Capture beads for the assay

Abstract

Disclosed herein are capture beads suitable for use in a detection device configured to associate the capture beads with a particular detection region within the device and to associate any captured nucleic acids or other cellular components produced during the detection process with the beads.

Description

[0001] Invention Field

[0002] This disclosure provides a capture bead suitable for an assay apparatus configured to correlate the capture bead with a specific detection region within the apparatus and to correlate any captured nucleic acids or other cellular components generated during the assay with the bead. This disclosure also provides methods, apparatus, and systems incorporating the capture bead.

[0003] Technical Background

[0004] Capture beads are used in assays to separate specific cellular components generated by the assay for analysis after the assay is complete. Typically, assay devices have multiple detection zones, allowing thousands to millions of assays to be performed simultaneously in a single device. Detection zones within the device can hold one or more target cells, and individual assays are performed in each detection zone. Assays typically include a perturbation element to disturb one or more cells, and then the effect of that perturbation on the cells is determined. Capture beads are contained within the detection zones. When the assay is complete and the cells have lysed, the nucleic acids released from the cells are captured by the beads. Typically (but not always), one perturbation element is combined with at least one cell to be disturbed. After capturing the nucleic acids, the capture beads are separated, the captured nucleic acids are released, and then sequencing is performed.

[0005] In a conventional system, the assay employs a perturbation bead containing multiple copies of the same perturbation element (e.g., the compound or perturbation compound described herein), which are releasably attached to the bead. Additionally, the perturbation bead contains multiple copies of an oligonucleotide releasably bound to the bead. This oligonucleotide is generated during the synthesis of the compound on the bead, and the reaction steps and / or building blocks used to synthesize the compound are recorded. This oligonucleotide is sometimes referred to herein as a “perturbation oligonucleotide.” In some embodiments, the perturbation element is a compound, and the perturbation oligonucleotide is sometimes referred to as a “compound oligonucleotide.” The assay is performed by perturbing the cells by releasing the perturbation compound from the perturbation bead. After the assay, the cells are lysed. When the compound oligonucleotide contains one or more nucleic acid capture elements, the nucleic acids released from the cells (after lysis) are captured, whether the compound oligonucleotide is released from the bead to the detection area or remains on the bead. In some embodiments, the nucleic acid is mRNA, and the nucleic acid capture element is polythymidine nucleotide (poly-T).

[0006] By attaching a poly-T to a perturbation oligonucleotide, mRNA released from lysed cells is captured by the perturbation oligonucleotide due to its polyadenine nucleotide (poly-A) tail. In some embodiments, the perturbation oligonucleotide comprises a poly-T and its bound mRNA, which can be sequenced on the bead in a single sequencing step. The resulting information helps to gain insights into changes in cellular function induced by perturbation, as well as the structure of the perturbation compound determined based on the reaction used to prepare the compound. More commonly, the compound oligonucleotide captures mRNA after release from the bead because hybridization capture via the solution phase is more efficient than hybridization involving solid-bound oligonucleotides with solution-phase mRNA.

[0007] To extract more meaningful information from these assays, such as changes in cellular function caused by perturbations during the assay (including both static and dynamic (e.g., real-time) changes), it is necessary to identify the cellular function changes that occur during the assay and correlate these changes with changes in mRNA expression determined only by sequencing after the assay is completed. However, this requires back-correlating the oligonucleotides used to capture the mRNA to the capture beads where they initially bound, and then back-correlating them to the specific detection region where the assay was performed, thereby back-correlating them to the cells within that detection region.

[0008] Limiting the information to static changes in cell function resulting from the perturbation, and further to mRNA and the perturbation compound that produced the perturbation, also limits the value of the collected information. Other unaddressed information regarding changes in cell function during the assay includes, by way of example only, the specific well from which the capture bead was taken, changes in cell morphology, how the assay conditions affected changes in cell function, and other information such as the technician performing the assay, the date of the assay, and other perturbation elements that may have been captured.

[0009] However, some or all of this additional information is often not collected during the assay for a variety of reasons, including the inability to retrospectively correlate the mRNA generated by the perturbing compound with the specific area being tested and any observations of the cells within it.

[0010] Information about specific detection regions that produce nucleic acids, as well as information about other functional changes within the cell itself, is lost. This hinders a more complete description of changes in cellular function and a more comprehensive understanding of the effects of perturbation elements on cells. Adding this information would provide a more complete picture of how perturbations alter cellular function and whether these changes are positive or negative.

[0011] Therefore, there is still a need to increase the amount of information available from one or more detection regions identified in the assays of diverse compound libraries. This additional information will enrich the knowledge gained from each assay and contribute to drug discovery. Summary of the Invention

[0012] This disclosure provides a capture bead configured to collect information on static and dynamic changes in cellular function induced by cell exposure to a perturbation element during an assay. In some embodiments, the information collected by the capture bead described herein correlates the changes in cellular function induced by the perturbation element with the detection region of the cell that was perturbed.

[0013] The ability to generate this kind of information depends on the ability to retrospectively correlate sequencing data generated by disturbed cells with the detection area of ​​cell deposition before the assay began.

[0014] In some embodiments, a capture bead for measurement is provided, the capture bead comprising: A detectable mark or set of detectable marks attached to the bead, the mark uniquely identifying the capture bead; Multiple capture bead indices are used to encode detectable tags or sets of tags, which are unique to the capture bead, and the indices are optionally attached to them via a connector; One or more capture elements are attached to the capture bead index.

[0015] In some embodiments, a mapping measurement device is provided, comprising a plurality of detection regions, and at least one capture bead in each detection region, wherein the mapping measurement device associates a given detection region with a specific location of the mapping and the capture bead contained in each detection region, wherein the capture bead comprises: A detectable mark or set of detectable marks attached to the bead, the mark uniquely identifying the capture bead; Multiple capture bead indices, used to encode detectable tags or sets of tags, are unique to the capture bead and optionally attached thereto via a connector; At least one capturing element attached to the capturing bead index.

[0016] In some implementations, the detectable markers or sets of detectable markers used in the capture beads are optically detectable.

[0017] In some implementations, the detectable markers or set of markers contain different colors and / or different intensities of one or more colors associated with different capture beads.

[0018] In some implementations, the detectable markers or set of markers are selected from: images, codes, shapes, colors, induced colors, text, numbers, symbols, barcodes, Universal Product Codes (UPCs), and materials associated with the capture bead.

[0019] In some implementations, the optional connector on the capture bead is a releasable connector.

[0020] In some implementations, the capture element comprises about 6 to about 2,000 nucleotides.

[0021] In some embodiments, the capture elements attached to multiple identical capture bead indices are selected from: receptors or ligands of the perturbation component to be captured, or complementary oligonucleotides of the corresponding oligonucleotide sequences contained in the perturbation oligonucleotide to be captured.

[0022] In some implementations, the capture element is an oligonucleotide.

[0023] In some implementations, the capture element (e.g., an oligonucleotide capture element) is attached to the proximal and / or distal ends of the oligonucleotide index (where the proximal end is adjacent to a releasable linker or bead).

[0024] In some implementations, attachment can be direct or via a connector.

[0025] In some implementations, the connector is a releasable connector.

[0026] In some implementations, the capture element is a receptor or ligand for the perturbation component to be captured. Such receptors include antibodies or antibody-binding fragments of the perturbation component; enzymes or enzyme receptors of the perturbation component; receptors for specific cytokines or chemokines, etc.

[0027] In some implementations, the trapping element on the trapping bead is avidin / streptavidin.

[0028] In some implementations, the capturing element is biotin, which captures perturbation components modified to contain avidin / streptavidin groups.

[0029] In some implementations, the capture bead index is attached to the oligonucleotide capture element, which is directly connected or connected via a releasable adapter.

[0030] In some implementations, the capture element is polythymine (poly-T), polyguanine (poly-G), or polycytosine (poly-C).

[0031] In some implementations, a capture bead as described above and represented by Formula I is provided: (W) r -CB-(LXQQ 1 ) n (I) in: CB stands for Capture Bead; L is an optional releasable connector; W is an optically detectable mark or a set of optically detectable marks that uniquely identify the capture bead; X is a key or capture element; Q is the capture bead index, which encodes the optically detectable mark or set of optically detectable marks on the capture bead; Q 1 It is a capture element, which targets n LXQQ. 1 Each member of the group is selected independently; n represents multiple such beads associated with the beads (LX-QQ) 1 ) group, and r is an integer from 0 to 100 (or larger, depending on the size and type / size of the beads).

[0032] In some implementations, r ranges from 10 to 6.02 x 10⁻⁶. 16 In some implementations, n ranges from approximately 10. 5 Up to 6.02 x 10 17 .

[0033] In some implementations, only multiple copies of the same single oligonucleotide index bind to the capture bead.

[0034] In some implementations, X and Q 1 It is a binding element that captures different targets (e.g., target perturbation components, such as those that may be released by cells during assays, and / or perturbation oligonucleotides discussed herein).

[0035] In some implementations, each X and Q 1 Both are for n LXQQ 1 Each individual choice within the group.

[0036] In some implementations, each X and Q 1 All are selected from functions that are complementary to those found on perturbed oligonucleotides or nucleic acids or other perturbed components released from lysed cells.

[0037] In some implementations, each Q 1 They are all oligonucleotides.

[0038] In some implementations, X and Q 1 One captures perturbed oligonucleotides, while the other captures perturbed components.

[0039] In some implementations, a capture bead with formula IA is provided: (W) r -CB-[L´-XQQ 1 -Y] n(IA) in: Q is the capture bead index; X is a key or capture element; Y is the perturbation component PC or the perturbation oligonucleotide PO; Q 1 It is a capturing element, where X and Q1 are capturing elements independently when X is not a key, where Q 1 The trapping element on each of X is complementary to the corresponding functional group on the target; Y is the perturbation component PC or a perturbation oligonucleotide; L is a releasable connector; And the definitions of n, r, and W are as described above.

[0040] In some implementations, oligonucleotides represented by formula IB are provided: AX 1 -QQ 1 -Y (IB) in: PC is the perturbation component; Q is the capture index; Q 1 It is a trapping element that is complementary to the corresponding functional group on Y; X 1 It is a trapping element that is complementary to the corresponding functional group on A; Y is either the perturbation component PC or the perturbation oligonucleotide PO; and A is the perturbation component PC or the perturbation oligonucleotide PO.

[0041] In some implementations, PC is mRNA, and Q 1 It is poly-T.

[0042] In some implementations, A is a perturbation oligonucleotide, and X 1 It is a complementary group to the functional group on the perturbed oligonucleotide.

[0043] In some implementations, A is a nucleic acid other than mRNA.

[0044] In some embodiments, a method is provided to correlate sequence data from a solution phase capture index with a specific detection region in a mapping assay apparatus, the solution phase capture index comprising one or more capture elements, wherein at least one of the elements is coupled with a perturbation component, the method comprising: a) Each capture bead is encoded by a releasable bond using an optically detectable tag or set of optically detectable tags (the tag being unique for the particular bead), wherein the capture bead contains an oligonucleotide index encoding the unique tag or set of tags and further contains a capture element thereon, wherein the capture index is releasably bound to the capture bead; b) Add the capture beads coded in a) to the detection area of ​​the measuring device to be measured; c) Generate a registry that records the unique beads in each detection area; d) Detection is performed in the presence of disturbed cells during the assay; e) Lyse cells and capture at least one nucleic acid perturbation component onto the capture element; f) Release the capture index from the capture bead; g) Sequencing of the capture index and nucleic acid perturbation components to provide correlation between the capture index and the markers on the capture beads; and h) Determine the detection area associated with the unique tag or set of tags by referring to the registry.

[0045] In some implementations, the correlation is performed via an image that aligns each optically detectable capture bead with a site on a map.

[0046] In some embodiments, the optically detectable markers on the capture beads may include additional information related to the assay to be performed in the detection area. This additional information includes (by example only): assay conditions, changes in cell function during the assay, synthetic reactions used in at least one step of the compound synthesis, identification of one or more reaction conditions used in the synthesis process, the technician performing the assay, the date of the assay, the pH of the assay solution, or a combination thereof. The unique optically detectable marker on each capture bead in each detection area may be recorded by means of photography, barcodes, QR codes, etc.

[0047] In some implementations, such additional information can be included on the perturbation beads by adding microcomponents to the detection region. Such microcomponents encoding this additional information are described in: U.S. Patent Application Serial No. 18 / 195,049, filed May 9, 2023, entitled “High-throughput single-cell assay for capturing genomic information for functional and imaging analysis and method of use thereof,” which claims priority to U.S. Provisional Patent Application Serial No. 63 / 339,782, filed May 9, 2024; International Patent Application Serial No. PCT / US24 / 23594, entitled “Cell transfer assembly for a multiwell plate assay device,” filed April 8, 2024; and / or U.S. Provisional Patent Application Serial Nos. 63 / 494,621, 63 / 494,628, and 63 / 494,636, all filed April 6, 2023. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0048] In some embodiments, micro-components can be of different shapes, or of the same shape but different colors, or with different images placed on them, such as different descriptions, barcodes, information, QR codes, etc. In some embodiments, different micro-components are used in combination. In some embodiments, different images are contained on the same micro-component, such that the combination of these images constitutes a unique micro-component. See, for example, Figure 5C The micro-component contains four quadrants, each containing an image, such as a picture or text information; the combination of these images is unique to the micro-component.

[0049] In some embodiments, a unique microcomponent or combination of unique microcomponents can provide a unique image that is uniquely associated with a given detection region. In some embodiments, each microcomponent used has multiple identical unique oligonucleotide chains associated with it. In some embodiments, the unique chain is reversibly attached to the microcomponent.

[0050] In some embodiments, the connector is used in combination with unique perturbation beads. In some embodiments, the connector is reversibly attached to the microcomponent, and the connector is released in a release manner orthogonal to that of the connector used on the perturbation beads.

[0051] In some embodiments, the perturbation oligonucleotides on the perturbation beads encode only the structure of the compounds synthesized therefrom. In some embodiments, the perturbation oligonucleotides contain a binding element at their distal end. In some embodiments, the oligonucleotide chain on the microcomponent may have a complementary oligonucleotide binding element to which it binds, thereby allowing hybridization with the binding element on the perturbation oligonucleotide.

[0052] In some embodiments, a single microcomponent having a unique label or combination of labels thereon has a unique oligonucleotide chain encoding that label, the oligonucleotide chain being attached to the microcomponent via a cleavable adapter that is cleaved orthogonally to an adapter used on a bead. In a preferred embodiment, the oligonucleotide chain encoding such a microcomponent includes a binding element complementary to a binding element at the distal end of a perturbation oligonucleotide, such that upon release, the oligonucleotide chain from the microcomponent hybridizes with the binding element at the distal end of the perturbation oligonucleotide.

[0053] In some embodiments, multiple microcomponents are used, such that the combination of these microcomponents is unique for detection on the assay device. In some embodiments, the oligonucleotide chains on the microcomponents can be released and sequentially hybridized with perturbation oligonucleotides by utilizing a unique combination of binding elements on each component or by utilizing orthogonal cleavage linkers.

[0054] In some embodiments, the aforementioned microcomponents may be used in combination with labeled or partially labeled perturbation beads that encode or partially encode the compound synthesized thereon. In some embodiments, one or more steps in the reaction sequence for synthesizing the compound are encoded by the microcomponent or a combination of microcomponents. In some embodiments, the unique set of microcomponents provides information regarding reaction conditions, etc., as described above.

[0055] In some implementations, unique micro-components or combinations of micro-components are recorded by photographing or video recording to associate the areas of examination where these unique micro-components or combinations of micro-components are found. In some implementations, the photographs or videos are recorded digitally.

[0056] Furthermore, the markers or unique set of markers on the capture beads can be further compiled into the library or registry, providing a complete set of information that allows technicians to associate the capture beads with specific detection areas. Additionally, perturbation components captured by the capture beads are identified as originating from that specific capture bead through a capture bead index. Conversely, by mapping detectable markers to detection areas, the detection areas used with the capture bead can be identified. This enables the tracking of captured perturbation components, tracing back to their specific detection area of ​​origin, and identifying the perturbation elements causing the perturbation.

[0057] In some implementations, only multiple copies of the same single capture bead index oligonucleotide bind to the capture bead.

[0058] In some implementations, X and Q 1 They are the same.

[0059] In some implementations, X and Q 1X is a binding element used to capture different targets. In some embodiments, the capture of perturbation components and / or perturbation oligonucleotides by X is achieved after cleavage of the releasable linker.

[0060] In some embodiments, the trapping element comprises avidin or streptavidin, and the perturbation component comprises biotin; or the trapping element comprises biotin, and the perturbation component comprises avidin or streptavidin. Similarly, the binding pair may be an enzyme and its binding partner, a cytokine or chemokine and its binding partner, an antibody / its binding moiety and its target.

[0061] When the trapping element on Q is an oligonucleotide, and it is already bound with perturbation oligonucleotides and / or nucleic acid perturbation components (such as mRNA), X, Q, and Q 1 Combinations and X 1 and Q 1 The captured components allow for the sequencing of multiple informational components in a single step.

[0062] In some implementations, the perturbation oligonucleotide also includes an mRNA capture element, such that after the assay is completed, Q... 1 The capture bead index -Q is attached above. 1 -Perturbation of oligonucleotide-mRNA. For example, the distal end of the oligonucleotide index (Q) is attached to a poly-C group (Q). 1 The perturbation oligonucleotide can be constructed with a poly-T group at one end and a poly-G group at the other end. When the perturbation oligonucleotide is released from the perturbation bead, the poly-G group can react with Q. 1 The poly-C group hybridizes. When cells lyse, the perturbed poly-T group of the oligonucleotide captures the poly-A tail of the mRNA released from the lysed cell.

[0063] In some implementations, capture can be performed on capture beads or in solution.

[0064] In some implementations, the capture of the disturbing component is performed on a capture bead. The capture bead has formula IA: (W) r -CB-[LXQQ 1 -Y] n (IA) in: Q is the capture bead index; X is a key or capture element; Y is the perturbation component PC or the perturbation oligonucleotide PO; Q 1 It is a capturing element, where X and Q 1 Both are independent capturing elements, where Q is a capturing element when X is a capturing element.1 The trapping elements of both X and Q are complementary to the corresponding functional groups on the target (in this case, for Q). 1 In other words, it is a perturbation component. in n、r The definitions of L and W are as described above.

[0065] In some implementations, the connector is a releasable connector.

[0066] In some implementations, when the adapter is cleaved, whether before or after capturing the perturbation oligonucleotide and / or mRNA, the resulting oligonucleotide is represented by the formula IB.

[0067] AX 1 -QQ 1 -Y (IB)

[0068] Among them, Q, Q 1 The definitions of Y and X are as described above. 1 For the capture element, Y and A are each independently a perturbation component or a perturbation oligonucleotide. In some embodiments, the perturbation oligonucleotide is subjected to Q before cell lysis. 1 Capture. Then, the releasable key is cut, making X 1 The perturbed component A can be captured in the solution phase. Alternatively, the capture can occur entirely in solution.

[0069] In either case, the captured disturbance components are associated with the capture bead via the capture bead index, thereby identifying the inspection area by referencing the registry.

[0070] In the above description, for n LXQQ 1 Each of the groups, X and Q 1 The identification can vary. However, in all cases, the capture bead index Q remains constant because it has a unique association with the markers or set of markers on the bead. In some embodiments, the perturbation element is a perturbation compound on the perturbation bead, which is encoded by a perturbation oligonucleotide. In some embodiments, a method is provided for identifying a specific detection region in an assay device, wherein a compound perturbation element is released, the method comprising: a) Determination is performed on at least a portion of a plurality of detection regions in a mapping assay apparatus, wherein each detection region comprises a cell, a perturbation bead (which contains a perturbation element (e.g., a compound)) and a perturbation oligonucleotide that is unique to the perturbation element (e.g., the compound), and an assay solution; b) Each detection region contains a capture bead, which comprises: An optically detectable mark or set of optically detectable marks attached to the bead, the mark uniquely identifying the capturing bead; Multiple identical capture bead indices, each index being unique to the capture bead and optionally attached thereto via a connector, wherein the index encodes a unique tag or set of tags; and Multiple capture elements attached to the oligonucleotide index; c) By associating the unique optically detectable marks on the capture beads with the mapping measuring device, the optically detectable marks or sets of marks in each detection area are associated and recorded, thereby providing a 1:1 relationship between the capture beads in each detection area; d) Inducing perturbation by releasing at least a portion of the perturbation element from the perturbation bead, thereby bringing the element into contact with the cell and inducing a functional change in the cell, the functional change including at least a change demonstrated by one or more perturbation components expressed by the cell; e) Release the unique perturbation oligonucleotide from the beads and capture the perturbation oligonucleotide onto the capture beads; f) Lyse the cells, release the perturbation component into the assay solution, and capture at least one of the components onto a capture bead index or a perturbation oligonucleotide; g) Sequencing of oligonucleotides containing capture bead indexes, perturbation oligonucleotides, and perturbation components; and h) By referring to the associations recorded in c) above, the capture bead index is associated with a unique marker or set of markers for a specific detection region, thereby identifying a specific detection region from which the perturbation component is retrieved.

[0071] In some implementations, the above method further includes: i) Acquire images of each detection area during the measurement period; j) Associate the image with the specific detection region defined in h) above; and k) Assess changes in cell morphology and / or cell function caused by cell perturbation during the assay.

[0072] In some implementations, the perturbation element need not contain a perturbation bead. Such perturbation elements may include, but are not limited to, one or more conditions in the assay, such as, but not limited to, the presence of buffers, salts, pH, temperature, nutrients, oxygen levels, oxidants, physical stress and / or antibodies, immune cells, siRNA, viruses, bacteria, fungi, etc. In this case, due to the absence of a perturbation bead, there is no need for a perturbation oligonucleotide. Instead, a unique oligonucleotide can be associated with one or more different non-compound perturbation elements (e.g., associated with a perturbation bead via a releasable adapter, and / or associated with the perturbation element itself in the case of immune cells, viruses, etc., and / or by adding it to the corresponding detection region), thereby keeping the basic principle unchanged—that is, releasing (if releasably attached) the perturbation oligonucleotide (i.e., the oligonucleotide encoding the perturbation element and / or condition), which is then captured by a capture bead, thereby identifying the perturbation element.

[0073] In some embodiments, the set of markers on the capture bead comprises two or more markers. In some embodiments, the set of markers comprises two to fifteen members.

[0074] In some implementations, the tag set contains multiple members that combine to identify the capture beads to which they are attached.

[0075] In some implementations, the perturbation element is a compound released from the perturbation bead.

[0076] In some implementations, the capture beads are positioned within the detection area before, during, or after the measurement is completed.

[0077] In some implementations, the capture beads are removed from the detection area before the oligonucleotides are removed from the beads.

[0078] In some embodiments, capture beads are added to the detection area prior to the assay. In some embodiments, capture beads are added to the detection area during the assay. In some embodiments, capture beads are added to the detection area after the assay is completed.

[0079] In some implementations, a registry is provided for a given assay device, which contains a record of each unique marker or set of markers for each capture bead, the specific detection region in which the capture bead is located in the assay device, and the unique oligonucleotides found on the capture bead.

[0080] In some implementations, the registry contains one or more digital photographs that record codes associated with each detection area and optically detectable markings on the capture beads.

[0081] In some implementations, the tag set contains tags that have the same color but different intensities.

[0082] In some implementations, digital photography employs megapixels, where the record for each detection area contains a different set of pixels than that for other detection areas. The use of pixels enables the differentiation of subtle color hues and the representation of the same color at different intensities.

[0083] In some embodiments, a method for performing a measurement in a measuring apparatus comprising multiple detection areas is provided, the method comprising: A. Integrate into each detection area to be used: a) A perturbation bead comprising multiple copies of the same perturbation compound unique to the bead, and multiple identical perturbation oligonucleotides encoding the perturbation compound; b) A capture bead with formula IA: (W) r -CB-[L´-XQQ 1 -Y] n (IA) in: Q is the capture bead index; X is the second capture element on the key or Q; Q 1 It is a capturing element, where X and Q 1 Both are independent capturing elements, where Q is a capturing element when X is a capturing element. 1 The trapping elements on X are complementary to the corresponding functional groups on the target; Y is a target, such as a perturbation component and / or a perturbation oligonucleotide (e.g., encoding a perturbation element, such as a perturbation compound released from a perturbation bead). L' is a releasable connector; W is an optically detectable mark or a set of optically detectable marks that uniquely identify the capture bead; n represents multiple such (L´-XQQ) beads. 1 ) group, and r is an integer from 0 to 100 (or larger, depending on the size of the capture bead and the type / size of the marker).

[0084] c) Cells; and

[0085] d) Determine the solution; B. The assay is initiated by severing at least a portion of the releasable connectors on the perturbation beads, thereby releasing at least a portion of the perturbation compound and perturbation oligonucleotide on each bead in each detection region; C. Capture a perturbation oligonucleotide on a capture element on the capture bead, wherein the perturbation oligonucleotide optionally includes or is modified to include a binding element for the perturbation component; D. After the assay is completed, the cells are lysed to release the perturbed components from the cells, at least a portion of which pass through X (when it is a trapping element) or Q. 1 Or the perturbation oligonucleotides are captured on the capture beads; F. Separate the trap beads and cleave the releasable bonds, wherein the trap bead index and the target trapped on it are released into the solution; and G. Sequencing of the capture bead index and the oligonucleotide targets captured on it.

[0086] In some implementations, the releasable connector is not released from the capture bead until the assay is complete (including cell lysis).

[0087] In some implementations, the cutting of the connector on the capture bead is performed after cutting the connector on the perturbation bead, but before lysing the cells.

[0088] In some embodiments, the cleavage of the junctions on the capture beads is performed before or simultaneously with the release of the perturbation oligonucleotides from the perturbation beads in order to capture the perturbation components and / or perturbation oligonucleotides in the assay solution.

[0089] In some embodiments, a method is provided for preparing a population of captured beads, wherein each bead in the population comprises a unique marker or set of markers and a captured bead index specific to that marker or set of markers, the method comprising: a) Select bead populations functionalized with oligonucleotide binding sites; b) Distribute the beads into a plurality of reaction containers in approximately equal quantities, wherein the number of reaction containers can be any integer (e.g., up to 12). c) In each reaction vessel, the same first mark is applied to each bead in a given reaction vessel, wherein the mark used in each vessel is different from the mark used in the other vessels; d) Attaching an oligonucleotide chain to the oligonucleotide binding site, wherein the oligonucleotide chain is identical for each bead in a common container, but the oligonucleotide chains differ between different containers; e) Gather the beads and mix until homogeneous; f) Repeat steps b) to e) a sufficient number of times such that almost every bead in the population is uniquely labeled and almost every bead contains a unique capture bead index, which, for each individual reaction step, contains an oligonucleotide chain attached to the previous oligonucleotide chain.

[0090] In some implementations, the population size of such beads ranges from 10,000 to 10,000,000. Attached Figure Description

[0091] Figure 1A and 1B Two different mapping measurement devices are shown, both of which have directional components.

[0092] Figure 2A-2D The exhibit showcases a perturbation bead (circle) with a set of optical markers attached to it, used to uniquely identify each capture bead from the other capture beads.

[0093] Figure 3 Showing Figure 1B The detection region subset shown Figure 2A-2D The disturbed beads.

[0094] Figure 4 It showcases mixed bead groups, including beads of different colors, as well as beads of the same color but with different color intensities.

[0095] Figures 5A-5D Examples of beads with markings and markings with micro-components are shown.

[0096] Figure 6 The marking area of ​​the marking device is shown.

[0097] Figures 7A-7D An example of a labeled bead with microcomponents corresponding to compounds (e.g., perturbation compounds) attached to the bead is shown.

[0098] Figures 8A-8B The results of assays used to identify compounds in the combinatorial library that can actively perturb A549 cells are shown. Detailed Implementation

[0099] This disclosure provides a capture bead suitable for an assay apparatus configured to correlate the capture bead with a specific detection region within the apparatus and to correlate any captured nucleic acids or other cellular components generated during the assay with the bead.

[0100] 1. the term

[0101] For ease of understanding, all terms below are defined. The terms explained use definitions accepted by the scientific community.

[0102] As used in this article, the singular forms “a” and “the” are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0103] As used herein, the term “about” (when placed before a numerical value, such as temperature, time, amount, concentration, etc., including numerical ranges) refers to an approximate value that can vary by (+) or (-) 10%, 5%, 1%, or any subrange or sub-value in between. In one embodiment, the term “about” when used to describe a dose indicates that the dose can vary by + / - 10%.

[0104] As used herein, the term "comprising" is intended to mean that a composition and method include the said elements, but does not exclude other elements.

[0105] As used herein, the term "consistently of..." when used to define compositions and methods should mean excluding other elements that have any substantial significance for the combination of said purposes. Therefore, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not substantially affect the claimed basic and novel features disclosed.

[0106] As used herein, the term "composed of" should mean excluding other components and material method steps beyond trace elements. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0107] In addition, the following chemical / biological and related terms were used: a) Measuring apparatus and mapping measuring apparatus; b) Related to it; c) Capture the bead; d) Capturing elements or combining elements; e) Capture bead index, capture index, capture oligonucleotide, or Oligonucleotide index; f) Cells; g) Cell components; h) Cell morphology; i) Functional changes; j) Complementary functions; k) compounds; l) Detectable tags or a set of detectable tags; m) Detection area; n) Connectors and releasable connectors; o) Multiple or multiple perturbation beads; P) Multiple disturbance elements; Q) Multiple oligonucleotides or capture / binding elements; r) Nucleic acid; S) oligonucleotides; t) Oligonucleotide chains; u) Optically detectable; v) or its precursor; w) Disturbance beads; x) Disturbance components; y) Disturbance complex; z) Perturbation element; aa) Disturbance oligonucleotides; ab) Releasable attachment; and ac) unique.

[0108] The meanings of the terms used in this article are as follows (not in alphabetical order). Terms not listed above have their generally accepted scientific meanings.

[0109] As used herein, the term "oligonucleotide chain" refers to an oligonucleotide having about 6 to about 20,000 nucleotides or 16 to about 50,000 nucleotides. These chains can attach to each other to form oligonucleotides.

[0110] As used herein, the term "oligonucleotide" refers to an oligonucleotide containing about 6 to about 500,000 nucleotides; however, if the oligonucleotide comprises two or more oligonucleotide chains attached together, then the oligonucleotide comprises about 12 to about 500,000 individual nucleotides. It should be understood that if an oligonucleotide is intended to be attached to another oligonucleotide, then, as described above, the oligonucleotide is referred to as an oligonucleotide chain. However, if the oligonucleotide is used and / or discussed as a separate component, it is not referred to as a chain.

[0111] As used herein, the term "capture bead" or "CB" refers to any and all beads used in an assay to capture perturbed oligonucleotides and one or more cellular components after perturbing and subsequently lysing cells to release cellular components (i.e., "perturbation components"). In some embodiments, the perturbation component includes nucleic acids, such as mRNA, DNA, etc. In some cases, the perturbation component can be a chemokine, cytokine, enzyme, protein, glycoprotein, peptide, hormone, metabolite, protein complex, protein-nucleic acid complex, endogenous cellular ligand, etc. The detection area may contain one or more capture beads. Multiple capture beads may be used when different perturbation components are to be captured. Alternatively, a single capture bead may be used so that different perturbation components can be captured by that bead.

[0112] As used herein, the term "detectable tag" refers to any source of information that can be detected visually, chemically, biologically, audioly, or otherwise to demonstrate the presence of the information generated by the tag. In some cases, detectable tags generate or are stimulated to generate visually and / or optically detectable images / signals, such as colors, visible light, text, numbers, symbols, barcodes, fluorescent particles, light-emitting diodes (LEDs), microcomponents such as microchips, UPCs, tags, or combinations thereof. These images / signals can be observed with the naked eye or with the aid of a microscope and are sometimes referred to herein as "optically detectable tags." In some cases, detectable tags generate chemical signals that optionally require detection by suitable instruments, such as quantum dots, fluorescence (e.g., fluorescent particles, laser particles, luminescent particles), mass spectrometry, and nuclear magnetic resonance spectroscopy (e.g., H2O). 1 Or C 13 Spectroscopy, oligonucleotides, etc. In some cases, detectable signals generate audio or electromagnetic signals, such as those generated by radio frequency identification (RFID), WiFi, or Bluetooth devices. In other cases, detectable tags generate biological or bio-inducible signals, such as nucleic acids, bioluminescence, etc. The specific detectable tags used in the embodiments disclosed herein are within the scope of conventional technical capabilities of those skilled in the art, depending on the limitations of the assay to be performed and the need to uniquely identify one or more components in that assay. Combinations of different tags may be used.

[0113] In this paper, the term "unique" refers to the unique combination of a perturbation element and the tags generated on the bead for a given perturbation bead having a perturbation element releasably attached to it. However, this does not mean that every bead in the population has a different perturbation element, because for probabilistic considerations in pool synthesis, there is a possibility of duplicate beads having the same unique perturbation element and tags or sets of tags.

[0114] For example, in the pooled reaction schemes shown herein (e.g., reaction schemes A and B described herein), a population of 100,000 beads is divided into 10 groups of 10,000 beads each, and then reacted with a different label in each of the 10 independent reaction containers in step 1, resulting in 10 sets of beads with different labels. In step 2, when the beads are collected and redistributed to the 10 reaction containers, the probability that a bead will enter the same reaction container as in step 1 in step 2 is 10%.

[0115] Therefore, after step 2, the number of beads with the same two labels in each reaction vessel is reduced to 1,000, or a total of 10,000 replicates.

[0116] In steps 3, 4, and 5, the probability that the bead remains in the same reaction vessel throughout steps 1 through 5 is also 10%.

[0117] Therefore, for step 3, the probability is 10% of that for step 2 (10,000), that is, 1,000 out of 100,000.

[0118] For step 4, the number of beads with the same probability of repetition becomes 100; for step 5, this number becomes 10 out of 100,000; and for step 6, the probability is 1 out of 100,000. If necessary, steps 7 and 8 can reduce this number to less than 0.01 out of 100,000. After step 5, at any time, the probability of repetition has been reduced to such an extreme that the vast majority of beads are unique. For completeness, the repetition rate can be less than one in a thousand, or one in ten thousand, or even one in a hundred thousand.

[0119] As used herein, the term "detection area" or "area" refers to a specific region (e.g., coordinates, region, location, space, orifice, compartment, etc.) within or part of a measuring apparatus that is used for a single measurement at a given time. Multiple detection areas may be employed in one or more measuring apparatuses. Detection areas may include droplets, orifices, channels, etc. Each area is capable of accommodating at least one cell, at least one trapping bead, and at least one perturbing element, and is isolated from other areas and / or sets of areas (e.g., other locations on the measuring apparatus). In some embodiments, the detection area does not include points within the apparatus that are not involved in the measurement. That is, only the area where the measurement is performed is considered a "detection area" as described herein.

[0120] As used herein, the terms “capture bead index,” “capture index,” “capture oligonucleotide index,” and “oligonucleotide index” are used interchangeably and all refer to an oligonucleotide that encodes a unique marker or set of markers on a capture bead, thereby associating the oligonucleotide index with that unique marker or set of markers, and subsequently with a unique, specific capture bead having that unique marker. In some embodiments, the oligonucleotide index is optionally attached to the capture bead in a releasable manner. In some embodiments, the oligonucleotide index may be attached to a “capture element” that can capture perturbed oligonucleotides and / or perturbed components (as defined above). In some embodiments, the capture element may be a complementary group, such as an oligonucleotide that can hybridize with a subset of nucleic acids recovered from lysed cells or with a portion of a perturbed oligonucleotide. In some embodiments, the capture element is a complementary functional group that recognizes and binds to a corresponding complementary functional group. These complementary functional groups include avidin or streptavidin and biotin, enzymes and their binding partners, cytokines or chemokines and their binding partners, antibodies or their binding moieties and their targets. The complementary group captured by the capture element on the capture bead is sometimes referred to as a “target.”

[0121] In some embodiments, the capture bead index may optionally be attached to the capture bead via a adapter (including a releasable adapter). In some embodiments, the oligonucleotide index contains a capture element at both its proximal and distal ends. In some embodiments, the capture bead and the oligonucleotide index may be represented by Formula I as defined above.

[0122] In some embodiments, the capture bead index is attached to the perturbation component (e.g., mRNA). In some embodiments, the capture bead index is attached to the perturbation oligonucleotide. In some embodiments, the capture bead index is attached to the perturbation component (e.g., mRNA) and the perturbation oligonucleotide in any order. In some embodiments, capture occurs during the assay; in other embodiments, capture occurs during and / or after the assay. In some embodiments, a single capture bead is used for each detection region. In some embodiments, multiple capture beads are used for each detection region. In some embodiments, each capture bead in a single detection region is indexed with the same unique oligonucleotide specific to that capture bead. In some embodiments, each capture bead in a single detection region is indexed with a unique oligonucleotide index, all of which are associated with that capture bead. For example, one or more of the additional capture beads are indexed with unique capture elements and unique oligonucleotide indexes to capture different perturbation components generated by perturbation.

[0123] As used herein, the term "perturbed oligonucleotide" refers to an oligonucleotide that uniquely encodes all or part of the perturbing elements on a perturbed bead. In some embodiments, the perturbed oligonucleotide also encodes a unique marker or set of markers on the perturbed bead, thereby uniquely identifying the perturbed bead. The perturbed oligonucleotide also includes a complementary binding domain for capturing binding elements on the bead. This complementary binding domain is preferably a proximal or distal portion of the perturbed oligonucleotide. In some embodiments, the perturbed oligonucleotide also includes, or can be modified to include, a complementary binding domain for perturbed components released from lysed cells. In some embodiments, the perturbed oligonucleotide is releasably attached to the perturbed bead. For example, see U.S. Provisional Patent Application Serial No. 63 / 624,167 and U.S. Provisional Patent Application Serial No. 63 / 542,760, both of which relate to encoding and / or partially encoding via oligonucleotide perturbing. These applications are incorporated herein by reference in their entirety.

[0124] As used herein, the term "perturbation element" refers to any source capable of perturbing cells. Examples of such sources include, but are not limited to: peptides, compounds (e.g., drug-like small molecules), antibodies, immune cells, siRNA, viruses, bacteria, fungi, and variations in one or more of the assay conditions (including, but not limited to, buffer solutions, salts, pH, temperature, nutrients, oxygen levels, oxidants, physical stress, etc.). In some embodiments, the perturbation element is a compound released from the perturbation bead. In some embodiments, a combination of two or more perturbation elements may be used. Such compounds are sometimes referred to herein as "perturbation compounds."

[0125] As used herein, the term "compound" or "perturbed compound" is an example of a perturbed element, referring to small molecules (molecular weight less than about 15,000 Daltons), peptides, and / or oligonucleotides; these substances are synthesized and then bound to the perturbed beads via releasable (cleavable) bonds located between the compound and the beads. The term "perturbed bead" is sometimes used in conjunction with the term "perturbed bead" when the perturbed element is a compound on the perturbed bead containing multiple copies of the same compound that can be released from the bead, as well as multiple copies of a perturbed oligonucleotide identifying the compound structure or identifying the perturbed oligonucleotide.

[0126] The terms “perturbation component,” “perturbation product,” or “PC” (or “PC” for integer i) used in this paper i"" refers to a cellular component whose quantity, expression, location, labelling, or other properties may be altered upon exposure to a perturbation element. Such perturbation components may include mRNA, DNA, epigenetically modified mRNA, alternatively spliced ​​mRNA, cytokines, chemokines, proteins (protein levels), protein isotypes, precursor mRNA, metabolites, lipids, glycoproteins, secretory proteins, and other such components constituting the cell. For ease of explanation, this document primarily discusses specific embodiments of perturbation components such as mRNA. However, any perturbation component that can be represented by a nucleic acid barcode sequence (e.g., proteins, metabolites, etc., as described above) may be used as a nucleic acid perturbation component of this disclosure to supplement or replace mRNA. Nucleic acid barcode sequences attached to antibodies or aptamers can be used to monitor protein and / or metabolite levels, where the selected antibody or aptamer binds to a target protein or target metabolite, and the barcode is specific to the target protein or target metabolite. See, for example, Stoeckius, M., et al., (2017), Large-scale simultaneous measurement of epitopes and transcriptomes in single cells. Nature methods, 14(9), 865. Nucleic acid barcodes representing various perturbation components can be captured in a manner similar to mRNA (as described herein), for example, by hybridization of a poly-A sequence on the barcode with a poly-T segment on the bead serving as a capture element, or by hybridization of other complementary sequences with other capture elements. In embodiments of this disclosure, after cells are exposed to perturbation elements and perturbation components are captured on the capture bead (or, in some embodiments, on the perturbation bead), the perturbation components from the cells are analyzed. In some embodiments, different types of perturbation components can be analyzed simultaneously. In these embodiments, different and / or different types of perturbation components are denoted as PC. 1 PC 2 etc. are used to represent the diversity of perturbation components and / or perturbation component types.

[0127] As used herein, the term "perturbation bead" refers to a bead carrying a perturbation element (e.g., in a releasable form). During assay in a detection area, the perturbation element carried by the bead is released, allowing interaction with cells in the detection area (e.g., cell exposure to the perturbation element). Typically, the perturbation bead carries multiple copies of the same perturbation element (e.g., a perturbation compound) in a releasable form, which constitutes the perturbation element. In some embodiments, the assay does not include a perturbation bead because the perturbation element constitutes one or more conditions of the assay described above; alternatively, the perturbation element may be a virus, antibody, or cell (e.g., an immune cell).

[0128] In some embodiments, the perturbation beads also contain perturbation oligonucleotides that encode perturbation elements on the beads.

[0129] As used herein, the term "measuring device" refers to a device having multiple detection zones for use in measuring with the capture beads described herein.

[0130] As used herein, the term "mapping measurement device" refers to any measuring device capable of uniquely identifying a detection area; such identification can be achieved through the geometry of the device (e.g., X and Y coordinates, or triangles, trapezoids, etc., that allow for the orientation of each detection area), or through images, text, numbers, etc., associated with each detection area. In some cases, the measuring device may have markers, labels, or other indications to help technicians correctly orient the mapping measurement device.

[0131] As used herein, the term "multiple perturbation beads" refers to more than one bead, such as at least 90 beads, at least 1,000 beads, at least 10,000 beads, or at least 100,000 beads. In all cases, the maximum number of beads can be as high as 5,000,000 or more, but is typically around 2,000,000.

[0132] When the term "multiple" is used in conjunction with perturbation elements, oligonucleotides, binding elements, etc., it means that the number of each is sufficient to achieve the following objectives: perturbation of cells; capture of perturbation components from lysed cells; and evaluation of the identifier of the perturbation component, the perturbation element that generates the perturbation component, and the detection region that generates the perturbation component. In some embodiments, this multiplicity ranges from approximately 1 x 102 5 To approximately 6.02 x 10 17 .

[0133] As used herein, "releasably bound or attached" means that the specific component is releasably attached to the capture bead, such that at an appropriate time during the assay (e.g., an anticipated, predetermined, or controlled time), a process of releasing / cleaving at least a portion of such compound, oligonucleotide, or nucleic acid from the bead can be initiated (e.g., by a technician). This controlled release can be achieved using cleavable covalent bonds—covalent bonds that are cleaved in response to appropriate stimuli such as light (e.g., ultraviolet light), heat, pH changes, enzyme activity, electromagnetic stimulation, sound waves, salinity, changes in oxidation state, etc. Such cleavable bonds are well known in the art.

[0134] As used herein, “cell morphology” refers to any combination of one or more characteristics of a cell or its components, such as the size, shape, structure, and / or form of the cell or its components. Cell components are well known in the art and include the nucleus, mitochondria, and ribosomes.

[0135] As used in this article, “functional changes” in cells refer to one or more changes in cell morphology compared to cells before exposure to the perturbation element, as well as changes in the cellular expression of nucleic acids, proteins, chemokines, cytokines, enzymes, peptides, hormones, etc. Other functional changes include the generation of apoptosis markers or other biomarkers induced by cell perturbation. These apoptosis markers include chromatin condensation, caspase activation, vesicle formation, DNA fragmentation, etc., some of which are readily observable.

[0136] As used herein, the term "cell" refers to a eukaryotic or prokaryotic cell. In some embodiments, the cell is a bacterial or yeast cell. In some embodiments, the cell is a primary cell or cell line used for assays. In some embodiments, the cell is a fish cell, amphibian cell, reptile cell, avian cell, or mammalian cell. In some embodiments, the cell is, for example, a mammalian cell, such as a cell derived from a primate. In some embodiments, the cell is a human cell.

[0137] In this document, the term "linker" refers to a group that attaches one entity to another. Such a linker can be releasable or non-releasable. In some embodiments, the linker is a nucleotide or oligonucleotide having 1 to 2000 nucleotide units. In some embodiments, the oligonucleotide incorporates a uracil (U) group, which can be cleaved by a User enzyme. In some embodiments, the oligonucleotide can be cleaved by a restriction endonuclease. In some embodiments, the oligonucleotide can be cleaved by a cleaving enzyme. In some embodiments, clustered regularly spaced short palindromic repeats (CRISPR®) technology is used to cleave the oligonucleotide. Chemical linkers, such as carbonate, ester, and carbamate groups, can be cleaved by pH, esterases, lipases, etc. Photolytically cleavable linkers are well known in the art and include coumarin groups, o-nitrobenzyl groups, and others found in (e.g.) Johan, people, Pharmaceuticals (Basel), 15(6): 655 and subsequent (June 2022) groups in Figure 2. The selection of a suitable linker is not critical and is well known to those skilled in the art.

[0138] In some embodiments, the linker is a non-nucleotide containing at least one and at most about 40 non-hydrogen atoms, including carbon, nitrogen, oxygen, sulfur, and phosphorus. Where appropriate, these atoms include hydrogen (including all isotopes), hydroxyl groups, oxo groups (=O), amino groups, or halogens, to satisfy the valence of the atoms. Unless otherwise stated or clearly inferred from its use, when the term "linker" is used, it means the direct connection of two groups without introducing any additional atoms.

[0139] The term "releasable" or "cleavable connector" refers to a connector that contains a cleavable functional portion—that is, the functional portion or covalent bond can be readily cleaved into a first component and a second component under the influence of a stimulus capable of breaking the bond. Stimuli capable of cleaving releasable bonds include enzymes, ultraviolet light, heat, pH changes, salts, and other components known in the art.

[0140] The term "static information" refers to one or more cellular components that change and / or whose expression changes during the assay, and this information is collected after the assay. Such static information may include / be based on the expression of cellular components (e.g., mRNA), or the upregulation or downregulation of one or more cellular components (e.g., chemokines, cytokines, hormones, enzymes, peptides, peptide fragments, etc.) captured by one or more capture beads. Static information typically includes and / or describes one or more molecular states of the cells. Static information also includes perturbed components captured by capture elements on the capture beads, as well as perturbed components that can be analyzed directly in the lysed cellular environment. For example, protein degradation components may be observed at the end of the assay, but perturbed components can be evaluated without capture. In some embodiments, if the perturbed and lysed cells are not from stable cell lines containing intrinsic fluorescence, protein degradation components can be evaluated by immunofluorescence staining after the assay.

[0141] The term "dynamic information" refers to information about the state of cells during the assay. This dynamic information can include images of cells undergoing perturbation, such as photographs or videos of perturbed cells taken during part or all of the assay. These images demonstrate changes in cell function, such as cell morphology, motility, size, shape, adhesion, division, apoptosis induction, gene expression (e.g., expression of fluorescent proteins or fluorescently labeled proteins), intracellular organization / structure (e.g., cytoskeleton organization / structure, organelle structure), extracellular expression, and other changes in cell structure / behavior.

[0142] For example, such dynamic information may include evidence detectable by optical means, involving the presence of apoptosis markers or other markers, upregulation or downregulation of proteins (such as fluorescent proteins) that can be monitored in vivo and / or in real time, T cell activation (from which their proliferation and size changes can be observed), T cell killing of target cells, etc. Such changes are considered "in vivo" or "real-time" because they are observed during the assay and can be recorded through photographs, video recordings, three-dimensional cell analysis, etc.

[0143] 2. General Discussion

[0144] This disclosure relates to assays comprising multiple independent measurements performed on the same assay apparatus using multiple detection regions to determine specific properties or characteristics exhibited by cells in response to perturbation elements used in the assay.

[0145] Such assays and assay devices are common in the prior art, but they often have limitations: they tend to provide only collective, population-level, and / or static information about changes in cellular contents (e.g., nucleic acids expressed after perturbation of cells following the assay). However, dynamic information about cellular behavior during the assay, static information at the level of individual / few / small (e.g., dozens) cells, and / or static information clearly correlated with the dynamic information of the corresponding single cell, or a direct link between the perturbation library and the cellular molecular response, are unavailable. This is because the previously existing correlation information about the detection region and perturbation factors is lost when the nucleic acids are sent for sequencing.

[0146] This disclosure provides methods and compositions for collecting static information as well as dynamic information occurring in real time, which, when combined, can provide a more complete picture of the substantial effects of perturbation elements on cells.

[0147] Perturbations that can alter cell function include not only compounds, but also one or more changes in pH, temperature, and different assay conditions (e.g., different buffers, nutrients, salts, hypoxia, assay duration, etc.). Furthermore, perturbation elements can be antibodies, cells (e.g., immune cells), or cell collectives whose interactions affect each other and the target cell.

[0148] This method and composition enable technicians to determine the precise detection area for the assay that generates the static information by correlating static and dynamic information captured by the capture beads described herein. Technicians can then correlate the dynamic information collected for that detection area with the static information generated during the assay. When combined, the static and dynamic changes in cell function provide a more complete analysis of the overall functional changes resulting from the perturbation. This is achieved by including on the capture beads: a unique detectable marker or set of detectable markers; a binding element; and a capture bead index reversibly associated with the capture bead, which both captures the perturbed component and, when used with a mapping assay device, retrospectively correlates the coded index to a specific detection area.

[0149] By coupling these information components, technicians can use the indexes reversibly bound to the capture beads to correlate the perturbation components captured by the beads. Subsequently, technicians can use the mapping on the assay apparatus to associate the capture bead with a specific detection region. This allows for the observation of dynamic changes in cell function (e.g., morphological changes) during the assay and attribution of these changes to a specific capture bead by referring to photographic or video recordings of the detection region taken during the assay. Combining all or some of this information using knowledge of which detection region was used, what perturbation element was used in that region, what capture bead was used in that region, and what perturbation components the capture bead recovered from that region provides substantial static evidence of functional changes. Furthermore, photographs or videos of the assay apparatus taken during the assay also provide information on the dynamic changes in cell function that occur during the assay.

[0150] In some implementations, if the perturbation element is a compound, the compound binds to the perturbation bead via a releasable connector. Additionally, perturbation oligonucleotides may also be releasably attached to the perturbation bead, identifying the compound bound to the bead. One or more capture beads included in the detection region capture the compound oligonucleotide as well as the perturbation component released from lysed cells, enabling a technician to determine the structure of the perturbation compound causing cell perturbation (based on static and real-time captured cells), and the static changes in cell function caused by that perturbation compound (e.g., the captured perturbation component).

[0151] Specifically, a measuring device comprising multiple detection regions is mapped to easily define each detection region. This mapping may include the X and Y coordinates of each detection region. Alternatively, each detection region in the device can be labeled with a unique image or code. However, this is usually unnecessary when mapping the X and Y coordinates is sufficient. For measuring devices where the X and Y coordinates are ambiguous due to a lack of uniformity among the detection regions, labeling each region eliminates this ambiguity. Furthermore, indicators or markings can be applied to the measuring device to orient it along its correct X and Y axis directions. See, for example, Figure 1A and 1B ; Furthermore, the method described herein allows for the use of multiple perturbations within the detection area. For example, multiple perturbations may include a first perturbation designed to generate a second perturbation, and the assay measures the effect of the second perturbation on the target cells. This can be illustrated by contacting and perturbing T cells with a perturbation compound (the first perturbation), and then having the T cells contact tumor cells to determine the effect of the perturbed T cells on the tumor cells.

[0152] In some implementations, when the capture element on the oligonucleotide index attached to the capture bead is an oligonucleotide (“capture oligonucleotide”), the capture oligonucleotide will capture nucleic acids from perturbed and lysed cells or perturbed oligonucleotides released from the perturbed bead.

[0153] In some embodiments, the capture bead index on the capture bead contains at least one capture oligonucleotide that captures the perturbation oligonucleotide. If the capture bead index and / or the compound oligonucleotide contains a second capture element or binding element, the perturbation component can also be captured after cell lysis.

[0154] In some embodiments, the capture bead CB comprises a capture bead index (e.g., oligonucleotide index Q in Formula I) and a first binding element (e.g., Q1 in Formula I) and an optional second binding element (e.g., X in Formula I), as shown in Formula I above.

[0155] By combining these previously independent information components, it is possible to link real-time information generated during the assay with the cells used in the detection area. This allows technicians to combine real-time and static information to provide crucial information about changes in cell function that occur during the assay.

[0156] 3. Perturbation element containing perturbation compound

[0157] In some embodiments, the perturbation element is a compound releasably bound to a perturbation bead. These perturbation compounds are synthesized on the perturbation bead using conventional split-pool synthetic methods well-known in the art, thereby generating a library of perturbation compounds. As shown in Reaction Scheme A below (for illustrative purposes only), the figure depicts a three-step synthetic route for preparing a combinatorial library of 1000 members. It should be noted that a four-step synthesis will provide 10,000 possible compounds in the combinatorial library, while a five-step synthesis will generate up to 100,000 compounds.

[0158]

[0159] Reaction scheme A

[0160] In step 1 of reaction scheme A, a plurality of beads (e.g., 1000) are divided into 10 containers, numbered 1-10 (e.g., approximately 100 beads per container). These beads contain cleavable linkers for reacting with the target component to form the compound to be prepared. Additionally, each bead has an oligonucleotide site for adding oligonucleotide chains that at least define a portion of the structure of the perturbation compound to be generated or the reaction steps for preparing that compound (the perturbation oligonucleotide—as described above). In some embodiments, the oligonucleotide site on the bead may contain up to 2000 nucleotides of poly-C, poly-G, or poly-T. This oligonucleotide site is located at the distal end of the linker, allowing the attachment of the following oligonucleotide chains.

[0161] If the capture bead captures a perturbation oligonucleotide that defines the structure of a compound or a reaction step, the oligonucleotide site binds to the bead via a releasable linker. In some embodiments, the releasable linkers for the perturbation compound and the perturbation oligonucleotide are identical, so that when the perturbation compound is released from the bead, the oligonucleotide is also released. The releasable linker can be cleaved by any means that would cause the specific linker to break, recombine, or otherwise release the attached compound / component (e.g., UV irradiation, pH, etc.). In some embodiments, the releasable linker for the perturbation oligonucleotide site is orthogonal to the releasable linker for the perturbation compound prepared on the bead. In some embodiments, the linker for the oligonucleotide binding site is not releasable under normal conditions, including the linker for cleaving the perturbation compound from the bead.

[0162] In Scheme A, the first component of the perturbation compound to be synthesized can be added to the beads using a high-yield chemical method (e.g., click chemistry). The reaction can be carried out under normal conditions and maintained until the reaction is considered complete. At this point, the beads contain 10 different building blocks (e.g., the beads in container 1 contain the first building block of container 1, the beads in container 2 contain the second building block of container 2, and so on, up to the beads in container 10 containing the tenth building block of container 10). Oligonucleotide chains can be introduced into each container before, during, or after the reaction to memorize the reaction performed in step 1 (e.g., the oligonucleotide chains are encoded by a unique sequence for each container, and each chain is linked to its preceding chain). The oligonucleotide chain with the unique sequence in each reaction container can then be added to the beads to memorize and recognize the reaction performed in that container.

[0163] In some embodiments, one or more first unique markers are added to the perturbation beads in each reaction vessel by conventional methods such as click chemistry, amide bond formation, electrostatic binding, or other suitable coupling chemistry methods familiar to trained chemists. Alternatively, the markers may be added according to the methods described in International Patent Application Serial Nos. PCT / US24 / 23594 (titled "Molecular Library Encoding System and Method," filed April 3, 2021) and PCT / US24 / 23594 (titled "Cell Transfer Components for Multiwell Plate Assay Apparatus," filed April 8, 2024). The entire contents of the foregoing applications and the applications claiming priority are incorporated herein by reference. Alternatively, if the perturbation beads are provided in subsets, where each subset contains beads with unique markers different from those in other subsets, then in the first step of synthesis, a single unique subset can be added to each vessel using approximately the same number of beads in each reaction vessel. Thus, each of the 10 reaction vessels contains a set of beads, where the beads in a given vessel have the same unique markers as the beads in other vessels. In some implementations, the oligonucleotide encoding the first step of the reaction in a given container (which is unique to that container) will also encode a unique tag used in that container.

[0164] In some embodiments, not all reaction steps in a fractional-cell synthesis require labeling. For example, in an eight-step synthesis, a unique set of labels may be added to the perturbation beads in step six. In this case, steps seven and eight do not require additional labeling and are therefore unnecessary. Thus, in this embodiment, the labeling process in the fractional-cell synthesis terminates once the unique label or set of labels has been added to the perturbation beads.

[0165] Typically, a unique oligonucleotide chain can be introduced into each reaction vessel before, during, or after each reaction step and attached to a precursor chain to remember the reaction that has occurred. In this step, a tag or set of tags is added to each bead in a given vessel, and an oligonucleotide chain is attached to the oligonucleotide precursor chain formed therein, encoding the tag and the reaction that has occurred or will occur in that vessel. Alternatively, as described above, individual oligonucleotide chains can be used to individually identify one or more added tags. In this case, it makes no difference whether the first unique oligonucleotide chain encoding the tag is added before or after the oligonucleotide encoding the reaction step.

[0166] Further in scheme A, the first component of the perturbation compound to be synthesized can be added to the beads using a high-yield chemical method (e.g., click chemistry). The reaction can be carried out under normal conditions and maintained until the reaction is considered complete. At this point, the beads contain 10 different building blocks (e.g., the beads in container 1 contain the first building block in container 1, the beads in container 2 contain the second building block in container 2, and so on, until the beads in container 10 contain the tenth building block in container 10).

[0167] In step 2, all beads in each reaction vessel are typically washed, then combined and mixed (e.g., shaken, stirred, etc.) to ensure homogeneity. The beads are then distributed to a second group of reaction vessels as described above (e.g., to make the number of beads in each vessel approximately equal). Each reaction vessel will receive a portion of the beads that were previously reacted in vessels 1-10. Thus, each reaction vessel will receive beads containing each of the 10 building blocks (e.g., beads containing the first building block, beads containing the second building block, etc.). The reaction vessels from step 1 can be used in step 2 (e.g., after washing the vessels and / or otherwise removing any reagents remaining from previous steps). The reaction on the beads in each vessel is memorized by adding another unique oligonucleotide chain to each reaction vessel. This oligonucleotide chain attaches to the previous oligonucleotide chain to form a single chain. The beads are then combined and mixed as described above. At this point, the beads contain up to 100 different building blocks (e.g., all combinations of the first 10 building blocks and the second 10 building blocks, provided that the first 10 building blocks and the second 10 building blocks are mutually exclusive). Step 2 can be repeated multiple times as needed to form a combined library.

[0168] In step 3 (e.g., the final synthesis step), the beads are separated again and added to different reaction vessels, as described above. The reaction in step 3 will generate 1000 compounds on the beads (e.g., if different components / reactions are carried out in each vessel in each step). The activity of the compound library generated on the beads can be tested using cells, for example in assays combining a single bead with at least one cell (e.g., up to 250 cells, up to 50 cells, about 20-30 cells, or even a single cell).

[0169] In some embodiments, the final step may utilize oligonucleotide chains to identify components added to the compound to be synthesized, thereby identifying the compound or reaction step used. In another embodiment, unlike steps 1-2 above, step 3 does not involve adding oligonucleotides to memorize the reaction. Instead, the beads in each reaction vessel are washed and (optionally) dried, and then placed in separate locations on the assay apparatus (i.e., beads from each vessel are transferred to independent locations on a single assay device, or to separate assay devices; wherein these independent locations or separate assay devices each contain an inspection area, such as a well or droplet, for performing the assay). Alternatively, the beads from each reaction vessel are placed in an independent assay device labeled with their originating reaction vessel. In this way, a technician can immediately identify the final step of the reaction used for those detection areas with abnormal results.

[0170] 4. Other disturbance elements

[0171] Other perturbation elements that can be used in the assays described herein include antibodies, immune cells, cancer cells, antigen-presenting cells (APCs), synthetic engineered cells, siRNA, peptides, viruses, bacteria, fungi, and other active agents; as well as changes in one or more of the assay conditions (including but not limited to buffer solutions, salts, pH, temperature, nutrients, oxygen levels, oxidants, physical stress, etc.). Each perturbation element can be used alone or in combination with one or more of these active agents (including perturbation compounds).

[0172] As for immune cells, adding them to the detection area can assess whether the cells can therapeutically perturb cells such as tumor cells. Furthermore, the detection area may also contain perturbation compounds to assess whether the compounds can perturb previously unresponsive or weakly responsive immune cells, thereby enabling them to exert a therapeutic effect on tumor cells. Moreover, immune cells can be used alone or in combination with perturbation compounds to assess whether such combination weakens or enhances the activity of the cells.

[0173] For antibodies, adding a weakly reactive antibody to the detection region containing tumor cells allows for the introduction of a perturbation compound that disrupts tumor cells. Subsequently, it can be assessed whether the perturbation induced in the tumor cells enhances the therapeutic activity of the antibody.

[0174] As for engineered cells, they may comprise cells engineered to secrete membrane-associated activators capable of interacting with test cells in the detection region. In some embodiments, engineered cells may be engineered to secrete synthetic proteins, cytokines, or antibodies. In some embodiments, engineered cells may be engineered to produce unique small molecules or metabolites. Engineered cells are fundamental to biotechnology, and various methods are well known to those skilled in the art of biotechnology and / or synthetic biology.

[0175] As for other perturbation elements, perturbing cells under specific conditions can provide valuable information to determine whether such perturbation is beneficial or harmful to the cells. If the conditions are harmful, then therapeutic agents that can induce similar perturbations in diseased cells are suitable candidates for inducing similar changes in vivo.

[0176] As mentioned above, the perturbation effect of such a combination can be determined using a combination of perturbation elements and compared with the case of individual perturbation elements applied separately.

[0177] A key feature of perturbation beads is their ability to recognize associated perturbation elements via perturbation oligonucleotides. Furthermore, or in other words, when a perturbation bead includes an optically detectable marker that uniquely identifies the perturbation bead and / or its position within the detection area of ​​the measuring device, the perturbation bead may optionally contain a labeled oligonucleotide; this labeled oligonucleotide may be part of or in combination with the perturbation oligonucleotide, such that the oligonucleotide is released from the perturbation bead and captured by the trapping bead (e.g., via trapping elements X, Q). 1 After capture (e.g., A), sequencing can be performed to identify the unique markers and / or sets of markers on the perturbation beads and / or the perturbation elements they provide. In turn, this allows the unique markers or sets of markers to be associated with the detection region via a reference registry.

[0178] 5. Capture Beads

[0179] As described herein, capture beads are used to capture perturbation components and perturbation oligonucleotides produced by disturbed cells during assays. Furthermore, each capture bead contains a unique marker or set of markers encoded onto the bead using unique oligonucleotides (“capture bead index”). To capture perturbation components and perturbation oligonucleotides, one or more capture elements are attached to the capture bead. These capture elements can capture perturbation components such as mRNA, DNA, nucleic acids, cytokines, chemokines, enzymes, proteins, hormones, and more. This diverse selection of perturbation components allows for the assessment not only of changes produced by perturbed cells compared to undisturbed cells but also of whether these changes are beneficial. In conclusion, by assessing as many components as possible, technicians can more comprehensively evaluate perturbation-induced changes in cellular function.

[0180] In some embodiments, the trapping beads contain multiple acid copies of the same oligonucleotide, which encodes a unique tag or set of tags on the bead. Such trapping beads can be prepared as shown in reaction scheme B:

[0181] Reaction scheme B

[0182] For example, in a six-step process using a similar pooling method for labeling beads, 10 reaction vessels and 100,000 beads are used, where each bead contains a precursor oligonucleotide binding site, which may contain up to 2,000 members of poly-C, poly-G, or poly-T. This oligonucleotide site is located at the distal end of the linker, allowing the following oligonucleotide chains to be attached to it.

[0183] In the first step of this scheme, the beads are divided into approximately 10,000 of each, roughly equal numbers. These beads are placed into individual reaction vessels labeled 1 to 10, with each vessel using a different label to mark the beads. In some embodiments, one or more first unique labels are added to the perturbed beads in each reaction vessel by click chemistry and / or any method (including the conventional methods described in International Publication No. WO 2021 / 042011, the entire contents of which are incorporated herein by reference).

[0184] Before step 2, the beads are homogenized and then evenly distributed into 10 reaction containers, each containing approximately 10,000 beads. Given that there are 10 containers and the bead set is completely homogenized, the probability that beads from the same reaction container in step 1 will enter the same reaction container in step 2 is 10%; in other words, in each second reaction container, it is expected that approximately 1,000 beads will also be found in the same first reaction container. Using the same 10% theoretical reduction factor to assess the probability of repetition in steps 3-6, it can be inferred that step 3 will reduce the total to 1,000 (for all 10 containers), step 4 will reduce the total to 100 (i.e., 1 / 1,000), step 5 will reduce the total to 10 (i.e., 1 / 10,000), and step 6 will reduce the number to 1 (i.e., 1 / 100,000). Furthermore, at each step of the labeling process, a unique oligonucleotide chain that exists only in the same reaction vessel can be attached to the existing oligonucleotide chain on the bead; thus, after labeling is completed, each unique set of labels on the bead can be identified by similar unique oligonucleotides.

[0185] After step 6, there will be 10 groups of beads, approximately 10,000 in each group, each bead bearing the same mark used to index a specific set of marks on a given bead. At this point, these beads can be merged and attached to a common capture element. These beads can then be represented by equations I and IA above.

[0186] Furthermore, if, during the assay, the perturbation oligonucleotide (PO) is also released when the perturbation element is released into the detection region, the perturbation oligonucleotide can also be captured by the capture element of the capture index on the capture bead, as shown in Equation II below: AXQQ 1 -PC (II) Among them, A, PC, Q, Q 1 The definitions of X and X are as above.

[0187] In some embodiments, the capture bead is present throughout the entire detection process. If so, at least one capture element on the capture bead is selected to capture the perturbed oligonucleotide. In some embodiments, one of the capture elements (X or Q) 1 This includes functions complementary to those found on the perturbation oligonucleotide. In some embodiments, the perturbation oligonucleotide also has a secondary function complementary to that on one or more perturbation components (e.g., perturbation nucleic acids) generated during the assay. More information is available to those skilled in the art regarding the generated perturbation components and the combinations of perturbation compounds that result in these components.

[0188] When the perturbation component is mRNA, the capture element can be a poly-T; for cytokines and chemokines, binding agents specific to these molecules (such as antibodies or aptamers) can be attached to the capture beads. Similarly, enzymes may also be captured by substances such as their substrates. It is important to note that the target range that the capture beads can capture is limited only by the recognition of target-specific binding elements. In some embodiments, binding elements can be constructed targeting specific perturbation components. For example, monoclonal antibodies can be generated using conventional methods and then bound to specific perturbation components generated during the assay.

[0189] In some implementations, multiple capture beads can be used within a single detection region. Each capture bead is used to capture different perturbation components from cells within the detection region. For illustrative purposes only, a first capture bead binds multiple copies of the same capture element targeting mRNA. A second capture bead binds multiple copies of the same capture element targeting a specific cytokine. Other capture beads can be used to bind other perturbation components. Each capture bead binds an oligonucleotide index unique to that bead. In some implementations, this capture index contains more than one capture element, thus allowing simultaneous binding of two different targets. In some implementations, multiple capture beads can capture the same perturbation component, and additional capture elements can improve capture efficiency.

[0190] For “W”, optically detectable markers include one or more colors, inducing colors, the same color in different intensities, text, numbers, images, logos, barcodes, quantum dots, light-emitting diodes (LEDs), QR codes, etc. A sufficient number of markers (used individually or in combination) are used to uniquely identify the capture bead, distinguishing it from other capture beads used in the assay. This means that each capture bead used in the assay will be uniquely identified. In some embodiments, a single optically detectable marker, such as a barcode or QR code, may be used. In another embodiment, the capture bead may contain multiple markers that, when combined, provide a unique code or image. Such embodiments include mixtures of colors, text, and / or numbers, etc. In some embodiments, “r” is an integer from 1 to 100 or from 1 to 10. In some embodiments, the capture bead is not marked (r=0). Instead, the perturbation bead and / or detection area may contain an optically detectable marker or set of markers, and a marked oligonucleotide encoding that marker or set of markers, which can be captured by the capture bead with the perturbation oligonucleotide and / or perturbation component.

[0191] As for n (as described above), it represents multiple copies of the component bound to the capture bead capture index. As used herein, the terms "multiple copies" or "multiple" when used to describe bead-bound components refer to a group of 2 or more, 10 or more, 100 or more, and up to approximately 1 x 10^6. 5 6.02 x 10 moles or 6.02 x 10 17 Individual molecules. The specific number is not important, as long as there is a sufficient amount of bead-binding material to be measured and a sufficient amount of perturbation component to be recovered to assess the cellular perturbation induced during the assay. In some embodiments, the attachment number ranges from approximately 1 x 10⁻⁶. 5 Up to 6.02 x 10 17 .

[0192] The specific type of capture beads used in this article is not critical, as long as these beads can be marked and modified, including -LQQ as needed. 1 or -LXQQ 1 The group name is acceptable. In some embodiments, the trapping beads can be polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic materials, ceramics, plastics, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination of the above materials. The specific material used for the trapping beads only needs to meet the following condition: the material is inert to the determination performed (including the solvents and reagents used).

[0193] In some implementations, the size of the capture bead corresponds to a percentage of the detection area size, with a larger detection area accommodating a larger capture bead. Typically, the size of the capture bead ranges from about 5% to about 50% of the detection area size (e.g., by volume). In terms of absolute size, the size of the capture bead along its longest axis is preferably from about 10 micrometers to about 250 micrometers, more preferably from about 20 micrometers to about 200 micrometers, and even more preferably from about 30 micrometers to about 180 micrometers.

[0194] In some implementations, the capture beads are added after the assay, wherein each capture element is releasable and binds to the capture beads. By adding capture beads after the assay, any cleavable bonds used with the captured components can be the same as those used during the assay. Once the capture beads are recovered, the captured components bound to the beads are released, and the assay can then be performed using conventional methods.

[0195] Furthermore, alternatively, the capture bead may optionally incorporate multiple capture elements, such as biotin or avidin / streptavidin groups, via a connector comprising a cleavable linker. For example, a perturbation oligonucleotide may be terminally linked to an oligonucleotide chain having a biotin or avidin / streptavidin group optionally linked via a linker. The purpose of selecting each group is such that if a biotin group is present on the compound oligonucleotide or the capture bead, an avidin / streptavidin group is also present on the capture bead or the compound oligonucleotide, thereby making them functionally complementary.

[0196] In some alternative embodiments, it is not necessary to use oligonucleotides to encode the perturbation element (e.g., unperturbation oligonucleotides), nor is it necessary to use oligonucleotides in the capture bead index. Instead, compounds with known molecular weights and / or some other known / detectable characteristics can be used as indexes on the capture beads. The molecular weight of such a compound is sufficient to identify the capture bead and is unique to compounds used as indexes on other capture beads. In some embodiments, the compound index is attached to the capture bead via a releasable connector. After the index compound is released, it can be analyzed by mass spectrometry. In some embodiments, each index compound on different capture beads can bind to the same substance, thereby increasing its molecular weight. For example, the index compound can be attached with a biotin group. After release, a compound with an avidin group can bind to each released compound index. The resulting conjugate has increased mass compared to the index compound, thus making it easier to obtain a mass spectrum of the conjugate. In some embodiments, the index compound can be attached to an oligonucleotide using a dendritic polymer to increase its molecular weight.

[0197] 6. Real-time analysis

[0198] As described above, this disclosure enables a person skilled in the art to associate a perturbed component generated by a perturbed cell with a specific capture bead that captures the perturbed component, and then, by referring to a mapping on a mapping assay device, to trace back to the detection area where the perturbed cell was located. In turn, by generating a record covering each cell in each detection area during the assay period, the record can be accessed and the visible cellular changes caused by the perturbed can be evaluated.

[0199] These cellular changes include variations in morphology, motility, size, shape, adhesion, division, apoptosis induction, gene expression (such as the expression of fluorescent proteins or fluorescently labeled proteins), intracellular organization / structure (such as cytoskeleton organization / structure, organelle structure), extracellular expression, and other cellular structural / behavioral changes. These changes are considered "real-time" because they are observed during the assay and can be recorded through photographs, video recordings, three-dimensional cellular analysis, and other methods.

[0200] Technicians can combine real-time functional changes with static changes to provide an overview of significant improvements in cellular function.

[0201] 7. system

[0202] In some embodiments, a system for providing information about measurements is provided, the system comprising: a) A mapping measurement device comprising multiple individually identifiable detection areas; b) A perturbation bead comprising multiple copies of a perturbation compound releasably bound thereto and a perturbation oligonucleotide encoding the compound, said compound being unique to other perturbation beads used in the assay device; c) Capture beads as described herein; and d) The registry that associates each capture bead with a specific inspection region.

[0203] The system described herein may also include one or more additional capture beads, one or more cells, or a combination of synergistic perturbation elements (a first perturbation element perturbs a second perturbation element, which then perturbs the target cell). In some embodiments, sequencing of the oligonucleotides bound to the capture beads can be performed on the beads themselves. In another embodiment, sequencing is performed after releasing the oligonucleotides by cleaving the releasable bonds.

[0204] 8. Measurement Procedure

[0205] The assays described herein are performed in a mapping assay apparatus having multiple detection regions. For illustrative purposes only, the assays described herein employ perturbation beads having multiple copies of the same perturbation compound releasably bound thereto, and an oligonucleotide that memorizes all or part of the reaction steps for preparing such compounds on multiple beads. Again, for illustrative purposes only, the oligonucleotide has a poly-T component attached to its proximal end (relative to the releasable linker), enabling it to be captured by a poly-A capturing element on the capturing bead. Alternatively, the oligonucleotide may have a first binding element at its proximal end and a second binding element at its distal end. For example, a poly-C compound may be attached proximally and a poly-T component at the distal end. When the releasable bond is cleaved, the poly-C can hybridize with a poly-G group capturing element attached to the capturing index. In turn, the poly-T group can bind to mRNA upon cell lysis.

[0206] In some embodiments, all or part of the detection area in the assay device includes the perturbing beads as described above; at least one cell to be perturbed (e.g., up to 250 cells, up to 50 cells, about 20-30 cells, or even a single cell); a capture bead as described herein, comprising multiple copies of a capture bead index with a capture element attached thereto; and an aqueous solution suitable for performing the assay. As described above, the capture bead index and at least one capture element are attached to each other and to the capture bead, wherein each attachment may optionally be achieved via a connector.

[0207] The identifier of each perturbation bead and the capture bead itself in each detection area is recorded by identifying the mark on each capture bead and / or perturbation bead and mapping that mark to the detection area mapped by the measuring device. This establishes a 1:1 association between each detection area and each perturbation bead / capture bead maintained therein, which is achieved simply by associating the unique mark or set of marks on the capture bead with the mapped detection area.

[0208] The assay is initiated by releasing at least a portion of the perturbation compound bound to the perturbation beads into an aqueous solution, in an amount sufficient to perturb the cells. In some embodiments, a release mechanism (e.g., ultraviolet light) also releases the compound oligonucleotide from the perturbation beads, as well as any releasable bonds on the capture beads that respond to the same release stimulus. In some embodiments, the perturbation oligonucleotide has a complementary function that can be captured by a capture element on the capture index. For example, complementary functions could be poly-A and poly-T, poly-G and poly-C, avidin / streptavidin and biotin, etc. When the compound oligonucleotide is released, it is captured by the complementary capture element on the capture index. The assay is then performed for a sufficient duration to induce one or more cell perturbations. In some embodiments, the cell response to the perturbation can be photographed or videotaped. After the assay is complete, the cells are lysed to release the cell contents.

[0209] In some implementations, the perturbation oligonucleotide also includes a functional group complementary to the nucleic acid released from the lysed cell. For example, the capture bead may have formula IA: (W) r -CB-[L´-XQQ 1 ] n (IA) As stated above.

[0210] Upon release, the released nucleic acids (e.g., mRNA of released cellular components and / or oligonucleotides released from the perturbation beads, such as perturbation oligonucleotides and / or labeled oligonucleotides with optically detectable tags on the perturbation beads) are captured by complementary capture elements on the capture beads. A capture index on the capture beads contains the captured perturbation oligonucleotide, which encodes the perturbation element. In some embodiments, the perturbation oligonucleotide contains a capture element that captures the perturbation component released from the lysed cells. In some embodiments, the capture beads are separated from the detection region, washed, and prepared for sequencing. In some embodiments, the releasable bonds to which the capture index is attached to the capture beads are cleaved, thereby releasing the capture index and the attached captured perturbation oligonucleotide, as well as the captured nucleic acid, wherein each capture index can be sequenced, the sequence identifying the capture bead, the perturbation compound, and the nucleic acid recovered from the lysed cells. In some embodiments, X is a capture element capable of capturing the same or different perturbation components released from the lysed cells. Conversely, by referencing the registry of a specific capture bead in a specific detection area, the identifier of the marker on the capture bead index can be directly associated with the detection area from which the bead originated.

[0211] In some implementations, some or all of the capture beads are merged, and the cleavable adapters on each bead are cleaved, thereby releasing a variety of different oligonucleotides. Furthermore, the oligonucleotides on the capture beads can also be sequenced without being released from them—using polymerase to replicate the oligonucleotides attached to the capture beads into complementary strands, followed by sequencing. Since each oligonucleotide encodes a perturbation compound and one or more markers on the capture bead (and / or the perturbation bead, or other locations in the detection region), each sequenced oligonucleotide can be associated with a specific capture bead, and consequently, with the detection region. Therefore, each oligonucleotide to be sequenced is self-identified.

[0212] Once the capture bead identifier is associated with the detection area, the functional changes of cells in that detection area can be directly linked to static changes demonstrated by sequencing of the lysed cell contents.

[0213] In some embodiments, the attachment order of the nucleic acid and the perturbation oligonucleotide is reversed, such that the nucleic acid released from the lysed cell is first captured by the capture element on the capture index. In this embodiment, the perturbation oligonucleotide binds to a perturbation bead via a releasable group orthogonal to the releasable group on which the perturbation compound is attached. When X is a capture element complementary to the capture element on the perturbation oligonucleotide, the perturbation oligonucleotide (poly-T) is released and captured by X after the nucleic acid is captured. Subsequently, the perturbation oligonucleotide is released by cleaving the releasable bond, thereby enabling it to be captured by the modified nucleic acid.

[0214] In some implementations, recording or memorization is done via digital photography or digital video recording. The digital nature of such recordings makes it possible to quickly evaluate the color tags used to uniquely identify specific captured beads.

[0215] As mentioned earlier, capture beads can be added to the detection area before, during, or after the measurement. However, it is preferable to add capture beads to the detection area before the measurement begins, as this makes it easier to record the detection area address onto a specific capture bead and at least avoids measurement fragmentation during recording.

[0216] 9. Example

[0217] The following examples illustrate the use of the capturing beads described herein. In this embodiment, the following terms used have the following meanings: bp = base pairs poly-A = polyadenine poly-T = polythymine mRNA = messenger RNA UV = Ultraviolet Example 1 - Measuring Apparatus A mapping measurement device is employed, comprising multiple detection regions (e.g., micropores, nanopores, droplets, etc., as described herein). Each detection region is arranged in rows and columns, with the number of columns exceeding the number of rows. To determine the orientation of the measurement device, a mark, symbol, or other distinguishing feature may be added to one corner of the device to indicate the "up" position and / or standard orientation. Figure 1A The arrangement of detection areas in a portion of the mapping measurement device is shown. Specifically, for example, a mark 12 is located at one corner of the mapping measurement device 10, which guides the user to identify the upper left corner of the device 10. Any mark, indentation, or other feature can be used, as long as it enables the orientation of the device, so that the individual detection areas can be identified by columns 14, 16 and rows 18, 20. Figure 1B Another orientation of the device 10 is shown, in which the groove 22 distinguishes one corner of the measuring device 10 from the other corners. In any case, a mapping measuring device is provided, in which each detection area is identified by columns (e.g., 14, 16) and rows (e.g., 20, 18). Any other distribution of detection areas can be used as long as the mapping can be clearly defined.

[0218] Example 2 - Cassette of Disturbed Beads

[0219] Step 1. A population of 100,000 beads, available from Rapp Polymere GmbH in Tübingen, Germany (Ernst-Simon-Strasse 9, 72072 Tuebingen), was used. These beads had multiple copies of the oligonucleotide binding site and multiple copies of the compound synthesis site bound thereto. The beads were distributed approximately evenly into 10 different reaction vessels. A unique first step of compound synthesis was performed in each vessel, ensuring that the building blocks generated in each vessel differed from those generated in other vessels. Before, during, or after the reaction, a unique first oligonucleotide chain for each reaction vessel was added to the oligonucleotide binding site, ensuring that all beads in the same reaction vessel had the same first oligonucleotide chain, distinct from the oligonucleotide chains in other vessels. After the first step of compound synthesis was completed, the beads were washed, then assembled and homogenized.

[0220] Step 2. Repeat the steps of Step 1, but in each of the 10 reaction vessels, synthesize the compound using a unique second building block and a unique second oligonucleotide chain for each reaction vessel. After this step of compound synthesis is complete, wash the beads, then assemble and homogenize.

[0221] Steps 3-5. Repeat the steps of step 2, but using unique third, fourth, and fifth compound building blocks and unique third, fourth, and fifth oligonucleotide chains. After completing step 5, 100,000 different compounds were prepared on 100,000 different beads.

[0222] A single perturbation bead is then added to a single detection area of ​​the measuring device. This single perturbation bead can be represented as shown in Equation III: (III) In this context, the circle containing PB represents a perturbation bead, PE is a perturbation element, and L... 3 and L 4 It is an independent release bond (cleavable bond), PO is a perturbed oligonucleotide, and V is an optional optically detectable tag (e.g., in the case where the capture bead does not have such a tag and / or r=0 in Formula I), and s , t and u These are independent integers, representing the number of each group attached to the perturbation bead. In some implementations, s and u The value range of is the same as the value range of n mentioned above. In some implementations, t The range of values ​​for can be the same as the range of values ​​for r described in this article.

[0223] Example 3 - Capturing Beads

[0224] Each capture bead can be made from any starting material suitable for cell assays (e.g., any material that is cell-inert and capable of attaching or embedding, for example, the group / label shown in Formula I), as described herein. For example, each capture bead can have the structure shown in Formula I. In some embodiments, each capture bead binds to or embeds a set of labels, including, for example, different colors, the same color with different intensities, fluorescent particles that produce one or more colors, microchips, etc., or combinations thereof. A cleavable adapter is attached to the bead or is already present on the provided bead. In some embodiments, a leader group for forming a capture bead index on the bead is attached to the adapter. The leader group can be designed to be complementary to the capture element on the capture bead index. The capture bead index uniquely encodes the label on the bead and is attached to the capture bead using methods known in the art. As mentioned above, each capture bead index is unique for capture beads in a given detection region. Furthermore, although multiple copies of the index exist on the bead, no other oligonucleotides with different sequences encode the label or combination of labels on the bead. In this embodiment, the resulting capture bead can be represented by the following structure shown in Formula IV: (IV) Here, CB represents the capture bead, which has multiple capture elements (Q). 1 These capturing elements are attached to the capturing bead index Q, which in turn is connected via a cuttable connector L. 5 (This can be any cutable / releasable connector described herein) attached to the bead. L 5 It may also include X as described herein, thus obtaining Formula I. W represents one or more markers used to uniquely identify the capture beads to be used in the measuring device from other capture beads. r and n The definitions are as described in this article.

[0225] In other embodiments, a second capture element may be included on the oligonucleotide index, as shown in Formula V: (V) Among them, CB, L 5 Q and Q 1 The definition is as described above. In equations IV and VI, for n capture groups, each Q 1 Can be used with other Q 1 They can be the same, or they can be different.

[0226] Example 4 - Marking of capture beads and / or disturbance beads

[0227] Figure 2A–2D illustration shows different optically detectable tags placed on the capture bead, which uniquely distinguish the bead from other capture beads. (Similar tags can also be added to perturbation beads, as described herein). Specifically, a unique combination of fluorescent dyes and / or their signal intensity can be covalently attached to the capture bead, allowing the generated fluorescent signal to specifically associate with that bead. Figure 2A In this method, the intensity of the first Y can be one-third that of the second Y, thus distinguishing the two Y values. Many quantum dots are commercially available (e.g., Thermo Fisher products) for this purpose. The number of quantum dots required per capture bead depends on the number of detection areas to be used in the measuring device. For example, ten uniquely identifiable quantum dots can provide over 3,000,000 unique combinations and / or signals. Figure 2A-2D In this diagram, B represents blue, G represents green, O represents orange, R represents red, and Y represents yellow. In each case, using only these five colors provides 120 unique combinations; however, adding five more colors as described above significantly increases the number of combinations.

[0228] Figure 3 An example is shown illustrating how to... Figure 2A-2D The marked beads are placed in such a way as Figure 1B The various independent detection areas of the measuring device 10 shown.

[0229] Alternatively, the perturbation beads and / or capture beads may be impregnated with dyes and / or bound dyes (e.g., covalently bound) such that each perturbation bead and / or capture bead has a distinguishable color or fluorescence-generating color as a marker to distinguish it from other perturbation beads. Figure 4 A measuring device is shown, in which a bead is placed in each well, wherein each well contains optically distinguishable beads of different colors (not visible in grayscale images) and intensities. In short, these beads are TENTAGEL™ beads, made by suspending one of the following substances: 1. 14 mg S30902 TENTAGEL™ resin, 10 μL QD600-WS quantum dots 2. 11 mg M30202 resin, 10 μL QD540-WS quantum dots, or 3. 11 mg M30352 resin, 10 μL QD680-WS quantum dots The quantum dots (from NanoOptical Materials—CuInZnS / ZnS core-shell quantum dots with central emission peaks at 600 nm, 540 nm, and 680 nm, respectively) were placed in 400 μL of water and equilibrated for 20 minutes in 1.5 mL Eppendorf tubes. Then, 4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) dissolved in 100 μL of water was added to each tube, and the reaction was carried out with intermittent shaking at room temperature for 16 hours. This allows the TENTAGEL™ beads embedded with quantum dots (QDs) to emit light of different colors and / or intensities, thus uniquely distinguishing each bead from others by its color and / or intensity.

[0230] Digital cameras record color and color intensity in the form of pixels. Therefore, two points of the same color but different intensities can be easily distinguished by using pixels to correlate the intensity differences. This can be easily achieved by analyzing pixels using a computer.

[0231] Alternatively, the perturbed beads and / or trapping beads may be tagged with one or more micro-components, which either have detectable codes (e.g., Figure 5A -B), or form and / or constitute detectable code (e.g., Figure 5D This allows for the unique identification of the corresponding beads.

[0232] Figure 5A A schematic diagram illustrates the use of micro-components (in this case, silicon micropucks) labeled with beads (in this case, 90-micron TENTAGEL™ microbeads, which can be used as perturbation or trapping beads), the micro-components bearing visually identifiable codes. Under ambient conditions, a layer of silicon oxide is deposited on the silicon micropuck, thereby forming acidic silanols (SiOH) on the surface of the micropuck. The silanols react rapidly with the amine groups on the TENTAGEL™ beads (e.g., 90-micron TENTAGEL™ beads) to form stable electrostatic interactions that remain stable under various solvents and reaction conditions. Figure 5B Three beads stably attached to coded silicon microdisks are shown. This was done to test the stability of the attachment between the microcomponents and the beads (i.e., as shown in the diagram). Figure 5A Formed as shown Figure 5B In the process, beads with attached micro-components were incubated in various chemicals / solvents for 24 hours. The bead preparation was as follows: Figure 5A As shown, the beads were incubated for 24 hours in 10% aqueous acetic acid, dimethylformamide, 10% aqueous ammonium hydroxide, dimethyl sulfoxide, phosphate buffer, methanol, water, or isopropanol, respectively. Under all conditions, the microcomponents maintained their attachment to the beads, indicating that these beads are suitable for the assay method described herein.

[0233] Because the bead (and the hole it contains) are transparent, they can be imaged using a bright-field microscope regardless of orientation. Aside from the size of the micro-components and the manufacturing process, there are no other particular limitations to this visually discernible code. Figure 5C An example of a four-quadrant code is shown, where each quadrant can contain different information, which may be related to the experiment and / or the bead or its conditions.

[0234] Figure 5D An example of a microbead with one or more optically detectable microchips attached to each detection area (well) in a porous measuring device is shown. Figure 5D An example is shown where the set of optically detectable microchips on each bead and their specific distribution are unique to that bead. Figure 5D The optically detectable microchips shown have various shapes and sizes and can be randomly attached to the beads, forming a distinguishable random distribution of microchips of different sizes and shapes attached to the beads. Both the beads and the holes are transparent, thus allowing imaging of the microchips on the beads using bright-field microscopy or other standard imaging techniques.

[0235] For preparation Figure 5D The beads were first prepared by resuspending microfabricated silica microchips in isopropanol at a concentration of 250,000 per milliliter. In a 15 mL Falcon centrifuge tube, 2 mL of the microchip suspension was gently mixed with 3.5 mg (approximately 168,000) of NH₂-Tentagel agitated / captured beads with a particle size of 30 μm, and the mixture was incubated with shaking for 12 hours. The tube was then centrifuged at 20,000 relative centrifugal force (rcf) for 2 minutes, and the supernatant was discarded. 2 mL of fresh microchip suspension was added to the beads, and the incubation and centrifugation steps were repeated. After the second centrifugation, the beads with randomly attached microchips were resuspended in pure isopropanol. The resulting optically detectable microchip-bead complex was stored in + Under C, the material is further loaded into the detection area. Once inside the detection area, due to the transparency of the beads, regardless of their orientation, the beads and the specific distribution of different microchips that uniquely identify these beads can be imaged using a bright-field microscope.

[0236] Figures 7A-7C Examples of adding markers (e.g., microchips) and corresponding compounds and / or compound building blocks to beads are shown (e.g., for preparing perturbed beads, for preparing capture beads containing perturbed elements, etc.). Figures 7A-7B The beads shown are manufactured so that optically detectable microchips of different shapes are randomly attached to them. Figure 7AWide-angle (left) and close-up (right) images of these beads in a reaction vessel (one well of a 96-well plate) are shown. Approximately 3.5 mg (~168,000) of 30 μm NH2-TENTAGEL™ trapping beads were added to each well of the 96-well plate. To prepare the resin containing the photolytically degradable beads, 10 mg of TENTAGEL™ M NH2 (10 µm in diameter, dry; 0.23 mmol / g) was loaded into a centrifuge column and swollen in DMF (room temperature, 1 h). The resin was then reacted with 4-(Fmoc-aminomethyl)-3-nitrobenzoic acid (20 mM, 3.6 equivalences) and N,N'-diisopropylcarbodiimide (DIC) (20 mM, 3.6 equivalences) (70 °C, 3 h, repeated twice) and washed with dimethylformamide (DMF) (3 × 0.6 mL). Remove the Fmoc-protected resin with a 20% piperidine DMF solution (15 minutes, room temperature) and wash with dimethylacetamide (DMA).

[0237] To prepare the microchip-bead conjugate, microfabricated silica microchips of a specific shape were first resuspended in isopropanol at a concentration of ~250,000 units / mL. A suspension of the uniquely shaped microchip was prepared in each test tube. Then, 2 mL of the microchip suspension was added to each well of a 96-well plate and gently shaken for 2 hours to mix with the beads. The plate was centrifuged at 20,000 rcf for 2 minutes, and the supernatant was discarded. This process was repeated twice. The culture medium was replaced with dimethylacetamide (DMA). The previously added one or more unique microcomponents optically encode building block information.

[0238] For peptide conjugation used to assemble building blocks, standard conjugation conditions and washing procedures were employed; for example, conjugation was performed using DIC (N,N'-diisopropylcarbodiimide), with the building block suspended in DMF (N,N-dimethylformamide). Detailed experimental protocols for the conjugation reaction can be found in the supplemental information section of the paper https: / / doi.org / 10.1021 / ac500693r. An example building block used is tetramethylrhodamine (TRITC) (… Figures 7B-7C); Dasatinib metabolite m6, namely dasatinib carboxylic acid – 2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)acetic acid; JQ1 acid – ((S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl)tert-butyl acetate); HPK1 inhibitor derivative – 4-{[2-fluoro-6-(trifluoromethyl)phenyl]amino}-2-[(6-methoxy-2-methyl-1,2,3,4-tetrahydro-7-isoquinolinyl)amino]-5-pyrimidin-5-carboxylic acid; and LNI, lenalidomide – 2 -(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-carboxylic acid ( Figure 7D ).

[0239] Figure 7B Bright-field and fluorescence images of beads formed as described above are shown, where triangular or circular microchips serve as optically detectable markers, and tetramethylrhodamine (TRITC) is added to the marked beads as an example building block. Figure 7C As shown. Figure 7B It demonstrates sequential labeling of beads with optically detectable markers, and then adding compounds as example building blocks without losing the optically detectable markers.

[0240] Figure 7D The results of adding beads prepared as described above, bearing optically distinguishable markers corresponding to one of four building blocks / compounds (dasatinib, JQ1, HPK, and LNI), are presented. Individual beads were added to the wells of a multi-well assay apparatus. Genetically engineered Jurkat cells (with their enhanced green fluorescent protein (eGFP) reporter fused to the activated T cell nuclear factor (NFAT) transcription factor) were added to each well (cells purchased from BPSBioscience, catalog number #78384). Irradiation with light at a wavelength of 365 nm and a power of 60 mW / cm² for 10 seconds released the compounds from the beads, allowing them to interact with the cells in the corresponding wells. Cells exposed to JQ1, HPK, and LNI (identified by the corresponding optically detectable markers on the perturbed beads imaged in each cell) showed minimal changes in NFAT-eGFP expression levels, while eGFP expression in cells exposed to dasatinib was almost completely downregulated. This example demonstrates the use of optically detectable markers on beads to encode interfering compounds and / or building blocks, and maps this visible code to cellular interference.

[0241] Example 5 - Preparation for Measurement

[0242] Select cells (e.g., human cells) as the cells to be perturbed in the assay. Combine the cells, perturbing beads, and trapping beads into a mapping assay device (e.g., as shown in the image). Figure 1A In a single detection area (or as shown in 1B), the detection area contains a suitable aqueous composition for performing the determination. A combination of the following components is provided in a single detection well, as shown in Reaction VI:

[0243] (VI)

[0244] Among them, L 3 L 4 ,L,PB,CB,PE,PO,V,W,X,Q,Q 1 , s , t , u , r and n As described in this article, x, y, and z each independently represent the number of beads in their respective categories. In one example, x, y, and z are each independently selected from 1 to 10. Furthermore, ellipses labeled "cell" inside represent undisturbed cells. The perturbed beads, captured beads, and cells correspond to their respective detection areas and to each other.

[0245] After these components are incorporated into a detection area of ​​the mapping apparatus (which also has other similarly filled detection areas), a registry is established at any appropriate time during the measurement process, preferably before the measurement begins, by capturing digital images of the apparatus including each detection area; this registry is preferably in electronic form, recording the uniquely marked capture beads within each detection area. In some embodiments, for example, confocal microscopy is used to capture images at a given depth and / or multiple depths and / or from multiple angles. In some embodiments, the measuring apparatus is transparent, allowing imaging from all angles.

[0246] The assay is initiated by releasing at least a portion of the perturbing compound from the perturbing bead into the assay solution, enabling the perturbing compound to interact with the cells. When the oligonucleotide compound is attached to the perturbing bead via a releasable bond (for illustration, this bond is the same as the releasable bond that attaches the perturbing compound to the perturbing bead), applying an appropriate stimulus (e.g., ultraviolet light) will cause at least a portion of the perturbing compound and the oligonucleotide compound to be released into the assay solution.

[0247] Example 6 - Measurement

[0248] Each assay performed in the mapping assay apparatus is incubated under standard conditions to allow the released compounds to disturb the cells. During the assay, photographs of each assay well are taken periodically, or the entire assay process is recorded via video / digital recording. These photographs or video / digital recordings can confirm changes in cell function or morphology, such as alterations in cell shape, as well as other possible properties (i.e., dynamic information) discussed elsewhere in this document. Possible changes in cell morphology are represented by Formula VII:

[0249] (VII)

[0250] The cells shown here, initially (for example only) elliptical in shape, have been distorted into other shapes, exhibiting features such as increased bubbling and / or spreading; this is likely caused by perturbations induced by released perturbing compounds. These changes, captured during the assay, are thought to reflect dynamic functional changes in the perturbed cells and can be recorded in real time, for example, through video and / or image acquisition.

[0251] After the assay, cells are lysed in the presence of the capture beads (dotted cells in reaction protocol C below). These capture beads capture perturbation components (PCs) generated by the cells during the assay, as well as perturbation oligonucleotides (POs) encoding perturbation elements (PEs). Using these capture beads, the target perturbation components in the cellular environment are captured on the beads, as are the perturbation oligonucleotides, as shown in reaction protocol C below:

[0252] Reaction scheme C

[0253] All symbols are defined as in this document, and among them... a , b and c All are integers, such that a , b and c The sum is less than or equal to n .

[0254] In the above reaction scheme, the perturbation compound and the perturbation oligonucleotide are captured by elements X and Q, respectively. 1Release and capture. The perturbation oligonucleotide is released and captured by capture elements indexed by oligonucleotides attached to the capture beads. Assay continues until completion, with cell lysis in each detection region, thereby releasing the perturbation component. Reaction scheme C illustrates an example where L is uncut and / or uncleducible, and capture occurs on the capture beads. In other embodiments, L is cleaved before, during, or after cell lysis, thereby allowing the perturbation component and / or the perturbation oligonucleotide to interact unimpeded (e.g., entirely in solution, without steric hindrance from the capture beads) with the capture elements X and Q. 1 Combined. Note that if the perturbation component is mRNA, the final product as a whole is a solution-phase oligonucleotide suitable for sequencing.

[0255] In one example, A549 cells were screened for a DNA-encoded (poly-T tailed) combinatorial library of ~10,000 members targeting bromodomain protein 4 (BRD4), the library being synthesized at the end of a nitrobenzyl linker attached to a bead.

[0256] Individual perturbation beads are assigned to corresponding micropores (130 μm in diameter and 130 μm in depth), which are distributed on a chip containing ~47,000 pores.

[0257] Then add 5-10 cells to each well and seal with oil.

[0258] The compound was cleaved from the beads using ultraviolet (UV) light to achieve a concentration of ~5 μM, and the chip was then incubated in a 37°C incubator for 24 hours.

[0259] After 24 hours of incubation, cells were lysed using a freeze-thaw cycle: treated at -80°C for 30 minutes, followed by thawing for 45 minutes. The released mRNA was hybridized to barcode dT oligonucleotides on the same bead (the perturbed bead also served as a capture bead). The oil cap on the chip was removed by a series of low-salt washes, protease digestion, and high-salt washes. The mRNA-hybridized beads were removed from the chip and reverse transcribed. The beads were then divided into eight portions for whole transcriptome amplification. Libraries were constructed via fragmentation of Nextera-B transposons and subsequent indexed polymerase chain reaction (PCR). Nucleic acids captured from cells were sequenced, ~25,000 reads per well.

[0260] Positive results were identified in parallel microarrays using fluorescence microscopy. Briefly, cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 30 minutes, followed by permeabilization with 0.5% Triton X-100. Cells were blocked with 5% bovine serum albumin (BSA) containing 0.2% Triton for 40 minutes, then stained with BRD4 and β-tubulin antibodies at room temperature for 2 hours, followed by staining with secondary antibody and Hoechst (nucleus) at room temperature for 2 hours. The microarray was imaged, and positive results were identified by the BRD4 / tubulin to BRD4 / nucleus ratio. These “positive results” were picked from the microarray and amplified using Illumina P5 / P7 primers. The library was sequenced, ~10,000 reads per bead, to identify the bead barcode / compound identifier oligonucleotides.

[0261] Figure 8A and 8B The sequencing results of the captured oligonucleotides in this embodiment are shown. When analyzing the mRNA-seq library, it was first aligned to the hg38 annotated genome using STAR (Systems Transcriptional Activity Reconstruction, e.g., Wang et al., Bioinformatics, 29(24): 3204-3210, December 2013, doi.org / 10.1093 / bioinformatics / btt558). Unique molecular identifiers (UMIs) were calculated to ensure accurate quantification of gene expression levels. Preprocessing steps included removing beads with high mitochondrial and ribosome content and normalizing gene expression to housekeeping genes. Dimensionality reduction techniques were applied to visualize the results on a two-dimensional graph based on transcriptome similarity ( Figure 8A (t-distributed random neighborhood embedding map in the image). The positive results identified in the fluorescence microscopy imaging are overlaid (see...). Figure 8A (Hollow circles in the image), and the TUBB / BRD4 mRNA ratio was calculated through sequencing. Figure 8B It was observed that compounds that led to decreased BRD4 levels upregulated BRD4 mRNA levels, suggesting the existence of a feedback loop that compensates for protein degradation by first increasing mRNA expression.

[0262] In some embodiments, the beads used may be doped with a ferromagnetic material, which can be used as a directional component. For example, Figure 6 The self-orientation characteristics of a perturbation bead 4 doped with ferromagnetic material are shown, which moves upward (or downward) to the marking position on the axis 1 of the marking device (not shown).

[0263] In addition, the perturbation beads added to the detection area can be oriented by placing a magnet below the measuring device. The magnet can readjust the direction of the beads so that the mark points roughly upwards for imaging.

[0264] Each assay performed in the mapping assay apparatus is incubated under standard conditions to allow the released compound to perturb the cells. During the assay, images covering each assay well are periodically captured. These images can confirm changes in cell morphology, such as alterations in cell shape, as well as other possible properties. Possible changes in cell morphology include distortion of cell shape. For example, if a cell was originally oval, it may be distorted into a shape after perturbation, which, while still resembling an oval, will exhibit ripples and raised areas due to the perturbation by the perturbing compound. These changes captured during the assay are considered to reflect dynamic functional changes in the perturbed cells.

[0265] After the assay, cells are lysed in the presence of perturbation beads, which capture perturbation components generated by the cells during the assay. Using these perturbation beads, target perturbation components in the cellular environment are captured on the beads, as are oligonucleotide compounds, as shown in reaction scheme 2 above. In some implementations, the mRNA, compound oligonucleotide, and labeled oligonucleotide (if separated from the compound oligonucleotide) can all be sequenced on the capture bead, or separated from the capture bead by cleaving the cleavable bond using an appropriate cleavage stimulus. As described above, the mRNA changes reflected by the perturbation can be combined with any dynamic changes in cellular function as previously described to enhance the cellular functional changes generated by the perturbation.

[0266] Changes in nucleic acid expression in lysed cells can be correlated with photographic or video recordings of the detection area where the cell lysis occurred (as detailed above).

[0267] 15. Implementation Plan

[0268] Each of the following implementation schemes represents a portion of the possible combinations that can be achieved in this paper, many of which have been explicitly provided above.

[0269] Implementation Plan 1: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device.

[0270] Implementation Plan 2: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measurement apparatus.

[0271] Implementation Plan 3: A capture bead for measurement in the detection area of ​​a mapping measurement device comprising multiple detection areas, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the mark uniquely identifying the capture bead in the measurement device, wherein the capture bead in the mapping measurement device is associated with a specific detection area, thereby identifying the detection area in which the capture bead is located.

[0272] Implementation Plan 4: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the mark uniquely identifying the capture bead in the measurement device and the detection area in the mapping measurement device being associated with the capture bead, wherein the association is memorized.

[0273] Implementation Plan 5: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the markers uniquely identifying the capture bead in the measurement device, and the detection area in the mapping measurement device being associated with the capture bead, the association being stored in a registry that records the position of each capture bead in each detection area of ​​the mapping measurement device.

[0274] Implementation Plan 6: A capture bead for measurement in a measuring apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the capturing apparatus further comprises multiple copies of a capture bead index encoding the unique mark or set of marks.

[0275] Implementation Plan 7: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead, the mark uniquely identifying the capture bead in the measurement apparatus, wherein the capture apparatus further comprises multiple copies of a capture bead index encoding the unique mark or set of marks.

[0276] Implementation Plan 8:A capture bead for measurement in the detection area of ​​a mapping measurement device comprising multiple detection areas, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead, the mark uniquely identifying the capture bead in the measurement device, wherein the capture bead in the mapping measurement device is associated with a specific detection area, thereby identifying the detection area in which the capture bead is located, wherein the capture device further comprises multiple copies of a capture bead index encoding the unique mark or set of marks.

[0277] Implementation Plan 9: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead, the mark uniquely identifying the capture bead in the measurement apparatus, and the detection area in the mapping measurement apparatus being associated with the capture bead, wherein the association is memorized, and wherein the capture apparatus further comprises multiple copies of a capture bead index encoding a unique mark or set of marks.

[0278] Implementation Plan 10: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead, the mark uniquely identifying the capture bead in the measurement device, and the detection area in the mapping measurement device being associated with the capture bead, the association being stored in a registry recording the position of each capture bead in each detection area of ​​the mapping measurement device, wherein the capture device further comprises multiple copies of a capture bead index encoding the unique mark or set of marks.

[0279] Implementation Plan 11: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the markers uniquely identifying the capture beads in the measuring apparatus, the indices being unique to other capture bead indices used in the measurement, wherein the beads do not contain other capture bead sequences containing coded markers or sets of markers.

[0280] Implementation Plan 12: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the markers uniquely identifying the capture beads in the measurement apparatus, the indices being unique to other capture bead indices used in the measurement, wherein the beads do not contain other capture bead sequences containing coded markers or sets of markers.

[0281] Implementation Plan 13:A capture bead for measurement in a detection area of ​​a mapping measurement device comprising multiple detection areas, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the markers uniquely identifying the capture bead in the measurement device, wherein the capture bead in the mapping measurement device is associated with a specific detection area, thereby identifying the detection area in which the capture bead is located, the indices being unique to other capture bead indices used in the measurement, wherein the bead does not contain other capture bead sequences containing coded markers or sets of markers.

[0282] Implementation Plan 14: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the markers uniquely identifying the capture bead in the measurement device and the detection area in the mapping measurement device being associated with the capture bead, wherein the association is memorized, the indices are unique to other capture bead indices used in the measurement, wherein the bead does not contain other capture bead sequences containing coded markers or sets of markers.

[0283] Implementation Plan 15: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the marker uniquely identifying the capture bead in the measurement device, and the detection area in the mapping measurement device being associated with the capture bead, the association being stored in a registry recording the position of each capture bead in each detection area of ​​the mapping measurement device, the index being unique for other capture bead indices used in the measurement, wherein the bead does not contain other capture bead sequences containing coded markers or sets of markers.

[0284] Implementation Plan 16: A capture bead for measurement in a measuring apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices, the mark uniquely identifying the capture bead in the measuring apparatus, the indices being unique to other capture bead indices used in the measurement, wherein the bead does not contain other capture bead sequences containing coded marks or sets of marks.

[0285] Implementation Plan 17:A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices, the mark uniquely identifying the capture bead in the measurement apparatus, the index being unique to other capture bead indices used in the measurement, wherein the bead does not contain other capture bead sequences containing coded marks or sets of marks.

[0286] Implementation Plan 18: A capture bead for measurement in the detection area of ​​a mapping measurement device comprising multiple detection areas, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices, the mark uniquely identifying the capture bead in the measurement device, wherein the capture bead in the mapping measurement device is associated with a specific detection area, thereby identifying the detection area in which the capture bead is located, the index being unique to other capture bead indices used in the measurement, wherein the bead does not contain other capture bead sequences containing coded marks or sets of marks.

[0287] Implementation Plan 19: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices, the mark uniquely identifying the capture bead in the measurement apparatus and the detection area in the mapping measurement apparatus associated with the capture bead, wherein the association is memorized, the index is unique to other capture bead indices used in the measurement, wherein the bead does not contain other capture bead sequences containing coded marks or sets of marks.

[0288] Implementation Plan 20: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices, the mark uniquely identifying the capture bead in the measurement device, and the detection area in the mapping measurement device being associated with the capture bead, the association being stored in a registry that records the position of each capture bead in each detection area of ​​the mapping measurement device, the index being unique to other capture bead indices used in the measurement, wherein the bead does not contain other capture bead sequences containing coded marks or sets of marks.

[0289] Implementation Plan 21: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture elements on the bead, the detectable mark uniquely identifying the capture bead in the measuring apparatus.

[0290] Implementation Plan 22: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture elements on the bead, the detectable mark uniquely identifying the capture bead in the measurement apparatus.

[0291] Implementation Plan 23: A capture bead for measurement in a detection area of ​​a mapping measurement device comprising multiple detection areas, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture elements on the bead, the mark uniquely identifying the capture bead in the measurement device, wherein the capture bead in the mapping measurement device is associated with a specific detection area, thereby identifying the detection area in which the capture bead is located in the device.

[0292] Implementation Plan 24: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture elements on the bead, the mark uniquely identifying the capture bead in the measurement device and the detection area in the mapping measurement device being associated with the capture bead, wherein the association is memorized.

[0293] Implementation Plan 25: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture elements on the bead, the mark uniquely identifying the capture bead in the measurement device, and the detection area in the mapping measurement device being associated with the capture bead, the association being stored in a registry that records the position of each capture bead in each detection area of ​​the mapping measurement device.

[0294] Implementation Plan 26: An optical bead for measurement in a measuring apparatus, the capturing bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capturing elements on the bead, the detectable mark uniquely identifying the capturing bead in the measuring apparatus.

[0295] Implementation Plan 27: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture elements on the bead, the detectable mark uniquely identifying the capture bead in the measurement apparatus.

[0296] Implementation Plan 28:A capture bead for measurement in the detection area of ​​a mapping measurement device comprising multiple detection areas, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture elements on the bead, the mark uniquely identifying the capture bead in the measurement device, wherein the capture bead in the mapping measurement device is associated with a specific detection area, thereby identifying the detection area in which the capture bead is located.

[0297] Implementation Plan 29: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture elements on the bead, the mark uniquely identifying the capture bead in the measurement apparatus and the detection area in the mapping measurement apparatus being associated with the capture bead, wherein the association is memorized.

[0298] Implementation Plan 30: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture elements on the bead, the mark uniquely identifying the capture bead in the measurement device, and the detection area in the mapping measurement device being associated with the capture bead, the association being stored in a registry that records the position of each capture bead in each detection area of ​​the mapping measurement device.

[0299] Implementation Plan 31: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the mark uniquely identifying the capture bead in the measuring apparatus, the indices being unique to other capture bead indices used in the measurement.

[0300] Implementation Plan 32: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the marks uniquely identifying the capture beads in the measurement apparatus, and the indices being unique to other capture bead indices used in the measurement.

[0301] Implementation Plan 33:A capture bead for measurement in the detection area of ​​a mapping measurement device comprising multiple detection areas, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the mark uniquely identifying the capture bead in the measurement device, wherein the capture bead in the mapping measurement device is associated with a specific detection area, thereby identifying the detection area in which the capture bead is located, and the indices are unique to other capture bead indices used in the measurement.

[0302] Implementation Plan 34: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the mark uniquely identifying the capture bead in the measurement apparatus and the detection area in the mapping measurement apparatus being associated with the capture bead, wherein the association is memorized and the index is unique for other capture bead indices used in the measurement.

[0303] Implementation Plan 35: A capture bead for measurement in a detection area of ​​a mapping measurement device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the mark uniquely identifying the capture bead in the measurement device, and the detection area in the mapping measurement device being associated with the capture bead, the association being stored in a registry that records the position of each capture bead in each detection area of ​​the mapping measurement device.

[0304] Implementation Plan 36: An optical bead for measurement in a measuring apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the marks uniquely identifying the capture bead in the measuring apparatus, the indices being unique to other capture bead indices used in the measurement.

[0305] Implementation Plan 37: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the marks uniquely identifying the capture bead in the measurement apparatus, and the indices being unique to other capture bead indices used in the measurement.

[0306] Implementation Plan 38:A capture bead for measurement in the detection area of ​​a mapping measurement device comprising multiple detection areas, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the mark uniquely identifying the capture bead in the measurement device, wherein the capture bead in the mapping measurement device is associated with a specific detection area, thereby identifying the detection area in which the capture bead is located, and the indices are unique to other capture bead indices used in the measurement.

[0307] Implementation Plan 39: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices and optionally a plurality of capture elements attached to the indices via connectors, the marks uniquely identifying the capture bead in the measurement apparatus and the detection area in the mapping measurement apparatus being associated with the capture bead, wherein the association is memorized and the indices are unique to other capture bead indices used in the measurement.

[0308] Implementation Plan 40: A capture bead for measurement in a detection area of ​​a mapping measurement apparatus, the capture bead comprising an optically detectable mark or set of optically detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical capture elements on the bead, the mark uniquely identifying the capture bead in the measurement apparatus, and the detection area in the mapping measurement apparatus being associated with the capture bead, the association being stored in a registry that records the position of each capture bead in each detection area of ​​the mapping measurement apparatus, the index being unique for other capture bead indices used in the measurement.

[0309] Implementation Plan 41: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the markers uniquely identifying the capture bead in the measuring apparatus, wherein the detectable markers are selected from visual, chemical, biological, audio, or other means to confirm the presence of information generated by the markers.

[0310] Implementation Plan 42: A capture bead for measurement in a measuring device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the markers uniquely identifying the capture bead in the measuring device, wherein the detectable markers generate color or can be stimulated to generate color.

[0311] Implementation Plan 43:A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark generates light or can be stimulated to emit light.

[0312] Implementation Plan 44: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark contains text.

[0313] Implementation Plan 45: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark comprises a number.

[0314] Implementation Plan 46: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark comprises a symbol.

[0315] Implementation Plan 47: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark comprises one or more barcodes.

[0316] Implementation Plan 48: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark comprises a UPC code.

[0317] Implementation Plan 49: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark comprises one or more tags.

[0318] Implementation Plan 50: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the capture bead in the measuring apparatus, wherein the detectable markers contain a chemical signal.

[0319] Implementation Plan 51: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises a quantum dot.

[0320] Implementation Plan 52: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the capture bead in the measuring apparatus, wherein the detectable markers comprise quantum dots and require an instrument.

[0321] Implementation Plan 53: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the capture bead in the measuring apparatus, wherein the detectable markers comprise fluorescence.

[0322] Implementation Plan 54: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the capture bead in the measuring apparatus, wherein the detectable markers comprise mass spectrometry.

[0323] Implementation Plan 55: A trapping bead for measurement in a measuring apparatus, the trapping bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the trapping bead in the measuring apparatus, wherein the detectable markers comprise nuclear magnetic resonance spectra (e.g., proton spectra or C-ray spectroscopy). 13 (Spectrum).

[0324] Implementation Plan 56: A capture bead for measurement in a measuring device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the capture bead in the measuring device, wherein the detectable markers comprise biosignals or bioinduced signals.

[0325] Implementation Plan 57: A capture bead for measurement in an assay apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the capture bead in the assay apparatus, wherein the detectable markers comprise nucleic acids.

[0326] Implementation Plan 58:A capture bead for measurement in a measuring device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the capture bead in the measuring device, wherein the detectable markers comprise bioluminescence.

[0327] Implementation Plan 59: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark comprises radio waves or electromagnetic signals.

[0328] Implementation Plan 60: A capture bead for measurement in a measuring device, the capture bead comprising a detectable tag or set of detectable tags attached to the bead, the detectable tag uniquely identifying the capture bead in the measuring device, wherein the detectable tag incorporates Bluetooth.

[0329] Implementation Plan 61: A capture bead for measurement in a measuring device, the capture bead comprising a detectable tag or set of detectable tags attached to the bead, the detectable tag uniquely identifying the capture bead in the measuring device, wherein the detectable tag comprises RFID.

[0330] Implementation Plan 62: A capture bead for measurement in a measuring device, the capture bead comprising a detectable tag or set of detectable tags attached to the bead, the detectable tag uniquely identifying the capture bead in the measuring device, wherein the detectable tag incorporates Bluetooth.

[0331] Implementation Plan 63: A capture bead for measurement in a measuring device, the capture bead comprising a detectable tag or set of detectable tags attached to the bead, the detectable tag uniquely identifying the capture bead in the measuring device, wherein the detectable tag includes WiFi.

[0332] Implementation Plan 64: A capture bead for measurement in an assay apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, the detectable markers uniquely identifying the capture bead in the assay apparatus, wherein the detectable markers comprise two or more markers selected from: color, code, shape, color, inducible color, text, number, symbol, barcode, UPC code, Bluetooth, WiFi, RFID, LED, bioluminescence, and nucleic acid.

[0333] Implementation Plan 65:A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the markers uniquely identifying the capture bead in the measuring apparatus, wherein the detectable markers are selected from visual, chemical, biological, audio, or other means to confirm the presence of information generated by the markers, and the indices are unique to other capture bead indices used in the measurement.

[0334] Implementation Plan 66: A capture bead for measurement in a measuring device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the markers uniquely identifying the capture beads in the measuring device, wherein the detectable markers generate color or can be stimulated to generate color, and the indices are unique to other capture bead indices used in the measurement.

[0335] Implementation Plan 67: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark generates light or can be stimulated to emit light, and the indices are unique to other capture bead indices used in the measurement.

[0336] Implementation Plan 68: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark contains text, and the indices are unique to other capture bead indices used in the measurement.

[0337] Implementation Plan 69: A capture bead for a measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark contains a number and the index is unique to other capture bead indices used in the measurement.

[0338] Implementation Plan 70: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises a symbol, and the index is unique to other capture bead indices used in the measurement.

[0339] Implementation Plan 71: A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring device, wherein the detectable mark comprises one or more barcodes, the indices being unique to other capture bead indices used in the measurement.

[0340] Implementation Plan 72: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark contains a UPC code, and the index is unique to other capture bead indices used in the measurement.

[0341] Implementation Plan 73: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises one or more tags, and the index is unique to other capture bead indices used in the measurement.

[0342] Implementation Plan 74: A capture bead for a determination in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the detectable markers uniquely identifying the capture beads in the measuring apparatus, wherein the detectable markers contain a chemical signal, and the indices are unique to other capture bead indices used in the determination.

[0343] Implementation Plan 75: A capture bead for a determination in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark optionally contains quantum dots requiring suitable instruments, and the indices are unique to other capture bead indices used in the determination.

[0344] Implementation Plan 76: A capture bead for a determination in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises a quantum dot, and the indices are unique to other capture bead indices used in the determination.

[0345] Implementation Plan 77: A capture bead for an assay performed in an assay apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the detectable markers uniquely identifying the capture beads in the assay apparatus, wherein the detectable markers contain fluorescence, and the indices are unique to other oligonucleotide indices used in the assay.

[0346] Implementation Plan 78: A capture bead for an assay performed in an assay apparatus, the capture bead comprising a detectable tag or set of detectable tags attached to the bead and a plurality of identical oligonucleotide indices, the detectable tag uniquely identifying the capture bead in the assay apparatus, wherein the detectable tag comprises a mass spectrometer, and the index is unique to other capture bead indices used in the assay.

[0347] Implementation Plan 79: A trapping bead for measurement in a measuring apparatus, the trapping bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical trapping bead indices, the detectable markers uniquely identifying the trapping beads in the measuring apparatus, wherein the detectable markers comprise nuclear magnetic resonance spectra (e.g., proton spectra or C-ray spectroscopy). 13 (Spectrum), the index is unique to the other capture bead indexes used in the determination.

[0348] Implementation Plan 80: A capture bead for a determination in a measuring device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the detectable markers uniquely identifying the capture beads in the measuring device, wherein the detectable markers contain a biosignal or bio-induced signal, and the indices are unique to other capture bead indices used in the determination.

[0349] Implementation Plan 81: A capture bead for an assay performed in an assay apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the detectable markers uniquely identifying the capture beads in the assay apparatus, wherein the detectable markers comprise nucleic acids, and the indices are unique to other capture bead indices used in the assay.

[0350] Implementation Plan 82: A capture bead for a determination in a measuring device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the detectable markers uniquely identifying the capture beads in the measuring device, wherein the detectable markers contain bioluminescence, and the indices are unique to other capture bead indices used in the determination.

[0351] Implementation Plan 83: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark contains radio waves or electromagnetic signals, and the indices are unique to other capture bead indices used in the measurement.

[0352] Implementation Plan 84: A capture bead for measurement in a measuring device, the capture bead comprising a detectable tag or set of detectable tags attached to the bead and a plurality of identical capture bead indices, the detectable tag uniquely identifying the capture bead in the measuring device, wherein the detectable tag incorporates Bluetooth, and the indices are unique to other capture bead indices used in the measurement.

[0353] Implementation Plan 85: A capture bead for measurement in a measuring device, the capture bead comprising a detectable tag or set of detectable tags attached to the bead and a plurality of identical capture bead indices, the detectable tag uniquely identifying the capture bead in the measuring device, wherein the detectable tag comprises RFID, and the indices are unique to other capture bead indices used in the measurement.

[0354] Implementation Plan 86: A capture bead for measurement in a measuring device, the capture bead comprising a detectable tag or set of detectable tags attached to the bead and a plurality of identical capture bead indices, the detectable tag uniquely identifying the capture bead in the measuring device, wherein the detectable tag incorporates Bluetooth, and the indices are unique to other capture bead indices used in the measurement.

[0355] Implementation Plan 87: A capture bead for measurement in a measuring device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the detectable markers uniquely identifying the capture bead in the measuring device, wherein the detectable markers include WiFi, and the indices are unique to other capture bead indices used in the measurement.

[0356] Implementation Plan 88:A capture bead for an assay performed in an assay apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices, the detectable markers uniquely identifying the capture beads in the assay apparatus, wherein the detectable markers comprise two or more markers selected from: color, code, shape, color, inducible color, text, number, symbol, barcode, UPC code, Bluetooth, WiFi, RFID, bioluminescence, and nucleic acid, the indices being unique to other capture bead indices used in the assay.

[0357] Implementation Plan 89: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, a plurality of identical capture bead indices, and a plurality of identical or different capture elements, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark is selected from visual, chemical, biological, audio, or other means to confirm the presence of information generated by the mark, the index is unique to other capture bead indices used in the measurement, and the elements optionally are attached to at least a portion of the index via a cuttable or non-cuttable connector.

[0358] Implementation Plan 90: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, a plurality of identical capture bead indices, and a plurality of identical or different capture elements, the marks uniquely identifying the capture beads in the measuring apparatus, wherein the detectable marks generate color or can be stimulated to generate color, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0359] Implementation Plan 91: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, a plurality of identical capture bead indices, and a plurality of identical or different capture elements, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark generates light or can be stimulated to emit light, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0360] Implementation Plan 92:A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, a plurality of identical capture bead indices, and a plurality of identical or different capture elements, the marks uniquely identifying the capture bead in the measuring apparatus, wherein the detectable marks contain text, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0361] Implementation Plan 93: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, a plurality of identical capture bead indices, and a plurality of identical or different capture elements, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark contains a number, and the index is unique to other capture bead indices used in the measurement.

[0362] Implementation Plan 94: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, a plurality of identical capture bead indices, and a plurality of identical or different capture elements, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises a symbol, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via a connector, the connector being identical or different from cuttable or incuttable.

[0363] Implementation Plan 95: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises one or more barcodes, the indices being unique to other capture bead indices used in the measurement, and the elements optionally being attached to at least a portion of the indices via cuttable or non-cuttable connectors.

[0364] Implementation Plan 96: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the marks uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark contains a UPC code, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0365] Implementation Plan 97: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the marks uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises one or more tags, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0366] Implementation Plan 98: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the marks uniquely identifying the capture beads in the measuring apparatus, wherein the detectable marks contain a chemical signal, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0367] Implementation Plan 99: A capture bead for a determination in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead, a plurality of identical capture bead indices, and a plurality of identical or different capture elements, the detectable mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises quantum dots optionally requiring suitable instruments, and the indices are unique to other capture bead indices used in the determination.

[0368] Implementation Plan 100: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the marks uniquely identifying the capture beads in the measuring apparatus, wherein the detectable marks comprise quantum dots, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0369] Implementation Plan 101: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the marks uniquely identifying the capture beads in the measuring apparatus, wherein the detectable marks contain fluorescence, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable connectors.

[0370] Implementation Plan 102: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the markers uniquely identifying the capture beads in the measuring apparatus, wherein the detectable markers comprise mass spectrometry, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0371] Implementation Plan 103: A trapping bead for measurement in a measuring apparatus, the trapping bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical trapping bead indices and a plurality of identical or different trapping elements, the markers uniquely identifying the trapping bead in the measuring apparatus, wherein the detectable markers comprise nuclear magnetic resonance spectroscopy (e.g., proton spectroscopy or C20 spectroscopy). 13 (Spectrum), the index is unique to other capture bead indices used in the determination, and the element is optionally attached to at least a portion of the index via a cuttable or non-cuttable joint.

[0372] Implementation Plan 104: A capture bead for measurement in a measuring device, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the markers uniquely identifying the capture beads in the measuring device, wherein the detectable markers contain a biosignal or bio-induced signal, the indices being unique to other capture bead indices used in the measurement, and the elements optionally being attached to at least a portion of the indices via a cuttable or non-cuttable connector.

[0373] Implementation Plan 105: A capture bead for an assay performed in an assay apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the markers uniquely identifying the capture beads in the assay apparatus, wherein the detectable markers comprise nucleic acids, the indices are unique to other capture bead indices used in the assay, and the elements are optionally attached to at least a portion of the indices via cleavable or incleavable adapters.

[0374] Implementation Plan 106:A capture bead for measurement in a measuring device, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the marks uniquely identifying the capture beads in the measuring device, wherein the detectable marks contain bioluminescence, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable connectors.

[0375] Implementation Plan 107: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the marks uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark contains radio waves or electromagnetic signals, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0376] Implementation Plan 108: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark incorporates Bluetooth, the index is unique to other capture bead indices used in the measurement, and the elements optionally attach to at least a portion of the index via a cuttable or non-cuttable connector.

[0377] Implementation Plan 109: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises RFID, the indices are unique to other capture bead indices used in the measurement, and the elements are optionally attached to at least a portion of the indices via cuttable or non-cuttable joints.

[0378] Implementation Plan 110: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark incorporates Bluetooth, the index is unique to other capture bead indices used in the measurement, and the elements optionally attach to at least a portion of the index via a cuttable or non-cuttable connector.

[0379] Implementation Plan 111: A capture bead for measurement in a measuring apparatus, the capture bead comprising a detectable mark or set of detectable marks attached to the bead and a plurality of identical capture bead indices and a plurality of identical or different capture elements, the mark uniquely identifying the capture bead in the measuring apparatus, wherein the detectable mark comprises WiFi, the index is unique to other capture bead indices used in the measurement, and the elements optionally attached to at least a portion of the index via a cuttable or non-cuttable connector.

[0380] Implementation Plan 112: A capture bead for an assay performed in an assay apparatus, the capture bead comprising a detectable marker or set of detectable markers attached to the bead, a plurality of identical capture bead indices, and a plurality of identical or different capture elements, the markers uniquely identifying the capture bead in the assay apparatus, wherein the detectable markers comprise two or more markers selected from: color, code, shape, color, inducible color, text, number, symbol, barcode, UPC code, Bluetooth, WiFi, RFID, bioluminescence, and nucleic acid, the indices being unique to other capture bead indices used in the assay, and the elements optionally being attached to at least a portion of the indices via cuttable or non-cuttable connectors.

[0381] Implementation Plan 113: A method for associating a capture bead with a specific detection region in a mapping assay apparatus, the method comprising: encoding each of the capture beads with an optically detectable marker or a set of optically detectable markers, the marker being unique to that bead; further encoding the capture bead with an oligonucleotide that uniquely encodes the marker or set of markers on the bead; adding the capture bead to at least a portion of the detection region in the apparatus; and associating each uniquely tagged bead with each detection region in the apparatus.

[0382] Implementation Plan 114: A method for associating a capture bead with a specific detection area in a mapping device, the method comprising: encoding each of the capture beads with an optically detectable mark or a set of optically detectable marks, the mark being unique to that bead; adding the capture bead to at least a portion of the detection area in the device; associating each uniquely marked capture bead with each detection area in the device; and memorizing each position of each capture bead to a specific detection area on the mapping device.

[0383] Implementation Plan 115: The methods of embodiments 113 and 114, wherein the markings are defined as in any of embodiments 41 to 66.

[0384] Implementation Plan 116:The method of embodiment 115, wherein the capture bead further comprises a capture bead index and a capture element attached thereto as defined in any of embodiments 89 to 113.

[0385] Implementation Plan 117: A method for performing an assay in at least a portion of a plurality of detection regions in a mapping assay apparatus, wherein each detection region comprises a cell, a perturbation bead containing a perturbation element and a perturbation oligonucleotide unique to the compound, and an assay solution, the method comprising: a) Determination is performed in at least a portion of a plurality of detection regions in a mapping assay apparatus, wherein each detection region comprises cells, perturbation beads (containing perturbation elements), and perturbation oligonucleotides unique to the compound, and an assay solution; b) Each detection region contains a capture bead, which comprises: An optically detectable mark or set of optically detectable marks attached to the bead, the mark uniquely identifying the capturing bead; Multiple identical oligonucleotide indexes, each index being unique to the capture bead and optionally attached thereto via a linker, wherein the indexes encode unique tags or sets of tags; and Multiple identical capture elements are attached to the oligonucleotide index; c) By associating the unique optically detectable marks on the capture beads with the mapping measuring device, the optically detectable marks or sets of marks in each detection area are associated and recorded, thereby providing a 1:1 relationship between the capture beads in each detection area; d) Inducing perturbation by releasing at least a portion of the perturbation element from the perturbation bead, thereby bringing the element into contact with the cell and inducing a functional change in the cell, the functional change including at least a change demonstrated by one or more perturbation components expressed by the cell; e) Release the unique perturbation oligonucleotide from the beads and capture the oligonucleotide onto the capture beads; f) Lyse the cells, release the perturbation component into the assay solution, and capture at least one of the components onto a capture bead index or a perturbation oligonucleotide; g) Sequencing of oligonucleotides containing oligonucleotide indexes, perturbation oligonucleotides, and perturbation components; and h) By referring to the associations recorded in c) above, oligonucleotide indexes are associated with unique markers or sets of markers for a specific detection region, thereby identifying a specific detection region from which perturbation components are retrieved.

[0386] Implementation Plan 118: The method of implementation scheme 117 further includes: i) Acquire images of each detection area during the measurement period; j) Associate the image with the specific detection region defined in H) above; and k) Assess changes in cell morphology caused by cell disturbance during the assay.

[0387] Implementation Plan 119: Multiple identical trapping elements on the trapping bead are receptors or ligands for the perturbed component to be trapped.

[0388] Implementation Plan 120: Multiple identical capture elements on the capture beads are antibodies or antibody-binding fragments targeting the perturbed compound.

[0389] Implementation Plan 121: Multiple identical trapping elements on the trapping bead are enzymes or enzyme receptors targeting the perturbed component.

[0390] Implementation Plan 122: Multiple identical trapping elements on the trapping beads are receptors for a given cytokine or chemokine.

[0391] Implementation Plan 123: Multiple identical capturing elements on the capturing beads contain avidin / streptavidin, and the avidin / streptavidin capture has been modified with a perturbation component containing a biotin group.

[0392] Implementation Plan 124: Multiple identical capturing elements on the capturing beads contain biotin, and the biotin capture has been modified with a perturbation component containing avidin / streptavidin groups.

[0393] Implementation Plan 125: Multiple identical oligonucleotide indices and multiple identical oligonucleotide capture elements are attached to the capture bead directly or via releasable adapters.

[0394] Implementation Plan 126: The multiple identical oligonucleotide capture elements on the capture beads are poly-T.

[0395] Implementation Plan 127: A library or registry containing references to each tag or set of detectable tags, the tags encoding unique capture beads and a specific detection area in a measuring device where each capture bead is located.

[0396] Implementation Plan 128:A capture bead comprising a detectable marker or set of detectable markers that uniquely associates the capture bead with a specific detection region, wherein the capture bead contains additional information relating to an assay to be performed in that detection region, including: assay conditions, changes in cell function during the assay, synthetic reactions employed in at least one step of the synthesis of the compound, identification of one or more reaction conditions employed during the synthesis, the technician performing the assay, the date of the assay, the pH of the assay solution, or a combination thereof.

[0397] Implementation Plan 129: Each capture bead in each detection area has a unique, optically detectable mark, recorded, for example, by photography, videography, barcodes, or QR codes.

[0398] Implementation Plan 130: A type of trapping bead, represented by the following formula I: (W) r -CB-(LXQQ 1 ) n. (I) in: CB stands for Capture Bead; L stands for connector; W is an optically detectable mark or a set of optically detectable marks that uniquely identify the capture bead; X is a key or capture element; Q is the capture bead index, which encodes the optically detectable mark or set of optically detectable marks on the capture bead; Q 1 It is a capture element, which targets n LXQQ. 1 Each member of the group is selected independently; n represents multiple such connectors (connectors - QQ) that are combined with beads. 1 ) group, and r It is an integer from 1 to 10.

[0399] Implementation Plan 131: Implementation Plan 130 The captured beads, wherein W is a marker as defined in any of embodiments 41 to 66.

[0400] Implementation Plan 132: Implementation Plan 130 The capture beads, wherein the capture beads further include a capture bead index and a capture element as defined in any one of embodiments 89 to 113.

[0401] Implementation Plan 133: Implementation Plan 130 The captured beads, where Q and Q 1 The attachment is either a direct connection or a connection via a connector.

[0402] Implementation Plan 134:The capturing bead of Formula I may include Formula IA, which is described as follows: (W) r -CB-[L´-XQQ 1 -PC] n (IA) in: Q is a bead-capturing oligonucleotide; X is a key or capture element; Q 1 It is a capture element, where X and Q are not keys when X is not a key. 1 Independently, it is a capture element, where Q 1 The trapping element on each of X is complementary to the corresponding functional group on the target; PC is the perturbation component; L' is a releasable connector; And the definitions of n, r, and W are as described above.

[0403] Implementation Plan 134: An oligonucleotide, represented by formula IB: AX 1 -QQ 1 -PC IB in: PC is the perturbation component; Q is the capture index; Q 1 It is a capture element that complements the corresponding functional groups on a PC; X 1 It is a trapping element complementary to the corresponding functional group on A; and A is a perturbation component or a perturbation oligonucleotide.

[0404] Implementation Plan 135: The oligonucleotide of implementation scheme 134, wherein PC is mRNA and Q 1 It is poly-T.

[0405] Implementation Plan 136: The oligonucleotide of implementation scheme 134, wherein A is a perturbation oligonucleotide and X 1 It is a complementary group to the functional group on the perturbed oligonucleotide.

[0406] Implementation Plan 137: The oligonucleotide of implementation scheme 136, wherein A is a nucleic acid other than mRNA.

[0407] Implementation Plan 138: A method for preparing a bead colony, wherein each bead in the colony comprises a unique marker or set of markers and an oligonucleotide specific to that marker or set of markers, the method comprising: a) Select bead populations functionalized with oligonucleotide binding sites; b) Distribute the beads in approximately equal quantities into multiple reaction containers, wherein the number of reaction containers ranges from 3 to 12; c) Affix a first label to each reaction vessel, wherein the label for each vessel is different from the labels for the other vessels; d) Attach the oligonucleotide chain to the oligonucleotide binding site; e) Gather the beads and mix until homogeneous; f) Repeat steps b) and e) a sufficient number of times such that each bead in the population is uniquely labeled and each bead contains a unique oligonucleotide, which, for each individual reaction step, comprises an oligonucleotide chain attached to a previous oligonucleotide chain.

Claims

1. A trapping bead for measurement, the trapping bead comprising: a) An optically detectable mark or set of optically detectable marks attached to the capture bead, the mark uniquely identifying the capture bead; b) Multiple capture bead indices, each encoding a detectable tag or a set of detectable tags, attached to the capture beads via a connector; and c) A plurality of one or more capture elements, each of which is attached to a capture bead index of a plurality of capture bead indices.

2. The capture bead of claim 1, wherein the detectable marker or the set of detectable markers is optically detectable.

3. The capture bead of claim 2, wherein the optically detectable marker comprises one or more of an image, code, shape, color, induced color, text, number, symbol, barcode, UPC code, or optically detectable material.

4. The capture bead as claimed in any one of claims 1-3, wherein the connector is a releasable connector.

5. The capturing bead as claimed in any one of claims 1-4, wherein the capturing bead index comprises about 6 to about 2,000 nucleotides.

6. The capture bead of any one of claims 1-5, wherein each of the one or more capture elements is selected from oligonucleotides, receptors, or ligands, wherein the capture element corresponds to a target perturbation component or a perturbation oligonucleotide encoding the perturbation element.

7. The capturing bead as claimed in any one of claims 1-6, wherein the capturing bead is represented by formula I: (W) r -CB-(L-X-Q-Q 1 ) n (I) in: CB stands for pearl; L stands for connector; W is an optically detectable mark or a set of optically detectable marks that identify the capture bead; X is a key or capture element; Q is the capture bead index, which encodes an optically detectable tag or a set of optically detectable tags; Q 1 It is a capture element, which targets n LXQQ. 1 Each member of the group is selected independently; n is a class of beads that are associated with (LX--QQ) 1 The number of groups, and r It is an integer from 1 to 100.

8. The capturing bead as claimed in claim 7, wherein n ranges from 10 to 6.02 x 10⁻⁶. 17 .

9. The capturing bead as described in any one of claims 7-8, wherein LXQQ 1 For n LXQQ 1 Each one in the group is the same.

10. The capturing bead as claimed in any one of claims 7-9, wherein X and Q 1 It is a binding element configured to capture different targets.

11. The capturing bead as claimed in any one of claims 7-10, wherein X and Q 1 For n LXQQ 1 Each of the groups is independently selected from up to 15 capture elements.

12. The capture bead as claimed in any one of claims 7-13, wherein L is a releasable connector.

13. The capturing bead as claimed in any one of claims 7-12, wherein X or Q 1 One is configured to capture perturbation oligonucleotides encoding perturbation elements, and the other is configured to capture one or more perturbation components released from the cell.

14. A mapping measurement device comprising multiple detection areas, wherein, Each of the plurality of detection regions, at least a portion thereof, contains at least one capture bead as defined in any one of claims 1-13, and The mapping measuring device is configured to associate the location of the detection area with at least one capture bead in the detection area.

15. A registry for use in the measuring apparatus of claim 14, indicating, for each optically detectable mark or set of optically detectable marks on at least one capture bead: Optically detectable markers or sets of optically detectable markers; as well as The corresponding detection areas of multiple detection areas detected optically detectable marks or sets of optically detectable marks.

16. The registry of claim 15, comprising one or more images, the images recording optically detectable markers or sets of optically detectable markers in corresponding detection areas.

17. A trapping bead, represented by formula IA: (W) r -CB-[L´-X-Q-Q 1 -Y] n (I-A) in: W is an optically detectable mark or a set of optically detectable marks that identify the capture bead; Q is a bead-capturing oligonucleotide; X is a key or capture element; Q 1 It is a capturing element, where X and Q 1 Both are independent capturing elements, where Q is a capturing element when X is a capturing element. 1 The trapping elements on X are complementary to the corresponding functional groups on the target; Y is one or more perturbation oligonucleotides PO that encodes a perturbation element PE or a perturbation component PC released from the cell; L' is a connector; and n It is combined with beads (LXQQ) 1 The number of PC groups, and r It is an integer from 1 to 100.

18. An oligonucleotide represented by formula IB: A-X 1 -Q-Q 1 -PC (I-B) in: PC is a perturbation component released by cells after they are exposed to perturbation elements; Q is the capture bead index; Q 1 It is a capture element that complements the corresponding functional groups on a PC; X 1 It is a trapping element that is complementary to the corresponding functional group on A; and A is a PC or perturbation oligonucleotide that encodes a perturbation element.

19. The oligonucleotide of claim 18, wherein PC is mRNA, and Q 1 Includes poly-T.

20. The oligonucleotide of claim 19, wherein A is a perturbative oligonucleotide, and X 1 It is a complementary group to the functional group on the perturbed oligonucleotide.

21. The oligonucleotide of claim 19, wherein A is a nucleic acid other than mRNA.

22. A method for identifying a specific detection region or multiple detection regions in a mapping measuring device, wherein a disturbance element has been released in the detection region, the method comprising: a) Provided in each of the multiple detection areas in the mapping measurement device: cell; Each of the plurality of perturbation beads comprises a perturbation element of one or more perturbation elements and a perturbation oligonucleotide of one or more perturbation elements encoding the perturbation element; The corresponding capture beads for multiple capture beads include: An optically detectable mark or set of optically detectable marks is attached to the capture bead, the mark uniquely identifying the capture bead; Multiple capture bead indices, which encode detectable tags or sets of detectable tags, are attached to the capture beads via connectors; and Multiple or one capture elements, each of which is attached to a capture bead index of multiple capture bead indices; c) For each of the multiple capture beads, memorize the information that associates the optically detectable mark or set of optically detectable marks on it with the position of the corresponding detection area, thereby providing a 1:1 relationship between each capture bead and each detection area of ​​the multiple detection areas; d) In each of the multiple detection areas, at least a portion of the perturbation element is released from the perturbation bead, thereby bringing the perturbation element into contact with the cell; e) In each detection area of ​​this section, the perturbation oligonucleotide is released from the perturbation bead, thereby capturing the perturbation oligonucleotide by the capture bead; f) In each detection region of this section, the cells are lysed to release perturbed components, wherein one or more capture elements or perturbed oligonucleotides are configured to capture perturbed components of the perturbed components; g) Sequencing the oligonucleotides generated from the capture described in f), wherein the oligonucleotides comprise a capture bead index, a perturbation oligonucleotide, and a perturbation component; as well as h) By referencing memory information, the capture bead index of sequencing oligonucleotides from sequencing is associated with optically detectable markers or sets of optically detectable markers associated with specific detection regions, thereby identifying specific detection regions.

23. The method of claim 22, further comprising: i) Acquire images of each detection area during the measurement period; j) Associate the image of the image with a specific detection region; as well as k) After releasing the perturbation element, evaluate the images of changes in cell morphology during the measurement process.

24. The method of any one of claims 22-23, wherein the perturbing element is a compound.

25. The method of any one of claims 22-24, wherein the perturbing element is selected from one or more of the following: Antibodies, immune cells, siRNA, viruses, bacteria, fungi, or Changes in one or more measurement conditions, such as buffer solution, salt, pH, temperature, nutrients, oxygen level, oxidant, or physical stress.

26. The method of any one of claims 22-25, wherein the corresponding capture beads are provided in the detection area before or during the determination.

27. The method of any one of claims 22-26, wherein each corresponding capture bead of the plurality of capture beads is represented by formula IA: (W) r -CB-[L´-X-Q-Q 1 -PC] n (I-A) in: Q is the capture bead index; X is the second capture element on the key or Q; Q 1 It is a capturing element, where X and Q 1 Both are independent capturing elements, where Q is a capturing element when X is a capturing element. 1 The trapping elements on X are complementary to the corresponding functional groups on the target; PC is the perturbation element of the perturbation component; L' is a connector; W is an optically detectable mark or a set of optically detectable marks that uniquely identify the capture bead; n represents multiple such (L´-XQQ) beads. 1 -PC) group, and r It is an integer from 1 to 100.

28. The method of any one of claims 22-27, wherein cutting the connector on the respective capture bead is performed after the perturbation element is released but before the cell is lysed, such that the capture of the perturbation oligonucleotide occurs on the capture bead, while the capture of the perturbation component released from the lysed cell occurs in solution.

29. The method of any one of claims 22-27, wherein the cleavage of the connector is performed before or simultaneously with the release of the perturbation oligonucleotide, thereby causing the capture of the perturbation component and / or the perturbation oligonucleotide to occur in solution.

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