels on said capture bead in said device is optically detectable.

5. The capture bead of claim 1, wherein the set of detectable labels has from 2 to 10 members.

6. The capture bead of claim 1, wherein the detectable label is a visible label selected from images, codes, shapes, colors, inducible colors, lettering, numbering, symbols, bar codes, UPC codes, and materials introduced onto the capture bead or electromagnetic signals such as radio waves selected from BlueTooth, WiFi, and RFID, or combinations thereof.

7. The mapped assay device of claim 2, wherein the set of detectable labels on said capture bead in said device has from 2 to 10 members.

8. The mapped assay device of claim 2, wherein the detectable label(s) on said capture bead in said device is a visible label selected from images, codes, shapes, colors, inducible colors, lettering, numbering, symbols, bar codes, UPC codes, and materials introduced onto the capture bead or electromagnetic signals such as radio waves selected from BlueTooth, WiFi, and RFID, or combinations of thereof.

9. The capture bead of claim 1, wherein said the oligonucleotide index is attached to the bead through a linker.

10. The capture bead of claim 1, wherein said the oligonucleotide index is attached to the bead through a releasable linker.

11. The capture bead of claim 1, wherein said oligonucleotide index comprises from about 6 to about 30 individual nucleosides.

12. The mapped assay device of claim 2, wherein the oligonucleotide index on said capture bead in said device is attached to the bead through a linker.

13. The mapped assay device of claim 2, wherein the oligonucleotide index on said capture bead in said device is attached to the bead through a releasable linker.

14. The mapped assay device of claim 2, wherein said oligonucleotide index on said capture bead in said device comprises from about 6 to about 30 individual nucleosides.

15. The capture bead of claim 1, wherein the capturing element is an oligonucleotide.

16. The capture bead of claim 15, wherein the oligonucleotide capturing element is attached to the capture bead.

17. The capture bead of claim 16, wherein, the attachment is through a linker.

18. The capture bead of claim 17, wherein the linker is a releasable linker.

19. The capture bead of claim 1, wherein the capturing element is a receptor or ligand for a perturbation component to be captured.

20. The mapped assay device of claim 2, wherein the capturing element on said capture bead in said device is an oligonucleotide.

21. The mapped assay device of claim 20, wherein the oligonucleotide capturing element is attached to the capture bead.

22. The mapped assay device of claim 21, wherein the attachment is through an optionally releasable linker.

23. The mapped assay device of claim 2, wherein the capturing element on said capture bead in said device is a receptor or ligand for a perturbation component to be captured.

24. A method for associating a capture bead to a specific examination area in a mapped assay device which method comprises: a) coding each of said capture beads with an optically detectable label or a set of optically detectable labels unique to that bead; b) adding a capture bead to at least a portion of the examination areas in said device; and c) associating each unique labeled bead to each examination area in said device.

25. The method of claim 24, which further comprises: d) memorializing each position of each capture bead to the specific examination area on the mapped assay device.

26. The method of claim 25, wherein the capture bead further comprises a unique oligonucleotide index attached thereto.

27. The method of claim 26, wherein the capture bead further comprises a capturing element.

28. A system for providing information regarding an assay to be conducted in a mapped assay device, said system comprises: a) a mapped assay device comprising multiple examination areas that are individually identifiable; b) a perturbation bead comprising multiple copies of a perturbation compound releasably bound thereto which compound is unique to other perturbation beads to be used in the assay device; c) a capture bead as described herein; and d) a registry that correlates each capture bead to a specific examination area.

29. A method for identifying a specific examination area associated to a perturbation component released from a perturbed lysed cell in said area of an assay device comprising a multiplicity of said examination areas, which method comprises: a) conducting an assay in each of a multiplicity of examination areas in a mapped assay device wherein each of said examination areas comprise a cell, a perturbating agent for said cell; an assay solution b) including in each of said examination areas a capture bead which comprises: an optically detectable label or a set of optically detectable labels attached to said bead which uniquely identifies the capture bead; a multiplicity of the same oligonucleotide index which is unique to said capture bead and is attached to thereto optionally through a linker; and a multiplicity of the same capturing element; c) correlating and memorializing the optically detectable label or set of labels in each examination area by associating the unique optically detectible label or set of 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 examination areas; d) inducing a perturbation on said cell 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) lysing said cell to release said perturbation components into said assay solution and capturing at least one of said component onto the capture bead;f) identifying the perturbation component(s) captured on said bead; g) sequencing the oligonucleotide index to identify the specific oligonucleotide index which correlates to a specific capture bead and then correlating the capture bead to the specific examination area by reference to the memorialized correlation in c) above thereby identifying the particular examination area from which the perturbation component was retrieved.

30. The method of claim 29, further comprising: h) obtaining images of each of the examination areas during the assay; i) correlating the images to the specific examination area defined in g) above; and j) evaluating changes in cellular morphology during the assay as a result of the perturbation of the cell.

Images