Multiplexed catalyst reporter deposition

Abstract

This invention provides a method for testing a sample for the presence of one or more targets, including multiplexed catalytic reporter deposition (CARD). [Solution] The method comprising: (1) contacting a sample to be tested with one or more target-specific binding partners, each target-specific binding partner being linked to a nucleic acid chain, with differently specific target-specific binding partners being linked to different nucleic acid chains; (2) contacting the sample with (a) an enzyme linked to a nucleic acid chain, and (b) an intermediate portion including a first domain that specifically binds to the nucleic acid chain linked to the target-specific binding partner and a second domain that specifically binds to the nucleic acid chain linked to the enzyme; (3) contacting the sample from step (2) with a substrate conjugate consisting of a detectably labeled substrate; (4) inactivating the bound enzyme; and (5) detecting a signal from the bound detectably labeled substrate.

Description

Technical Field

[0001] Specification Cross - References to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 682,765, filed Jun. 8, 2018, and U.S. Provisional Application No. 62 / 760,450, filed Nov. 13, 2018, the entire contents of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Field This application generally relates to the field of detection of analytes (e.g., targets), and more particularly to detection methods using catalytic reporter deposition (CARD).

Background Art

[0003] In research and medical applications, it may be desirable to detect multiple biologically - relevant molecules within a single cell or tissue sample. Immunological methods are generally used for this purpose. Such methods generally involve binding to an antibody against the molecule of interest in the sample, generating a detectable signal associated with the antibody such that each molecule of interest is distinguishable from another, and using an analytical method to detect the signal. Catalytic reporter deposition (CARD) has been used in such immunological methods to produce an enhanced signal associated with the antibody. A representative workflow for CARD is to apply a target - molecule - specific antibody to the sample, and then apply a secondary antibody that recognizes the primary antibody. The secondary antibody is linked to an enzyme (e.g., horseradish peroxidase (HRP), alkaline phosphatase) that converts a substrate to a reagent that binds to a nearby phenol residue (e.g., tyrosine). Since phenol residues are abundant in cell samples, as a result, there is a high density of labeling in the vicinity of the primary antibody.

[0004] One of the technical problems associated with CARD is the difficulty in removing secondary antibodies from a sample. This problem arises when it is desirable to probe for further molecules of interest in the same sample using additional primary antibodies. While it is possible to use heat or microwaves to remove secondary antibodies, this method can damage the sample and degrade the quality of subsequent tests. Furthermore, this process can often be time-consuming.

[0005] Therefore, a method that avoids harsh sample handling and enables the detection of multiple target molecules in a single sample would be a useful advance in the CARD methodology. [Overview of the Initiative]

[0006] According to this specification, a method for testing a sample for the presence of one or more targets is provided for multiplexing catalytic reporter deposition (CARD) and for achieving a higher level of multiplexed detection.

[0007] In one embodiment, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (provided that either (i) or (ii) or both of the following conditions are met: (i) The nucleic acid chain is linked to a target-specific binding partner by a first release linker, and (ii) The enzyme is linked to a complementary nucleic acid chain by a second release linker. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a detectably labeled substrate (the substrate and the detectable label are optionally linked by a third release linker). (6) Optionally, remove the unbound substrate conjugate. (7) optionally release the bound enzyme, (8) Optionally, imaging the sample to detect a conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (7) or any subset thereof before imaging step (8).

[0008] In some embodiments, this method further includes repeating steps (3) to (7) before the imaging step (8).

[0009] In some embodiments, in step (5), a substrate conjugate consisting of a detectably labeled substrate reacts with an enzyme to form an activated substrate conjugate, which then binds to a receptor for the activated substrate conjugate. Furthermore, the receptor for the activated substrate conjugate may be present in the sample immobilized on a solid support, leading to the deposition of the detectably labeled substrate.

[0010] In another embodiment, the method may further include (10) optionally emitting a coupled detectable label after step (8) by emitting a third emission linker, and optionally repeating steps (1) to (10) or any subset thereof.

[0011] In some embodiments, the method may further include amplifying the nucleic acid chain, or a portion thereof, that is linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0012] In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) The sample from step (1) or optionally from step (2) is brought into contact with the first member of the binding pair that is linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (i) or (ii) or both of the following conditions are met: (i) The nucleic acid chain is linked to a target-specific binding partner by a first release linker, and (ii) The first member of the bonding pair is linked to a complementary nucleic acid chain by a second release linker. (4) Optionally, remove the unbound first member of a binding pair linked to a complementary nucleic acid chain. (5) optionally bring the sample from step (3) or step (4) into contact with the second member of the binding pair linked to the enzyme by a third release linker, and optionally remove the unbound second member of the binding pair linked to the enzyme. (6) The sample from step (5) is brought into contact with a substrate conjugate consisting of a labeled substrate (the substrate and label are optionally linked by a fourth release linker). (7) Optionally, remove the unbound substrate conjugate. (8) optionally release the first or second member of the bonded pair, (9) Optionally, imaging the sample to detect a conjugated detectable label, and (10) Optionally, repeat steps (1) to (9) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before imaging step (9).

[0013] In some embodiments, the method may further include (11) optionally releasing a bound detectable label after step (9) by cleaving a third release linker between the label and the substrate, and optionally repeating steps (1) to (11) or any subset thereof.

[0014] In some embodiments, the method may further include amplifying the nucleic acid chain or a portion thereof that is linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0015] In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (provided that either (i) or (ii) or both of the following conditions are met: (i) The nucleic acid chain is linked to a target-specific binding partner by a first release linker, and (ii) The enzyme is linked to a complementary nucleic acid chain by a second release linker. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) Contacting the sample of step (3) or optionally step (4) with a substrate conjugate consisting of a nucleic acid-binding substrate (the substrate and the nucleic acid strand are optionally linked by a third releasable linker), (6) Optionally, removing unbound substrate conjugates, (7) Optionally, releasing the bound enzyme, (8) Contacting the sample of step (5) or optionally steps (6)-(7) with a nucleic acid-binding detectable label optionally linked by a fourth releasable linker (the nucleic acid strand is a specific binding partner member for the nucleic acid strand of step (5)), (9) Optionally, imaging the sample to detect the bound label, (10) Optionally, releasing the bound label of step (8), and (11) Optionally, repeating steps (1)-(10) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeating steps (1)-(8) or any subset thereof before step (9).

[0016] In some embodiments, the bound label is released with the use of a releasable linker. In some embodiments, in step (10), the bound label is released by cleaving the releasable linker of step (5) or optionally step (8) or optionally steps (5) and (8). In some embodiments, the bound label is released without the use of a releasable linker. In some embodiments, the bound label is released by nucleic acid strand dehybridization. In some embodiments, the bound label is released by dehybridization of the nucleic acid strand of step (8) that is bound to the nucleic acid strand of step (5).

[0017] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) linked to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0018] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) of the nucleic acid chain-bound substrate in step (5). In some embodiments, the amplification step is performed after step (5) or any step (6) or any step (7).

[0019] In some embodiments, the method may further include a first amplification of the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1), and a second amplification of the nucleic acid chain of the nucleic acid chain-bound substrate in step (5). In some embodiments, the first amplification step is performed before step (3), and the second amplification step is performed after step (5) or any step (6) or any step (7).

[0020] In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one target-specific binding partner (the target-specific binding partner is linked to the nucleic acid chain). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample with an HRP-binding nucleic acid chain that is complementary to the nucleic acid chain linked to the target-specific binding partner. (either (i) or (ii) or both are satisfied: (i) The nucleic acid chain is linked to a target-specific binding partner by a first release linker, and (ii) HRP is linked to a complementary nucleic acid chain by a second release linker. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a detectably labeled phenol-containing substrate (the substrate and the detectable label are optionally linked by a third release linker). (6) Optionally, remove unbound substrates. (7) Optionally, imaging the sample to detect a detectable label. (8) optionally releasing a bound label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (6) or any subset thereof before imaging step (7).

[0021] In some embodiments, after step (5), the phenolic portion is enzymatically converted to an activated state, resulting in the deposition of the label.

[0022] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0023] In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a detectably labeled substrate (the substrate and the detectable label are optionally linked by an evacuation linker). (6) Optionally, remove the unbound substrate conjugate. (7) Optionally, inactivate the bound enzyme. (8) Optionally, imaging the sample to detect a conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (7) or any subset thereof before imaging step (8).

[0024] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0025] In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) The sample from step (1) or optionally from step (2) is brought into contact with the first member of the binding pair, which is linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) Optionally, remove the unbound first member of a binding pair linked to a complementary nucleic acid chain. (5) Contact the sample from step (3) or optionally from step (4) with the second member of the binding pair linked to the enzyme, and optionally remove the unbound second member of the binding pair linked to the enzyme. (6) The sample from step (5) is brought into contact with a substrate conjugate consisting of a labeled substrate (the substrate and label are optionally linked by an evacuation linker). (7) Optionally, remove the unbound substrate conjugate. (8) Optionally, inactivate the bound enzyme. (9) Optionally, imaging the sample to detect a conjugated detectable label, and (10) Optionally, repeat steps (1) to (9) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before imaging step (9).

[0026] In some embodiments, the method further includes (11) optionally releasing a bound detectable label after step (9) by cleaving an emission linker between the label and the substrate, and optionally repeating any of steps (1) to (9) and (11).

[0027] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0028] In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a nucleic acid chain-binding substrate (the substrate and nucleic acid chain are optionally linked by a first release-type linker). (6) Optionally, remove the unbound substrate conjugate. (7) Optionally, inactivate the bound enzyme. (8) The sample from step (5) or optionally from steps (6)-(7) is brought into contact with a nucleic acid chain-bound detectable label, optionally linked by a second release linker (where the nucleic acid chain is a specific binding pair member to the nucleic acid chain from step (5)). (9) Optionally, imaging the sample to detect the conjugated label. (10) Optionally, release the bound label of step (8), and (11) Optionally, repeat steps (1) to (10) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before imaging step (9).

[0029] In some embodiments, the conjugated label is released with the use of an emission linker. In some embodiments, in step (10), the conjugated label is released by cleaving the emission linker in step (5) or optionally in step (8) or optionally in steps (5) and (8). In some embodiments, the conjugated label is released without the use of an emission linker. In some embodiments, the conjugated label is released by dehybridization of the nucleic acid chain. In some embodiments, the conjugated label is released by dehybridization of the nucleic acid chain in step (8) that is bound to the nucleic acid chain in step (5).

[0030] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0031] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) of the nucleic acid chain-bound substrate in step (5). In some embodiments, the amplification step is performed after step (5) or any step (6) or any step (7).

[0032] In some embodiments, the method may further include a first amplification of the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1), and a second amplification of the nucleic acid chain of the nucleic acid chain-bound substrate in step (5). In some embodiments, the first amplification step is performed before step (3), and the second amplification step is performed after step (5) or any step (6) or any step (7).

[0033] In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one target-specific binding partner (the target-specific binding partner is linked to the nucleic acid chain). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample with an HRP-binding nucleic acid chain that is complementary to the nucleic acid chain linked to the target-specific binding partner. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a detectably labeled phenol-containing substrate (the substrate and the detectable label are optionally linked by an evacuation linker). (6) Optionally, remove unbound substrates. (7) Optionally, inactivate HRP. (8) Optionally, imaging the sample to detect a conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (7) or a subset thereof before step (8).

[0034] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner of (1). In some embodiments, the amplification step is performed before step (3).

[0035] In some embodiments, the enzyme is inactivated using an enzyme inactivator, which includes dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, peroxides, cyanides, fluorides, or azides.

[0036] In some embodiments, the step of inactivating the conjugated enzyme (7) is carried out in less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, or 1 minute or less. In some embodiments, the step of inactivating the conjugated enzyme (7) is carried out in less than 20 minutes. In some embodiments, the step of inactivating the conjugated enzyme (7) is carried out in less than 10 minutes. In some embodiments, the step of inactivating the conjugated enzyme (7) is carried out in less than 5 minutes. In some embodiments, the step of inactivating the conjugated enzyme (7) is carried out in less than 2 minutes. In some embodiments, the step of inactivating the conjugated enzyme (7) is carried out in 1 minute or less.

[0037] Further objectives and benefits will be described in part in this specification, and in part will be apparent from this specification or may be revealed through practice. These objectives and benefits will be realized and achieved by the elements and combinations indicated in particular in the appended claims.

[0038] It should be understood that both the general statements above and the detailed statements below are merely illustrative and explanatory, and do not limit the scope of the claims.

[0039] The accompanying drawings incorporated herein and constituting part of this specification illustrate one (or more) embodiments and, together with the description, help illustrate the principles described herein.

[0040] The patent or application file shall contain at least one drawing performed in color. A copy of the published patent or patent application accompanied by the color drawing(s) shall be provided by the Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0041] [Figure 1] Two schematic diagrams of multiplexed catalytic reporter deposition (CARD) are provided. Diagram A shows a schematic without stacking. Diagram B shows a schematic with stacking. [Figure 2] A schematic diagram of multiplexed catalytic reporter deposition (CARD) using binding pairs, where the binding pair is a nucleic acid chain (a barcode or containing a barcode) and a complementary nucleic acid chain, is provided. [Figure 3] This specification illustrates an exemplary embodiment in which two target molecules are detected using the multiplexing method described herein. [Figure 4A] An exemplary embodiment of how the enzyme is cleaved is shown. [Figure 4B] An exemplary embodiment in which the enzyme is inactivated is shown. [Figure 5] An exemplary embodiment in which a substrate barcode is used is shown. [Figure 6] A scheme for synthesizing exemplary substrate barcodes is shown. [Figure 7] Quadriplex images of CD8, CD68, PD-L1, and CK targets (without nuclear counterstaining) obtained using sequential deposition of fluorescent tyramide reagent according to Example 1 are shown. CD8 is shown in green; CD68 in red; PD-L1 in cyan; and CK in magenta. Nuclear counterstaining is not shown. [Figure 8]The second example shows a comparison of HRP enzyme inactivation by TCEP treatment (Figure 8A) versus a control without TCEP treatment (Figure 9B). [Figure 9] Quadruple images of CD8, CD68, PD-L1, and CK targets obtained using sequential deposition of nucleic acid barcodes according to Example 3 are shown. CD8 is shown in green; CD68 in red; PD-L1 in cyan; and CK in magenta. Nuclear counterstaining is not shown. [Modes for carrying out the invention]

[0042] Description of the Embodiment I. Definition The term "deposition" refers to the direct binding of an activating substrate conjugate to a receptor, resulting from the formation of specific binding pair interactions, as described below.

[0043] The term "receptor" refers to a site that binds to an activating substrate conjugate via the formation of specific binding pair interactions, as described below.

[0044] The term "activated substrate conjugate" means that the substrate conjugate is primed by a reporter enzyme in order to bind to a receptor.

[0045] The term "detectably labeled" substrate conjugate means that the substrate can bind to either a detectable label (the term "reporter" may be used interchangeably) or an unlabeled first member of a specific binding pair to facilitate detection after deposition. If the substrate binds to the unlabeled member of the specific binding pair, after deposition, the substrate-specific binding pair complex reacts with the second member of the binding pair that binds to the reporter (or label). Alternatively, the substrate-specific binding pair complex can be pre-reacted with the detectably labeled second member of the specific binding pair before deposition.

[0046] Where used herein, the terms “amplification” or “amplification” refer to increasing the copy number of a nucleic acid sequence, such as a nucleic acid chain or a portion thereof (e.g., a barcode), so that multiple copies of the nucleic acid sequence are linked to their respective target-specific binding partners. Various amplification methods known in the art may be used to increase the copy number of a nucleic acid sequence. Examples of nucleic acid amplification methods include hybridization chain reaction (HCR) (Dirks et al., 2014, PMID: 15492210, 24712299), DNA hairpin-based dendritic reaction (HDR) (Yin et al., 2008, PMID 18202654), rolling circle amplification (RCA), primer exchange reaction (PER), and other nucleic acid amplification methods such as those described in WO2018 / 107054; WO2017 / 143006; WO2018 / 132392A2, the contents of which are incorporated herein by reference. This amplification is different from the signal amplification generated by the CARD.

[0047] II. Method for Deposition of Multiplexed Catalytic Reporters This application discloses a method for multiple catalytic reporter deposition (CARD). Schematic diagrams of multiple catalytic reporter deposition are shown in Figures 1A, 1B, 2, and 3.

[0048] According to this specification, in some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is optionally linked to a nucleic acid chain using an evacuation linker, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample with an enzyme or a member of a specific binding pair for subsequent enzymatic labeling, which is linked to a nucleic acid chain complementary to the chain on the target-specific binding partner (if the nucleic acid chain bound to the target-specific binding partner is not linked using a release linker, the enzyme or a member of the specific binding pair is linked to the complementary chain in step (3) using a release linker; if the nucleic acid chain bound to the target-specific binding partner is linked using a release linker, the enzyme or a member of the specific binding pair is optionally linked to the complementary chain in step (3) using a release linker). (4) Optionally, remove any unbound enzymes or specific binding pair members linked to complementary nucleic acid chains. (5) If a member of a specific binding pair is used in step (3) to bring the sample into contact with a second member of a specific binding pair that is optionally linked to the enzyme by a release linker, optionally remove the unbound enzyme linked to the second member of the specific binding pair. (6) The sample is brought into contact with a substrate conjugate consisting of a detectably labeled substrate (the substrate and label are optionally linked by an evacuation linker). (7) Optionally, remove the unbound substrate conjugate. (8) Optionally, release a conjugated enzyme, a first or second specific binding pair member, (9) Optionally, detect a combined detectable sign. (10) Optionally, repeat steps (1) to (9) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before step (9) of detection.

[0049] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0050] A. Multiple cards in which the enzyme is bound to the probe chain. In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (provided that either (i) or (ii) or both of the following conditions are met: (i) The nucleic acid chain is linked to a target-specific binding partner by a first release linker, and (ii) The enzyme is linked to a complementary nucleic acid chain by a second release linker. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a detectably labeled substrate (the substrate and label are optionally linked by a third release linker). (6) Optionally, remove the unbound substrate conjugate. (7) optionally release the bound enzyme, (8) Optionally, imaging the sample to detect a conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (7) or any subset thereof before imaging step (8).

[0051] In some embodiments, in step (5), a substrate conjugate consisting of a detectably labeled substrate reacts with an enzyme to form an activated substrate conjugate, which then binds to a receptor for the activated substrate conjugate.

[0052] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0053] In some embodiments, receptors for the activating substrate conjugate are present in the sample, leading to the deposition of a detectable label.

[0054] In some embodiments, all labels are removed, and the above step for labeling is restarted ("stacking") to allow for another round of multiplexing. In some embodiments, the method further includes (10) emitting the bound detectable label after the imaging step (8), and optionally repeating steps (1) to (10) or any subset thereof. In some embodiments, the bound label is emitted using an emission linker. In some embodiments, in step (10), the bound label is emitted by cutting a third emission linker. In some embodiments, the bound label is emitted without using an emission linker.

[0055] Figures 1A and 1B show embodiments of a multiplexed card method that does not include stacking (Figure 1A) and includes stacking (Figure 1B).

[0056] In the stacking-free multiplexed CARD shown in Figure 1A, a sample (tissue, 100) is first obtained in which a target-specific binding partner (antibody, 101) is bound to a nucleic acid chain containing a barcode (barcode, 102). This sample (tissue, 100) is then brought into contact with an enzyme (103) linked to a nucleic acid chain (probe, 104) complementary to the nucleic acid chain containing the barcode (barcode, 102) linked to the antibody (101). The nucleic acid chain containing the barcode (barcode, 102) is linked to the antibody (101) by a first release linker (105). Alternatively, the enzyme (103) is linked to a complementary nucleic acid chain containing a sequence complementary to the barcode (probe, 104) having a second release linker (106). Optionally, any unbound enzyme linked to the complementary nucleic acid chain is removed.

[0057] Next, a substrate conjugate containing a detectable label (108) (labeled substrate, 107) is brought into contact with the sample (100). The detectable label reacts with the enzyme to form an activated substrate conjugate, which then binds to receptors for the activated substrate conjugate present in the sample, resulting in the deposition of the detectable label on the sample (108). Unbound substrate conjugate (107) is optionally removed in a washing step. The enzyme is then released by cleaving the release linker with a cleavage agent. Depending on the use of the first release linker (105) or the second release linker (106), after cleavage of the release linker, either (bottom left) the enzyme (103) linked to the complementary nucleic acid chain (probe, 104) is removed, or (bottom right) the nucleic acid chain is cleaved, resulting in the removal of the enzyme, the nucleic acid chain containing the barcode (barcode, 102) linked to the antibody (101), and the complementary nucleic acid chain (probe, 104) linked to the enzyme. The above deposition of detectable labels can be repeated for each target. The sample is then imaged to detect the conjugated detectable labels of various targets.

[0058] In the multiplexed CARD with stacking shown in Figure 1B, the substrate (107) and label (108) can be linked by a third release linker (109), and after the imaging step, all of the bound label is removed by cleaving the third release linker (109), and the above step is repeated for label deposition for detection of more targets ("stacking").

[0059] Figure 3 also shows an exemplary embodiment in which two target molecules are detected using the method described herein. In a tissue sample, two targets 1 and 2 are bound to their respective antibodies, each antibody being linked to a different nucleic acid chain (Step A). ​​An enzyme (enzyme probe 1) is linked to the nucleic acid chain linked to the antibody of target 1, and the nucleic acid chain is linked to a nucleic acid chain complementary to the nucleic acid chain linked to the antibody of target 1. Unbound enzyme probes are optionally washed (Step B). Next, a labeled substrate is added to the sample (Step C) and reacts with the enzyme to form an activated substrate conjugate. The activated substrate conjugate binds to receptors for the activated substrate conjugate present in the sample, resulting in the deposition of a detectable label (Step D). Enzyme probe 1 is released by cleaving the release linker with a cleavage agent (Step E) or by inactivating the enzyme with an enzyme inhibitor (or enzyme inactivator) (Step F). In step 3, depending on the position of the release linker, the enzyme is removed, or the barcode and probe linked to the enzyme are also removed along with the enzyme (step G). The above deposition of the label can be repeated for target 2 (steps H and I). The sample is then imaged to detect the conjugated detectable label on both targets. Optionally, the label signal may be reduced as needed by using methods well known in the art (e.g., chemical fading, photobleaching, and / or photochemical fading).

[0060] B. Bonded pair as multiple cards In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) The sample from step (1) or optionally from step (2) is brought into contact with the first member of the binding pair that is linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (i) or (ii) or both of the following conditions are met: (i) The nucleic acid chain is linked to a target-specific binding partner by a first release linker, and (ii) The first member of the bonding pair is linked to a complementary nucleic acid chain by a second release linker. (4) Optionally, remove the unbound first member of a binding pair linked to a complementary nucleic acid chain. (5) optionally bring the sample from step (3) or step (4) into contact with the second member of the binding pair linked to the enzyme by a third release linker, and optionally remove the unbound second member of the binding pair linked to the enzyme. (6) The sample from step (5) is brought into contact with a substrate conjugate consisting of a labeled substrate (the substrate and label are optionally linked by a fourth release linker). (7) Optionally, remove the unbound substrate conjugate. (8) optionally release the first or second member of the bonded pair, (9) Optionally, imaging the sample to detect a conjugated detectable label, and (10) Optionally, repeat steps (1) to (9) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before imaging step (9).

[0061] In some embodiments, the method may further include amplifying the nucleic acid chain linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0062] In some embodiments, in the case of stacking, the method further includes (11) optionally releasing a bound-type detectable label. In some embodiments, the bound-type label is released by releasing a third release-type linker between the label and the substrate. In some embodiments, the bound-type label is released without using a release-type linker.

[0063] In some embodiments, the method further includes (11) optionally releasing a bound detectable label after the imaging step (9) by releasing a third release linker, and further optionally repeating steps (1) to (11) or any subset thereof. In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (provided that either (i) or (ii) or both of the following conditions are met: (i) The nucleic acid chain is linked to a target-specific binding partner by a first release linker, and (ii) The enzyme is linked to a complementary nucleic acid chain by a second release linker. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a nucleic acid chain-binding substrate (the substrate and nucleic acid chain are optionally linked by a third release linker). (6) Optionally, remove the unbound substrate conjugate. (7) optionally release the bound enzyme, (8) The sample from step (5) or optionally from steps (6)-(7) is brought into contact with a nucleic acid chain-bound detectable label, optionally linked by a fourth release linker (where the nucleic acid chain is a specific binding pair member to the nucleic acid chain from step (5)). (9) Optionally, imaging the sample to detect the conjugated label. (10) Optionally, release the bound label of step (8), and (11) Optionally, repeat steps (1) to (10) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before imaging step (9).

[0064] In some embodiments, the bound label is released along with the release linker. In some embodiments, the bound label is released by cleaving the release linker in step (5) or optionally in step (8) or optionally in steps (5) and (8). In some embodiments, the bound label is released without the use of a release linker. In some embodiments, the bound label is released by dehybridization of the nucleic acid strands bound to each other. In some embodiments, the bound label is released by dehybridization of the nucleic acid strand in step (8) that is bound to the nucleic acid strand in step (5). Dehybridization of nucleic acids may be achieved by conventional methods known in the art, including heating, addition of chaotropic agents (e.g., urea, formamide, and guanidinium chloride), and low ionic strength. The binding affinity between nucleic acid strands is broken by increasing the temperature or decreasing the ionic strength of the culture medium containing the nucleic acid strands. Heating, one or more chaotropic agents, and low ionic strength can each be used independently to dehybridize nucleic acids, or one or more of these conditions can be used in combination. As used herein, “low ionic strength” means the ionic strength of a medium as measured by the amount of salt concentration in the medium, which is less than 300 mM. In one embodiment, the salt concentration is below the concentration required for Tm to be lower than the ambient temperature, for example, below 70 mM. The hybridization rate decreases as the salt concentration decreases. Dehybridization at low ionic strength is not performed by conventional methods, such as adding water to remove salt (e.g., Na). + This may be achieved by reducing the concentration.

[0065] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1). In some embodiments, the amplification step is performed before step (3).

[0066] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) of the nucleic acid chain-bound substrate in step (5). In some embodiments, the amplification step is performed after step (5) or any step (6) or any step (7).

[0067] In some embodiments, the method may further include a first amplification of the nucleic acid chain (or a portion thereof) linked to the target-specific binding partner in step (1), and a second amplification of the nucleic acid chain of the nucleic acid chain-bound substrate in step (5). In some embodiments, the first amplification step is performed before step (3), and the second amplification step is performed after step (5) or any step (6) or any step (7).

[0068] Figure 2 shows an embodiment of a multiplexed catalytic reporter deposition (CARD) method using binding pairs, where the binding pair is a nucleic acid chain (a barcode or containing a barcode) and a complementary nucleic acid chain.

[0069] First, a sample (tissue, 200) is obtained in which a target-specific binding partner (antibody, 201) is bound to a nucleic acid chain containing a barcode (barcode, 202). This sample is then brought into contact with an enzyme (203) linked to a nucleic acid chain (probe, 204) complementary to the nucleic acid chain (barcode, 202) linked to the antibody (201). The nucleic acid chain (barcode, 202) is linked to the antibody (201) by a first release linker (205). Alternatively, the enzyme (203) is linked to a complementary nucleic acid chain (probe, 204) by a second release linker (206). Optionally, any unbound enzyme (203) linked to the complementary nucleic acid chain (202) is removed.

[0070] Next, the sample (200) is brought into contact with the nucleic acid chain-binding substrate (217), and the substrate (217) and nucleic acid chain (227) are deposited on the tissue. Then, the unbound substrate conjugate and / or binding enzyme are removed by cleaving the release linker with a cleavage agent (e.g., TCEP, DTT, or periodate). The above deposition of nucleic acid-binding substrate can be repeated for each target. A washing step in the final round of deposition is optional.

[0071] Next, a tissue sample (200) is brought into contact with a nucleic acid chain-bound detectable label (208) optionally linked to a fourth release linker (209), where the nucleic acid chain (probe, 208) is a specifically binding pair member to a nucleic acid chain (227) complementary to the probe. If four primary antibodies binding to four different targets are provided, then four different labeled probes (208) are provided. The tissue sample (200) is imaged to detect the detectable labels bound to various targets. To detect further targets, after the imaging step, all bound labels are removed in a washing step to cleave the third release linker (209) between the label and probe (208) using a cleavage agent, and the above steps are repeated for label deposition ("stacking") to detect more targets. The above steps or any subset thereof are repeated as needed to detect more targets.

[0072] C. Embodiments using phenolic substrates In some embodiments, the multiplexed CARD method is a multiplexed tyramide signal amplification (TSA) method. TSA amplification methods that allow for the easy decolorization of fluorophores may also be used. For example, many cyanine fluorophores and Alexa fluorophores can be easily decolorized with hydrogen peroxide under acidic or basic conditions (PMID: 26399630). Alternatively, TSA dyes containing cleavable bonds between the tyramide and fluorophores can be synthesized. In this case, the fluorophores can be inactivated by cleaving these bonds and washing.

[0073] In some embodiments, a method for testing a sample for the presence of one or more targets includes: (1) contacting the sample to be tested for the presence of one or more targets with one target-specific binding partner (the target-specific binding partner is linked to a nucleic acid chain); (2) optionally removing an unbound target-specific binding partner; (3) contacting the sample with an HRP-binding nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (i) or (ii) or both: (i) the nucleic acid chain is linked to the target-specific binding partner by a first release linker, and (ii) HRP is linked to the complementary nucleic acid chain by a second release linker); (4) optionally complementary (5) Remove the unbound enzyme linked to the target nucleic acid chain; (6) Contact the sample from step (3) or optionally step (4) with a substrate conjugate consisting of a detectably labeled phenol substrate (the substrate and detectable label optionally linked by a third release linker); (7) Optionally remove the unbound substrate; (8) Optionally image the sample to detect it; (9) Optionally release the bound label; and (1) to (2) Optionally repeat steps (1) to (3) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (4) or any subset thereof before imaging step (7).

[0074] In some embodiments, after step (5), the phenol substrate is enzymatically converted to an activated state, resulting in the deposition of the label. In some embodiments, the phenol substrate is a tyramide.

[0075] In some embodiments, the method may further include amplifying a nucleic acid chain (or a portion thereof) linked to a target-specific binding partner. In some embodiments, the amplification step is performed before step (3) of contacting the sample with an HRP-binding nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. Catalytic reporter deposition is a signal amplification method that utilizes an enzyme directly or indirectly bound to a target-specific binding partner, which catalyzes the conversion of a substrate conjugate consisting of a detectably labeled substrate to an activated substrate conjugate that binds to a receptor for the activated substrate conjugate, resulting in the deposition of a detectably labeled substance. Examples of conventional catalytic reporter deposition were described, for example, in U.S. Patents 5,196,306, 5,583,001, and 5,731,158.

[0076] Catalytic reporter deposition is used to enhance signals in immunohistochemistry, immunocytochemistry, in situ hybridization, ELISA, flow cytometry, electron microscopy, and other applications.

[0077] However, previous catalytic reporter deposition methods have several limitations. One limitation is the ability to multiplex, i.e., the ability to detect multiple assay targets in the same assay. This limitation worsens when using homogeneous antibodies in the assay. Detection of two targets using homogeneous antibodies has been achieved by using catalytic reporter deposition to detect one target and standard detection for the second target (Shindler J. Histochem Cytochem 1996 Vol 44 No 11 1331-1335).

[0078] Previous attempts to multiplex immunohistochemical assays using catalytic reporter deposition involved microwave irradiation of tissue sections between each round of reporter deposition (Toth J. Histochem Cytochem 2007 Vol 55(6)545-554). Microwave irradiation removes antibodies and reporter enzymes, but the detectable label remains intact. Microwave irradiation adds many steps, as each antibody and reporter must be added sequentially and then microwaved to remove them. As an example of microwave irradiation use, the Opal® 7 Solid Tumor Immunotherapy Kit (PerkinElmer) requires more than 90 minutes per cycle, and the entire procedure takes two days to complete. Microwave irradiation can also modify or damage the sample being imaged.

[0079] In another previous attempt, thermal inactivation was used to detect a quintuple assay (Zhang, W., Hubbard, A., Jones, T. et al. j. immunotherapy cancer (2015) 3(Suppl 2):P111). Four thermal inactivation cycles reduced the fluorescence of 12–50% of the five fluorescent labels used. The assay took 9 hours to complete. Other changes due to heating are described in U.S. Patents 9,689,875 and 9,874,571, incorporated herein by reference, regarding techniques of thermal changes. However, thermal inactivation may alter or damage the sample being imaged.

[0080] With microwave irradiation or thermal inactivation between cycles, the number of detectable targets is still limited by the number of labels that the reader and software can distinguish. In the field of immuno-oncology, the number of targets to be detected already ranges from 12 to 20, which far exceeds the limitations of the reader and software.

[0081] The method described above in this disclosure provides a signal amplification method that can be multiplexed in a simple and rapid manner, avoiding harsh sample processing such as heating and microwave irradiation, and having a higher level of multiplexing capability.

[0082] D. Kit In some embodiments, the kit comprises a composition comprising (1) one or more target-specific binding partners (each binding partner being bound to a nucleic acid chain), (2) an enzyme linked to a nucleic acid chain complementary to the nucleic acid chains bound to one or more target-specific binding partners (either (i) or (ii) or both are satisfied: (i) the nucleic acid chain is linked to each target-specific binding partner by a first release linker, and (ii) the enzyme is linked to the complementary nucleic acid chain by a second release linker), and (3) a substrate conjugate comprising a detectably labeled substrate (the substrate and label optionally linked by a third release linker).

[0083] In some embodiments, the kit comprises a composition comprising: (1) one or more target-specific binding partners (each binding partner being bound to a nucleic acid chain); (2) binding pairs comprising a first member linked to a nucleic acid chain complementary to the nucleic acid chain bound to each target-specific binding partner, and a second member linked to an enzyme (i) or (ii) or both of the following: (i) the nucleic acid chain is linked to each target-specific binding partner by a first release linker, and (ii) the first member of the binding pair is linked to the complementary nucleic acid chain by a second release linker); and (3) a substrate conjugate comprising a detectably labeled substrate (the substrate and label optionally linked by a third release linker, and the second member of the binding pair being linked to an enzyme by a fourth release linker).

[0084] In some embodiments, the kit further includes a fluid system for performing a fluid exchange step, software for controlling the fluid system and timing, and / or synchronizing the fluid step with an imaging step. In some embodiments, the kit further includes an imaging chamber fixed on the sample of interest on at least one optically transparent side to enable imaging of the sample.

[0085] III. Components of the Method Enzymes used in A.CARD Enzymes suitable for direct or indirect binding to target-specific binding partners include hydrolases, lyases, oxidoreductases, transferase isomerases, ligases, peroxidases, oxidases, phosphatases, esterases, and glycosidases. Specific examples include alkaline phosphatase, lipase, β-galactosidase, and horseradish peroxidase (HRP). Multiple enzymes can be used simultaneously, in combination with substrate conjugates specific to each enzyme, as needed.

[0086] B. Specific binding pairs Members of specific binding pairs suitable for use in the implementation of the present invention may be immunogenic or non-immunogenic. Immunogenic specific binding pairs are exemplified by antigen / antibody systems or hapten / anti-hapten systems. The antibody member of a binding pair, whether polyclonal, monoclonal, or its immunoreactive fragment, can be produced by conventional methods well known to those skilled in the art. The terms immunoreactive antibody fragment or immunoreactive fragment mean a fragment containing an antibody binding region. Such fragments may be Fab-type fragments, e.g., Fab, Fab', and F(ab')2 fragments, defined as fragments lacking an Fc moiety, obtained by reductive cleavage of disulfide bonds linking the heavy chain components of an intact antibody. The fragments may also be single-domain antibodies derived from heavy or light chain antibodies and may contain nanobodies. If the antigen member of a specific binding pair is non-immunogenic, e.g., a hapten, it can be covalently bound to a carrier protein to confer immunogenicity.

[0087] Non-immune binding pairs include systems in which two components share a natural affinity for each other but are not antibodies. Exemplary non-immune binding pairs include biotin-avidin or biotin-streptavidin, complementary probe nucleic acid folate-folate-binding proteins, etc. Non-immune binding pairs also include those that form covalent bonds with each other. Exemplary covalent pairs include sulfhydryl-reactive groups, e.g., maleimides and haloacetyl derivatives, as well as amine-reactive groups, e.g., isothiocyanates, succinimidyl esters, sulfonyl halides, and click chemistry, as well as coupler dyes, e.g., 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB).

[0088] Complementary probe nucleic acids can be used to enhance the signal by increasing the number of binding sites to the nucleic acid probe using methods such as rolling circle amplification (Paul M Lizardi, et al. Nature Genetics, 19(3):225-232, 1998) and primer exchange reactions (WO2017 / 143006 Al).

[0089] C. Target-specific binding - Partner Suitable target-specific binding partners for carrying out the present invention may be immunotype or non-immunotype. Immunotype-specific binding pairs are exemplified by antigen / antibody systems. The antibody member of a binding pair, whether polyclonal, monoclonal, or its immunoreactive fragment, can be generated by conventional methods well known to those skilled in the art. The terms immunoreactive antibody fragment or immunoreactive fragment mean a fragment containing the binding domain of an antibody. Such fragments may be Fab-type fragments, e.g., Fab, Fab', and F(ab')2 fragments, defined as fragments lacking an Fc moiety, obtained by reductive cleavage of disulfide bonds linking the heavy chain components of an intact antibody. Fragments may also be single-domain antibodies derived from heavy or light chain antibodies and may contain nanobodies. If the antigen member of a specific binding pair is non-immunogenic, e.g., a carbohydrate or phosphorylated amino acid, it can be covalently bound to a carrier protein to confer immunogenicity.

[0090] Table 1 shows a representative list of targets and their corresponding target recognition components. TIFF2026071202000002.tif206170

[0091] Table 2 provides a list of additional targets. Antibodies and other known binding partners for these targets may be used as target recognition moieties. TIFF2026071202000003.tif172170

[0092] Non-immune binding pairs include systems in which two components share a natural affinity for each other but are not antibodies.

[0093] Exemplary non-immune binding pairs include complementary probe nucleic acids and aptamers. Complementary probe nucleic acids are suitable target-specific binding partners for nucleic acid (e.g., DNA or RNA) targets.

[0094] 1. Nucleic acid chains used for binding pairs In some embodiments, the nucleic acids used in the binding pair are single-stranded nucleic acids such as single-stranded DNA, RNA, or nucleic acid analogs. Nucleic acid analogs (also known as non-natural nucleic acids) may include modified phosphate skeletons, modified pentose sugars, and / or modified nucleic acid bases. Nucleic acid analogs may include, but are not limited to, 2'-O-methylribonucleic acid, 2'-fluororibonucleic acid, peptide nucleic acids, morpholino and locked nucleic acids, glycolic acid, and threose nucleic acids.

[0095] In some embodiments, the nucleic acid chain comprises single-stranded nucleic acids and may be about 5–20 nucleotides long, about 8–15, or about 10–12 nucleotides long. In some embodiments, the nucleic acid chain is about 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, or 20 nucleotides long.

[0096] The nucleic acid chain may be an independent element or part of the target recognition region.

[0097] Nucleic acid chains may be provided in a liquid culture medium or buffer.

[0098] The target-specific binding partner may be provided in a liquid culture medium or buffer.

[0099] D. Binding of the activating substrate conjugate to the receptor The direct binding of an activating substrate conjugate to a receptor results from the formation of a specific binding pair interaction. Members of a specific binding pair suitable for use with an activating substrate that binds to a receptor may be immunotype or non-immunotype. Immunotype-specific binding pairs are exemplified by antigen / antibody systems or hapten / anti-hapten systems. The antibody member of a binding pair, whether polyclonal, monoclonal, or its immunoreactive fragment, can be produced by conventional methods well known to those skilled in the art. The term immunoreactive antibody fragment or immunoreactive fragment means a fragment containing an antibody binding region. Such fragments may be Fab-type fragments, e.g., Fab, Fab', and F(ab')2 fragments, defined as fragments lacking an Fc moiety, obtained by reductive cleavage of disulfide bonds linking the heavy chain components of an intact antibody.

[0100] Non-immune binding pairs include systems in which two components share a natural affinity for each other but are not antibodies. Exemplary non-immune binding pairs are pairs that form a covalent bond with each other. Exemplary covalent bonding pairs include, but are not limited to, dimerization of phenolic moieties (e.g., tyramine and tyrosine), sulfhydryl reactive groups (e.g., maleimide and haloacetyl derivatives), amine reactive groups (e.g., isothiocyanates, succinimidyl esters, sulfonyl halides, and click chemistry), and coupler dyes (e.g., 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB)).

[0101] E. Reporter or Sign A wide variety of reporters (or labels) are available to bind to substrates to form substrate conjugates or to members of specific binding pairs. Reporters (or labels) may be, but are not limited to, enzymes, fluorescent, colorimetric, chemiluminescent, mass tags, magnetic, plasmons, or electrochemical materials.

[0102] In some embodiments, but not limited to, fluorescent molecules such as fluorophores, for example, small organic molecules including fluorescein, rhodamine, cyanine dyes, Alexa dyes, DyLight dyes, Atto dyes, etc., may be used.

[0103] The color-developing, fluorescent, and chemiluminescent materials may include, but are not limited to, 3,3'-diaminobenzidine (DAB), nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-Gal).

[0104] In some embodiments, organic polymers such as p-dots may be used. In some embodiments, the observable portion may be a biomolecule, including, but not limited to, a fluorescent protein or fluorescent nucleic acid (including fluorescent RNA, including spinach and its derivatives). In some embodiments, the observable portion may be an inorganic portion, including a Q-dot. In some embodiments, the observable portion may be a portion that functions via either elastic or inelastic scattering, such as nanoparticles and surface-enhanced Raman spectroscopy (SERS) reporters (e.g., 4-mercaptobenzoic acid, 2,7-mercapto-4-methylcoumarin). In some embodiments, the observable portion may be a chemiluminescent / electrochemiluminescent emitter, such as a ruthenium complex and luciferase. The observable portion may produce an optical signal, an electromagnetic signal (across the entire electromagnetic spectrum), an atomic / molecular weight (e.g., detectable by mass spectrometry), a real mass (e.g., detectable by atomic force microscopy), an electric current, or a voltage.

[0105] F. Substrate The substrates used in substrate conjugates are specific to the selected enzyme and are known to those skilled in the art. Many phenolic substrates used with horseradish peroxidase (U.S. Patents 5,196,306, 5,583,001, 5,573,1158, 5,863,748, and 6,355,443) are described, each of which is incorporated by reference for teaching phenolic substrates. Substrates for hydrolases are also described (U.S. Patents 5,196,306, 5,583,001, 5,573,1158, 7,291,474, and Polaske, Bioconjugate Chemistry (2016)), each of which is incorporated by reference for teaching hydrolase substrates.

[0106] In some embodiments, the enzyme is horseradish peroxidase (HRP), and the substrate is a phenolic substrate (e.g., tyramine (Figure 6) and tyrosine). In some embodiments, the enzyme is a hydrolase, and the substrate is an ester, amide, or glycoside substrate.

[0107] G. Release-type linker Throughout this disclosure, first, second, or third emission linkers may be referred to, and in many embodiments, some or all of them may be optional. Simply referring to a second or third emission linker does not require the presence of two or three emission linkers. Instead, this numbering is used throughout this application to name the linkers and distinguish them from one another. Thus, embodiments including a second emission linker do not necessarily have two emission linkers, etc. This language indicates only which linkers are present, not how many linkers are present.

[0108] Release linkers incorporate functionalities that allow them to link two or more molecules, such as proteins, nucleic acids, proteins and nucleic acids, and substrates and detectable labels, and incorporate bonds that can be cleaved without heating or microwave irradiation. Exemplary cleavable bonds include disulfide bonds (cleaved by reducing agents such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP)), esters (cleaved by hydroxylamine), vicinal diols (cleaved by sodium metaperiodate), sulfones (cleaved under basic conditions), and photocleavable linkers containing a 2-nitrobenzyl group.

[0109] Another method using release linkers is to completely remove the enzyme or specific binding pair member bound to the nucleic acid chain. Removal can be facilitated by disrupting the binding affinity between nucleic acid chains or by enzymatically cleaving the specific nucleic acid sequence. Exemplary removal methods include the use of USER enzyme (New England Biolabs), a decrease in the ionic strength of the culture medium, an increase in temperature, and / or the use of chaotropic agents such as guanidine, ethylene carbonate, and / or formamide.

[0110] In some embodiments, the release linker includes a photocleavable linker that can be cleaved photochemically (e.g., by UV exposure, visible light, infrared, near-infrared, X-rays, microwaves, radio waves, or gamma rays). In some embodiments, the release linker includes a moiety that can be cleaved enzymatically. Examples of such enzymatically cleavable moieties include, but are not limited to, ribonucleotides that can be cleaved by various RNases; deoxyuridines that can be cleaved by combinations of enzymes such as USER (New England Biolabs); and restriction sites that can be cleaved by sequence-specific nicking enzymes or restriction enzymes. In some embodiments, the release linker includes a deoxyuridine whose uracil group can be cleaved by uracil-DNA glycosylase. In some embodiments, the release linker includes a debasic site that can be cleaved by an endonuclease.

[0111] 1. Nucleolytic enzyme Numerous enzymes can cleave covalent bonds within nucleic acid molecules. For example, some glycosylases can remove bases from the sugar portion of nucleotides, and endonucleases can cleave bonds within phosphodiester bridges within nucleic acid molecules, while exonucleases can similarly cleave phosphodiester bridges at the 5' or 3' end of nucleic acid molecules in a sequential manner. Another example includes DNAzymes or deoxyribozymes, oligonucleotides with catalytic activity capable of cleaving phosphodiester bonds in nucleic acid molecules. All of these types of enzymes may be manipulated to release release linkers, constituting enzymatic cleavage, modification, or degradation of release linkers.

[0112] Glycosylase. Glycosylases can reduce the strength of the interaction between two strands if they can specifically remove bases involved in base pairing. For example, deoxyuridine (dU) can be used to replace deoxythymidine (dT) in release linkers. dU can pair with dA in the same way that dT pairs, but can be removed in particular by uracil DNA glycosylase (UDG, commercially available from New England Biolabs, catalog number M0280S). This reaction will produce the debasement site(s) on the nucleic acid strand linked to the detectable label. Such debasement sites can be further cleaved by endonuclease VIII. Furthermore, this promotes the dissociation of the rest of the binding pair. Enzyme blends containing both UDG and endonuclease VIII are also commercially available (e.g., from New England Biolabs, under the USER brand name, catalog number M5505S). Approximately 1–20, 1–15, 1–10, or 1–5 dU nucleotides may be positioned in the nucleic acid chain linked to the detectable label. When using USER, the dU may be positioned such that, after U removal, the remainder is short enough (e.g., approximately 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide or less) to dissociate spontaneously and rapidly. When using UDG (i.e., endonuclease VIII) alone, removing the dU unit may make the chain unstable enough to facilitate removal. The total number of base pairs between binding pairs after dU removal may be 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide or less. Therefore, in some embodiments, the release linker may contain at least one U that can be cleaved by USER.

[0113] In addition to temperature, the remaining sequence influences the desired remaining length for spontaneous dissociation. For example, a shorter sequence with a higher GC content may have a higher binding affinity than another longer sequence. Thus, in some cases, the necaper may be sufficient for stable binding, and in other cases, it may be sufficient for dissociation. Those skilled in the art can evaluate the sequence, temperature, and affinity in the cleavage of non-natural nucleotides discussed here and below.

[0114] Restriction endonucleases and nickeling endonucleases. The restriction site of the release linker may be manipulated. This allows the use of a corresponding restriction endonuclease that cleaves such a restriction site, thereby disrupting the release linker. As an example, Cas9 (CRISPR-related protein 9) is an RNA-induced endonuclease that can be used to specifically cleave the release linker by manipulating a specific recognition site of the corresponding sequence. This cleaves both strands, preventing re-searching for the corresponding target. To solve this problem, nickeling endonucleases that cleave only one strand may be used. As an example, Cas9 nickase is a Cas9 enzyme designed to contain only one active cleavage site where a single-strand break occurs while maintaining the high specificity of Cas9. Other examples of site-directed endonucleases include, but are not limited to, zinc finger nucleases, activator-like effector nucleases (TALENs), and deoxyribozymes.

[0115] Rnase. Instead of DNA nucleotides (also called deoxynucleotides), some or all of the nucleotides may be converted into release linker RNA nucleotides (also called ribonucleotides). Such RNA nucleotides can be removed with Rnase.

[0116] Polymerase. Released linkers can also be removed by using polymerases that have chain displacement activity or 5'-to-3' exonuclease activity.

[0117] Cleavage of non-natural nucleotides. Non-natural nucleotides that function as substrates for specific enzymes may be used. For example, 8-oxoguanine may be cleaved by DNA glycosylase OGG1. Debase sites may also be incorporated into DNA strands, such as DNA strands linked to detectable labels that can be cleaved by endonucleases. For example, 1',2'-dideoxyribose, dSpacer, aprine / apyrimidine, tetrahydrofuran, or debase furan may be cleaved by endonuclease VIII. Thus, in some embodiments, the nucleic acid strand linked to the detectable label or intermediate strand may contain at least one debase site that can be cleaved by endonuclease VIII. In some embodiments, the nucleic acid strand linked to the detectable label or intermediate strand may contain at least one deoxyuridine and at least one debase site that can be cleaved by USER, UDG, or endonuclease VIII. Photocleavable spacers or RNA debase sites, such as ribospacers (rSpacer) or Abasic II modifications, may also be used. Other pairs of non-natural nucleotides and their paired enzymes may also be used.

[0118] H. Enzyme inactivation An alternative method for using a release linker or removing an enzyme or specific binding pair member bound to a nucleic acid chain is to inactivate the enzyme. Exemplary enzyme inactivators include, but are not limited to, peroxides, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and reducing agents such as reduced glutathione, diisopropyl fluorophosphate (DFP), α-difluoromethylornithine, fluoride salts, cyanide salts, azides, and specific binding pair members such as antibodies and aptamers.

[0119] In some embodiments, the enzyme is inactivated using an enzyme inactivator, which includes dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, peroxides, cyanides, fluorides, or azides.

[0120] In some embodiments, enzyme inactivation is carried out in less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, or 1 minute or less.

[0121] In some embodiments, enzyme inactivation is carried out by contacting the sample with an enzyme inactivator for less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, or 1 minute or less.

[0122] I. Intermediate section / Amplification / Preamplifier In some cases, the nucleic acid chain linked to the target-specific binding partner binds to the nucleic acid chain linked to the enzyme via an intermediate portion.

[0123] In some embodiments, the intermediate portion is an intermediate chain containing nucleic acid. In some embodiments, the nucleic acid is a single-stranded nucleic acid such as single-stranded DNA, RNA, or a nucleic acid analog. The nucleic acid analog (also known as a non-natural nucleic acid) may include a modified phosphate skeleton, a modified pentose sugar, and / or a modified nucleic acid base. The nucleic acid analog may include, but is not limited to, 2'-O-methylribonucleic acid, 2'-fluororibonucleic acid, peptide nucleic acid, morpholino and locked nucleic acid, glycol nucleic acid, and threose nucleic acid.

[0124] In some embodiments, the intermediate chain has a first region complementary to the nucleic acid chain linked to the target-specific binding partner and a second region complementary to the nucleic acid chain linked to the enzyme. In such embodiments, the nucleic acid chain linked to the target-specific binding partner does not need to be complementary to the nucleic acid chain linked to the enzyme.

[0125] In some embodiments, the intermediate and nucleic acid chains linked to the target-specific binding partner are not added in separate steps. In some cases, the intermediate and nucleic acid chains linked to the target-specific binding partner are hybridized together before being added in a single step.

[0126] In some embodiments, the intermediate chain and nucleic acid chain linked to the enzyme are not added in separate steps. In some cases, the intermediate chain and nucleic acid chain linked to the enzyme are hybridized together before being added in a single step.

[0127] In some embodiments, the intermediate chain is a pre-made amplicon containing repeat sequences, each of which is capable of binding to a nucleic acid chain linked to the enzyme. In some embodiments, the intermediate chain is a first region complementary to the corresponding region of the nucleic acid chain linked to the target-specific binding partner, and a second region containing repeat sequences, each of which is capable of directly or indirectly binding specifically to the corresponding region of the nucleic acid chain linked to the enzyme.

[0128] In some embodiments, the method described herein further includes amplifying a nucleic acid chain (or a portion thereof) that is directly or indirectly linked to the target-specific binding partner of step (1).

[0129] In some embodiments, the method described herein further includes amplifying the intermediate portion bound to the nucleic acid chain linked to the target-specific binding partner in step (1) by a primer exchange reaction (PER) as further described below. In some embodiments, labeled nucleotides are used in the amplification reaction.

[0130] In some embodiments, the method described herein further includes adding an intermediate portion containing a repeating sequence. In some embodiments, the repeating sequence is prepared using a primer exchange reaction (PER) as described herein. In some embodiments, the preamplicon is prepared using rolling circle amplification (RCA) as described herein.

[0131] 1. Primer exchange reaction (PER) PER can be used to prepare a nucleic acid product containing multiple probe-binding sites (i.e., intermediate portions as described herein), which can then bind to a nucleic acid (or its barcode domain) linked to a target-specific binding partner, thereby allowing the nucleic acid product to exhibit probe-binding sites. In such embodiments, the probe-binding sites may not be complementary to the barcode domain of the target-specific binding partner.

[0132] Various PER and PER-based signal amplification methods are described in Saka et al., “Highly multiplexed in situ protein imaging with signal amplification by Immuno-SABER” (2018; available as a preprint at www.biorxiv.org / content / 10.1101 / 507566v1 as of June 6, 2019); WO2017 / 143006; and WO2018 / 132392A2, the contents of which are incorporated herein by reference.

[0133] The PER reaction forms a concatemer (repeatable) sequence. The PER concatemer has a first domain complementary to the nucleic acid chain linked to the target-specific binding partner and a second domain containing the repeat sequence. The repeat sequence may be the same as the nucleic acid chain linked to the target-specific binding partner. The repeat sequence may be different from the nucleic acid chain linked to the target-specific binding partner.

[0134] IV. Support information on multiplex imaging methods A. Spectrum and successive multiplexing There are two main methods for creating multiplexing in exchange imaging: spectral multiplexing and successive multiplexing. Spectral multiplexing refers to the ability to use different labels (e.g., different fluorophores) in a single round of imaging. Spectral multiplexing does not require the signal of the first label to be extinguished before displaying the second label. For example, in the case of fluorophores, light of different excitation wavelengths can be used to excite different fluorophores individually. This does not require separate imaging rounds. Successive multiplexing refers to the ability to use the same label (e.g., the same fluorophore) in multiple rounds of imaging by extinguishing the signal of the first round of imaging before the second round of imaging. Spectral multiplexing and successive multiplexing can be used alone or in combination with each other. However, using multiple multiplexing techniques can significantly increase the number of targets that a user can visualize during a particular experiment.

[0135] In some embodiments, multiple rounds of imaging are performed using at least a portion of the same fluorophores. For example, in the first round of imaging, target A can be imaged with label X, target B can be imaged with label Y, and target C can be imaged with label Z. In the next step, the signals of these labels can be eliminated. Then, in the second round of imaging, target D can be imaged with label X, target E can be imaged with label Y, and target F can be imaged with label Z. In some embodiments, at least two targets are imaged using at least two labels, the signals are eliminated, and then at least one more target is imaged using at least one of the same labels, and the imaging steps may be performed in any order. This means that the order of the steps can be reversed, so that the first imaging step includes imaging at least one target, the signals are eliminated, and the second imaging step includes imaging at least two targets.

[0136] Combining both spectral and sequential multiplexing can improve the overall convenience of performing imaging for the user and reduce damage to the sample being imaged.

[0137] B. Control experiments and background removal Control experiments and background removal may be used to further improve the results of methods for testing a sample for the presence of one or more targets. None of these embodiments are necessary for a useful experiment, but both improve the quality of multiplexing and can be used individually or in combination with each other.

[0138] 1. Control experiment Control measurements may be added at multiple points in the multiplexing process by performing a control step, which involves contacting the sample with a binding pair having a nucleotide sequence that is not complementary to the nucleic acid chain, bound to a target-specific binding partner (or a part thereof, such as a barcode).

[0139] 2. Background removal In any of the embodiments considered throughout this application, the method may employ background removal. In some embodiments, the method includes imaging a sample to detect and / or measure a background signal, and removing the background signal from the image of the sample to detect a conjugated label. Such a background signal may include autofluorescence and / or residual fluorescence associated with the incomplete removal of the signal from the conjugated label. In some embodiments, the background signal is measured before imaging the sample to detect the conjugated label. In other embodiments, the background signal is measured after imaging the sample to detect the conjugated label.

[0140] C. Description of the sample 1. Type of sample Various types of samples may be imaged using these methods. In some embodiments, the sample is a fixed sample. In some embodiments, the sample is a cell, cell lysate, tissue, tissue lysate, and / or whole organism. In some embodiments, the sample is a cell or tissue sample, a cell or tissue lysate, or body fluid. In some embodiments, the sample is a tissue, and imaging includes intra-tissue multiplexing for immunostaining.

[0141] The sample may be provided in a liquid culture medium or buffer solution.

[0142] 2. Antigen recovery In some embodiments, staining a sample with a target-specific binding partner requires specific conditions, and not all target-specific binding partners bind to antigens under the same conditions. This may be because their target antigens are not available under the same conditions.

[0143] 3. Use in target description and biomarker identification In some embodiments, this method is useful for identifying biomarkers. In some cases, samples are imaged and data analysis is performed on these samples. In some embodiments, multiple targets are tested for the use of corresponding target-specific binding partners for each target. In some cases, relationships between different targets may be evaluated; for example, a user may attempt to determine the relationship between multiple markers and disease conditions, concluding that diseased samples have increased levels of A, decreased levels of B, and levels of C within a certain range compared to healthy tissue that does not have the distribution of that biomarker.

[0144] In some embodiments, at least 10, 96, 100, 384, or 500 samples are imaged, and data analysis is performed on those samples.

[0145] In some embodiments, at least 5, 10, 15, 25, 30, 50, 75, or 100 or more targets are tested for use with target-specific binding partners corresponding to each target.

[0146] D. Equipment and Software 1. Imaging chambers such as flow cells In some embodiments, an imaging chamber can be used. In some cases, the imaging chamber is a fixed chamber without an inlet and outlet. In some embodiments, the imaging chamber has a single inlet / outlet combination. In other examples, the imaging chamber allows flow and is referred to as a flow cell. The flow cell may consist of a first optically transparent support combined with a second optically transparent material (e.g., glass or plastic coverslip) to obtain a flow cell having an upper and lower surface and a fluid between them. The first and second optically transparent materials may be arranged parallel to each other, if used. With respect to parallelism, it includes geometric arrangements that are perfectly parallel, and geometric arrangements that are shifted by 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, or 10° from parallel. In some embodiments, the second optically transparent material is very close to the first optically transparent material, for example, about 5 microns to 5 mm, 50 microns to 500 microns, or 500 microns to 5 mm.

[0147] The imaging chamber may also consist of a first optically transparent support and a gasket (also called an isolator or spacer). The gasket may be open to air on its top surface, or it may be closed and have an optically transparent top surface. The gasket may have a mixed inlet / outlet, or it may have both an inlet and an outlet. The gasket may also not have an outlet. The gasket may be made of plastic, rubber, or adhesive. The gasket may include CoverWell chamber gaskets (Thermo Fisher), ultrathin sealed chambers for upright and inverted microscopes (Bioscience Tools), or incubation chambers (including Grace Bio-Labs, HybriSlip® hybridization cover, HybriWell® sealing system, CoverWell® incubation chamber, imaging spacer, SecureSeal® hybridization chamber, FlexWell® incubation chamber, FastWells® reagent barrier, and Silicone Isolators®).

[0148] In some cases, the gasket may be used together with a coverslip that forms the upper surface of the imaging chamber or flow cell.

[0149] Imaging chambers, such as flow cells, may be reusable or disposable, though not limited to these.

[0150] 2. Software for controlling fluid steps In some embodiments, all fluid exchange steps are performed using a fluid system including electronic and / or pneumatic and / or hydraulic and / or electrofluidic actuators and systems. In certain circumstances, the fluid system is controlled by software. In some embodiments, the fluid system is automatically controlled by software that synchronizes the steps(s) described above in the above paragraph with the imaging steps.

[0151] In some embodiments, the fluid system is controlled by software that synchronizes the above-described step(s) of the method with the imaging step by communicating with the imaging software with reference to the step numbers described in the paragraph above.

[0152] In some embodiments, all fluid steps are performed while the sample is on the imaging device.

[0153] The sample may be fixed in a disposable imaging chamber (e.g., a flow cell), a reusable imaging chamber (e.g., a flow cell), a slide, a slide with a coverslip, or any other configuration. [Examples]

[0154] Example 1. Deposition of fluorescent reporter by HRP cycling Four different targets, CD8, CD68, PD-L1, and CK, were labeled using sequential deposition of each fluorescent tyramide reagent. Specifically, formalin-fixed paraffin-embedded (FFPE) tissue slides were baked and then loaded into a BOND RX automated staining system (Leica Biosystems, Nussloch GmbH) in all subsequent steps. The slides were dewaxed, and the antigens were recovered using BOND Epitope Recovery Solution 2 (Leica Biosystems). Endogenous peroxidase activity was quenched with 3% H2O2 and then blocked at room temperature for 15 minutes with antibody diluent (Ultivue, Cambridge, Massachusetts). A mixture of four different primary antibodies (anti-CD8, anti-CD68, anti-PD-L1, and anti-CK), conjugated to nucleic acid barcodes and diluted with antibody diluent, was incubated with the tissue samples at room temperature for 30 minutes. The sample was washed with BOND washing solution (Leica Biosystems), then incubated with pre-amplification mix (Ultivue) at room temperature for 10 minutes, followed by a further washing step. The sample was incubated with amplification solution (Ultivue) for 15-20 minutes, then washed. A first set of nucleic acid probe strands complementary to the anti-CD8 antibody conjugate and linked to the HRP enzyme by disulfide bonds was added to the sample and hybridized to the partner strand for 15 minutes. Unbound HRP-bound probes were removed in the washing step. The CD8 target was labeled by applying the working solution of Alexa Fluor® 488 tyramide reagent (Invitrogen®) to the tissue sample according to the package instructions. The HRP enzyme linked to the first set of nucleic acid probe strands in the sample was inactivated by adding TCEP solution in Tris buffer for 1 minute. Following the inactivation of the CARD and associated HRP enzyme in the first round, a second set of nucleic acid probe strands complementary to the anti-CD68 antibody conjugate, linked to the HRP enzyme by disulfide bonds, was added to the sample and hybridized to the partner strand. Unbound HRP-conjugated probes were removed in a washing step. The CD68 target was labeled by applying the Alexa Fluor® 555 tyramide reagent (Invitrogen) working solution to the tissue sample according to the package instructions.The HRP enzyme linked to the second set of nucleic acid probe strands in the sample was inactivated by adding TCEP solution in Tris buffer for 1 minute. After the second round of inactivation of the HRP enzyme associated with CARD, a third set of nucleic acid probe strands complementary to the anti-PD-L1 antibody conjugate, linked to the HRP enzyme by disulfide bonds, was added to the sample and hybridized to the partner strand. Unbound HRP-conjugated probes were removed in a washing step. The PD-L1 target was labeled by applying the Alexa Fluor® 647 tyramide reagent (Invitrogen) working solution to the tissue sample according to the package instructions. The HRP enzyme linked to the third set of nucleic acid probe strands in the sample was inactivated by adding TCEP solution in Tris buffer for 1 minute. After the third round of inactivation of the HRP enzyme associated with CARD, a fourth set of nucleic acid probe strands complementary to the anti-CK antibody conjugate, linked to the HRP enzyme by disulfide bonds, was added to the sample and hybridized to the partner strand. Unbound HRP-conjugated probes were removed in the washing step. CK targets were labeled by applying the Alexa Fluor® 750 tyramide reagent (Invitrogen) solution to the tissue sample according to the package instructions. Nuclear counterstaining was performed on the slide, a coverslip was placed over the sample, and imaging was performed using a fluorescence microscope.

[0155] Figure 7 shows that four targets, CD8, CD68, PD-L1, and CK, were successfully labeled using sequential deposition of their respective fluorescent reporters. Box A shows the exemplary location of labeled CD8; Box B shows labeled CD68; Box C shows labeled PD-L1; and Box D shows labeled CK.

[0156] Example 2. Inactivation of HRP for periodic deposition To achieve periodic deposition of the label using an HRP-based enzymatic reaction, the HRP enzyme must be removed or inactivated between successive deposition rounds. The HRP enzyme may be inactivated by heating or pH-based denaturation, or it may be cleaved from the sample via an unstable linker (Figure 4A). In this example, the HRP enzyme was inactivated using a reducing agent in the absence of any unstable linker (Figure 4B).

[0157] FFPE tissue slides were baked at 60°C for 30 minutes, then dewaxed, and antigens were recovered using BOND Epitope Recovery Solution 2 (Leica Biosystems) in a BOND RX automated staining system (Leica Biosystems). The slides were removed from BOND RX for the subsequent steps. Endogenous peroxidase activity was quenched with 3% H2O2 for 20 minutes, washed with PBS, and then blocked at room temperature for 15 minutes with antibody diluent (Ultivue, Cambridge, Massachusetts). The primary antibody (from Example 1) conjugated to the nucleic acid barcode was diluted in antibody diluent and incubated with the tissue sample at room temperature for 1 hour. The sample was washed and incubated with pre-amplification mix (Ultivue) at room temperature for 25 minutes, followed by a further washing step. The sample was incubated with amplification solution (Ultivue) at 30°C for 15 minutes, and then washed with PBS. A first set of nucleic acid probe strands complementary to one of the nucleic acid barcodes conjugated with an antibody linked to an HRP enzyme (without disulfide crosslinking) was added to the sample and hybridized to the partner strand. Unbound HRP-conjugated probes were removed in a washing step. To inactivate HRP, tissue slides were exposed to TCEP solution for 5 minutes or to Tris buffer as a control. Alexa Fluor® 647 tyramide (Invitrogen) working solution was added to all slides, incubated at room temperature for 10 minutes, and washed with PBS. Finally, the slides were stained with nuclear counterstain, washed, and covered with coverslips. The slides were imaged using a fluorescence microscope.

[0158] Figures 8A-8B show that samples treated with TCEP solution before tyramide reagent deposition did not result in tyramide deposition, while the tyramide reagent successfully deposited in control samples not exposed to TCEP. This surprising result indicates that the TCEP solution inactivated the HRP enzyme.

[0159] Example 3. Deposition of binding pairs by HRP cycling FFPE tissue slides were baked and then loaded into a BOND RX automated staining system (Leica Biosystems) for all subsequent steps. The slides were dewaxed and the antigen was recovered using BOND Epitope Recovery Solution 2 (Leica Biosystems). Endogenous peroxidase activity was quenched using 3% H2O2 and then blocked at room temperature for 15 minutes with antibody diluent (Ultivue, Cambridge, Massachusetts). A mixture of four different primary antibodies (anti-CD8, anti-CD68, anti-PD-L1, and anti-CK), conjugated to nucleic acid barcodes and diluted with antibody diluent, was incubated with the tissue sample at room temperature for 30 minutes. The sample was then washed and incubated with pre-amplification mix (Ultivue) at room temperature for 10 minutes, followed by a further washing step. The sample was incubated with amplification solution (Ultivue) for 15–25 minutes and then washed. A first set of nucleic acid probe strands, complementary to the anti-CD8 antibody conjugate and linked to the HRP enzyme via disulfide bonds, was added to the sample and hybridized to the partner strand for 15 minutes. Unbound HRP-bound probes were removed in a washing step. A solution of nucleic acid barcodes linked to a phenol-HRP reactive substrate (Figure 6) was applied to the tissue sample to deposit additional nucleic acid barcodes at the CD8 target site (e.g., shown in Figure 5). The HRP enzyme linked to the first set of nucleic acid probe strands in the sample was inactivated by adding TCEP solution in Tris buffer for 1 minute. After the first round of inactivation of the HRP enzyme associated with CARD, a second set of nucleic acid probe strands, complementary to the anti-CD68 antibody conjugate and linked to the HRP enzyme via disulfide bonds, was added to the sample and hybridized to the partner strand. Unbound HRP-bound probes were removed in a washing step. A solution of nucleic acid barcodes linked to a phenol-HRP-reactive substrate was applied to a tissue sample to deposit additional nucleic acid barcodes at the CD68 target site. The HRP enzyme linked to the second set of nucleic acid probe strands in the sample was inactivated by adding TCEP solution in Tris buffer for 1 minute.Following the inactivation of the HRP enzyme associated with CARD in the second round, a third set of nucleic acid probe strands complementary to the anti-PD-L1 antibody conjugate, linked to the HRP enzyme by disulfide bonds, was added to the sample and hybridized to the partner strand. Unbound HRP-conjugated probes were removed in a washing step. A solution of nucleic acid barcodes linked to a phenol-HRP reactive substrate was applied to the tissue sample to deposit additional nucleic acid barcodes at the PD-L1 target site. The HRP enzyme linked to the third set of nucleic acid probe strands in the sample was inactivated by adding TCEP solution in Tris buffer for 1 minute. Following the inactivation of the HRP enzyme associated with CARD in the third round, a fourth set of nucleic acid probe strands complementary to the anti-CK antibody conjugate, linked to the HRP enzyme by disulfide bonds, was added to the sample and hybridized to the partner strand. Unbound HRP-conjugated probes were removed in a washing step. A solution of nucleic acid barcodes linked to a phenol-HRP-reactive substrate was applied to a tissue sample to deposit additional nucleic acid barcodes at the CK target site. The sample was washed, and a mixture of fluorescently labeled probe strands complementary to the nucleic acid barcodes was hybridized to the sample. Unbound fluorescently labeled probe strands were removed, and nuclear counterstaining was performed on the slide. The sample was covered with a coverslip and imaged using a fluorescence microscope.

[0160] Figure 9 shows that four targets, CD8, CD68, PD-L1, and CK, were successfully labeled using sequential deposition of their respective fluorescent reporters. Box A shows the exemplary location of labeled CD8; Box B shows labeled CD68; Box C shows labeled PD-L1; and Box D shows labeled CK.

[0161] Example 4. Additional Embodiments The following numbered clauses provide additional support and explanation for the embodiments described herein.

[0162] Article 1. A method for testing a sample for the presence of one or more targets, (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (provided that (i) or (ii) or both of the following are satisfied: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release linker, and (ii) The enzyme is linked to the complementary nucleic acid chain by a second release linker. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a detectably labeled substrate (the substrate and the detectable label are optionally linked by a third release linker). (6) Optionally, remove the unbound substrate conjugate. (7) optionally release the bound enzyme, (8) Imaging the sample to detect the conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof. The method, including the method described above.

[0163] Clause 2. The method according to Clause 1, wherein in step (5), the substrate conjugate comprising the detectably labeled substrate reacts with the enzyme to form an activated substrate conjugate, and the activated substrate conjugate binds to a receptor for the activated substrate conjugate.

[0164] Clause 3. The method according to Clause 2, wherein the receptor for the activating substrate conjugate is immobilized on a solid support, causing the deposition of the detectable label.

[0165] Clause 4. The method according to Clause 1, further comprising (10) optionally releasing the coupled detectable label after step (8) by releasing the third release linker, and optionally repeating steps (1) to (10) or any subset thereof.

[0166] Article 5. A method for testing a sample for the presence of one or more targets, (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) The sample from step (1) or optionally from step (2) is brought into contact with the first member of a binding pair linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (provided that (i) or (ii) or both of the following are satisfied: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release linker, and (ii) The first member of the bond pair is linked to the complementary nucleic acid chain by a second release linker. (4) Optionally, remove the unbound first member of a binding pair linked to a complementary nucleic acid chain. (5) optionally bring the sample from step (3) or step (4) into contact with the second member of the binding pair linked to the enzyme by a third release linker, and optionally remove the unbound second member of the binding pair linked to the enzyme. (6) The sample from step (5) is brought into contact with a substrate conjugate consisting of a labeled substrate (the substrate and label are optionally linked by a fourth release linker). (7) Optionally, remove the unbound substrate conjugate. (8) optionally release the first or second member of the coupling type of the coupling pair, (9) Imaging the sample to detect the conjugated detectable label, and (10) Optionally, repeat steps (1) to (9) or any subset thereof. The method, including the method described above.

[0167] Clause 6. The method according to Clause 5, further comprising (11) optionally releasing the bound-type detectable label after step (9) by cleaving the third release-type linker between the label and the substrate, and optionally repeating steps (1) to (11) or any subset thereof.

[0168] Article 7. A method for testing a sample for the presence of one or more targets, (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner (provided that (i) or (ii) or both of the following are satisfied: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release linker, and (ii) The enzyme is linked to the complementary nucleic acid chain by a second release linker. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a nucleic acid chain-binding substrate (the substrate and nucleic acid chain are optionally linked by a third release linker). (6) Optionally, remove the unbound substrate conjugate. (7) optionally release the bound enzyme, (8) The sample from step (5) or optionally from steps (6) to (7) is brought into contact with a nucleic acid chain-bound detectable label, optionally linked by a fourth release linker (where the nucleic acid chain is a specific binding pair member to the nucleic acid chain from step (5)). (9) Optionally, imaging the sample to detect the conjugated label, (10) optionally, releasing the bonded label of step (8) by releasing the release-type linker of step (5) or optionally step (8) or optionally steps (5) and (8), and (11) Optionally, repeat steps (1) to (10) or any subset thereof. The method, including the method described above.

[0169] Article 8. A method for testing a sample for the presence of one or more targets, (1) The method comprises bringing a sample to be tested for the presence of one or more targets into contact with a target-specific binding partner, wherein the target-specific binding partner is linked to a nucleic acid chain, and further, (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample with the horseradish peroxidase (HRP) enzyme-bound nucleic acid chain which is complementary to the nucleic acid chain linked to the target-specific binding partner. (The following conditions (i) or (ii) or both must be met:) (i) The nucleic acid chain is linked to the target-specific binding partner by a first release linker, and (ii) The HRP is linked to the complementary nucleic acid chain by a second release linker. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate comprising a detectably labeled phenol-containing substrate (the substrate and the detectably labeled substrate are optionally linked by a third release linker). (6) Optionally, remove unbound substrates. (7) Imaging the sample and detecting the conjugated detectable label, (8) optionally releasing the concatenated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof. The method, including the method described above.

[0170] Clause 9. The method of Clause 8, wherein, after step (5), the phenolic moiety is enzymatically converted to an activated state, resulting in the deposition of the label.

[0171] Clause 10. The method according to any one of Clauses 1 to 9, wherein the release linker comprises at least one disulfide bond, an ester, a vicinal diol, a sulfone, and a photocleavable linker.

[0172] Clause 11. The method according to any one of Clauses 1 to 10, wherein the enzyme is selected from oxidoreductase, hydrolase, lyase, transferase, isomerase, and ligase.

[0173] Clause 12. The method according to any one of Clauses 1 to 11, wherein the enzyme is selected from peroxidase, oxidase, phosphatase, esterase, and glycosidase.

[0174] Clause 13. The method according to Clause 12, wherein the enzyme is selected from horseradish peroxidase, glucose oxidase, alkaline phosphatase, and β-galactosidase.

[0175] Clause 14. The method according to Clause 13, wherein the enzyme is horseradish peroxidase.

[0176] Clause 15. The method according to any one of Clauses 1 to 14, wherein the binding pair is immunotyped or non-immunotyped.

[0177] Clause 16. The method according to Clause 15, wherein the immunotype binding pair is selected from antigen-antibody and hapten-antihapten.

[0178] Clause 17. The method according to Clause 16, wherein the antibody member of the binding pair is polyclonal, monoclonal, or an immunoreactive fragment thereof.

[0179] Clause 18. The method according to Clause 15, wherein the non-immunotype binding pair is selected from biotin-avidin, biotin-streptavidin, and complementary probe nucleic acid folate-folate binding proteins.

[0180] Clause 19. The method according to Clause 18, wherein the non-immunotype bond pair forms a covalent bond via sulfhydryl reactive groups (e.g., maleimide and haloacetyl derivatives), amine reactive groups (e.g., isothiocyanates, succinimidyl esters, sulfonyl halides, click chemistry), and coupler dyes (e.g., 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB)).

[0181] Clause 20. The method according to any one of Clauses 1 to 19, wherein the detectable label is selected from enzymes, radioisotopes, fluorescence, chemiluminescence, and electrochemicals.

[0182] Clause 21. The method according to any one of Clauses 1 to 20, wherein the enzyme is horseradish peroxidase (HRP) and the substrate is a phenolic substrate.

[0183] Clause 22. The method according to any one of Clauses 1 to 21, wherein the enzyme is a hydrolase and the substrate is an ester, amide, or glycoside substrate.

[0184] Clause 23. A composition, (1) A sample bound to multiple target-specific binding partners (each binding partner is bound to a nucleic acid chain), (2) Enzymes for nucleic acid chains complementary to the nucleic acid chain bound to the target-specific binding partner, (The following conditions (i) or (ii) or both must be met:) (i) The nucleic acid chain is linked to the target-specific binding partner by a first release linker, and (ii) The enzyme is linked to the complementary nucleic acid chain by a second release linker, and (3) The composition comprising a substrate conjugate comprising a detectably labeled substrate (the substrate and label optionally linked by a third release linker).

[0185] Clause 24. A composition, (1) A sample bound to multiple target-specific binding partners (each binding partner is bound to a nucleic acid chain), (2)(i) A first member linked to a nucleic acid chain complementary to the nucleic acid chain bound to the target-specific binding partner, and (ii) A binding pair including a second member linked to the enzyme (The following conditions (i) or (ii) or both must be met:) (i) The nucleic acid chain is linked to the target-specific binding partner by a first release linker, and (ii) The first member of the bond pair is linked to the complementary nucleic acid chain by a second release linker), and (3) A substrate conjugate comprising a detectably labeled substrate (the substrate and label optionally linked by a third release linker), The composition wherein the second member of the bond pair is linked to the enzyme by a fourth release-type linker.

[0186] Clause 25. A composition, (1) A sample bound to multiple target-specific binding partners (each binding partner is bound to a nucleic acid chain), (2) An enzyme linked to a nucleic acid chain complementary to the nucleic acid chain bound to the target-specific binding partner. (The following conditions (i) or (ii) or both must be met:) (i) The nucleic acid chain is linked to the target-specific binding partner by a first release linker, and (ii) The enzyme is linked to the complementary nucleic acid chain by a second release linker, and (3) A substrate conjugate consisting of a nucleic acid chain-bound substrate (the substrate and nucleic acid chain are optionally linked by a third release-type linker). (4) A nucleic acid chain-bound detectable label optionally linked by a fourth release linker (where the nucleic acid chain in (4) is a member of a specific binding pair to the nucleic acid chain in (3)). The composition comprising the above.

[0187] Article 26. A method for testing a sample for the presence of one or more targets, (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a detectably labeled substrate (the substrate and the detectably labeled substrate are optionally linked by an evacuation linker). (6) Optionally, remove the unbound substrate conjugate. (7) Optionally, inactivate the bound enzyme. (8) Optionally, imaging the sample to detect the conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof. The method, including the method described above.

[0188] Article 27. A method for testing a sample for the presence of one or more targets, (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with a first member of a binding pair that is linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) Optionally, remove the unbound first member of a binding pair linked to a complementary nucleic acid chain. (5) Contact the sample from step (3) or optionally from step (4) with the second member of the binding pair linked to the enzyme, and optionally remove the unbound second member of the binding pair linked to the enzyme. (6) The sample from step (5) is brought into contact with a substrate conjugate consisting of a labeled substrate (the substrate and label are optionally linked by an evacuation linker). (7) Optionally, remove the unbound substrate conjugate. (8) Optionally, inactivate the aforementioned bound enzyme. (9) Optionally, imaging the sample to detect the conjugated detectable label, and (10) The method comprising optionally repeating steps (1) to (9) or any subset thereof.

[0189] Clause 28. The method according to Clause 27, further comprising (11) optionally releasing the bound-type detectable label after step (9) by cleaving the release-type linker between the label and the substrate, and optionally repeating any of steps (1) to (9) and (11).

[0190] Article 29. A method for testing a sample for the presence of one or more targets, (1) A sample to be tested for the presence of one or more targets is brought into contact with one or more target-specific binding partners (each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains). (2) Optionally, remove any unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a nucleic acid chain-binding substrate (the substrate and nucleic acid chain are optionally linked by a first release-type linker). (6) Optionally, remove the unbound substrate conjugate. (7) Optionally, inactivate the bound enzyme. (8) The sample from step (5) or optionally from steps (6) to (7) is brought into contact with a nucleic acid chain-bound detectable label, optionally linked by a second release linker (where the nucleic acid chain is a specific binding pair member to the nucleic acid chain from step (5)). (9) Optionally, imaging the sample to detect the conjugated label, (10) optionally releasing the linked label in step (8) by releasing the linker in step (5) or optionally in step (8) or optionally in steps (5) and (8), and (11) Optionally, repeat steps (1) to (10) or any subset thereof. The method, including the method described above.

[0191] Article 30. A method for testing a sample for the presence of one or more targets, (1) A sample to be tested for the presence of one or more targets is brought into contact with a target-specific binding partner (the target-specific binding partner is linked to a nucleic acid chain), (2) Optionally, remove any unbound target-specific binding partners. (3) Contacting the sample with an HRP-binding nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner, (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The sample from step (3) or optionally from step (4) is brought into contact with a substrate conjugate consisting of a detectably labeled phenol-containing substrate (the substrate and the detectably labeled substrate are optionally linked by an evacuation linker). (6) Optionally, remove unbound substrates. (7) Optionally, the sample is imaged to detect the conjugated detectable label. (8) Optionally, inactivating the HRP, and (9) Optionally, repeat a subset of steps (1) to (8). The method, including the method described above.

[0192] Clause 31. The method according to any one of Clauses 26 to 30, wherein the enzyme is inactivated using dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, peroxide, cyanide, fluoride, or azide.

[0193] Equivalent The above written specification is considered to be sufficient for a person skilled in the art to implement the embodiments. The above description and examples detail specific embodiments and describe the best methods contemplated by the inventors. However, it will be understood that, however detailed the above may be presented in text, the embodiments may be implemented in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0194] As used herein, the term "about" refers to numerical values, including, for example, integers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values (e.g., + / -5 to 10% of the recited range) that a person skilled in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as "at least" and "about" precede a list of numerical values or ranges, the terms modify all of the numerical values or ranges presented in the list. In some cases, the term "about" may include values that are rounded to the nearest significant digit.

Claims

1. A method for testing a sample for the presence of one or more targets, (1) The method includes contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains, and further, the method (2) In one or two steps, the sample is brought into contact with (a) an enzyme linked to a nucleic acid chain, and (b) an intermediate portion comprising a first domain capable of specifically binding to the nucleic acid chain linked to the target-specific binding partner and a second domain capable of specifically binding to the nucleic acid chain linked to the enzyme, and (3) The method further includes bringing the sample from step (2) into contact with a substrate conjugate consisting of a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked by an evacuation linker, and the method further (4) Optionally, inactivate the bound enzyme. (5) Optionally, to detect a signal from the concatenated detectable sign, and (6) Optionally, repeat steps (1) to (5) or a subset thereof, and optionally, repeat steps (3) to (4) or any subset thereof before imaging step (5). The method, including the method described above.

2. The method according to claim 1, wherein the second region of the intermediate portion includes a repeating sequence, each of which is directly or indirectly specific to a corresponding sequence of the nucleic acid chain linked to the enzyme.

3. The method according to claim 1, further comprising a nucleic acid amplification reaction to amplify the intermediate portion bound to the nucleic acid chain linked to the target-specific binding partner of step (1) to form an amplicon of repeating sequences, wherein each of the sequences directly or indirectly specifically binds to the corresponding sequence of the nucleic acid chain linked to the enzyme.

4. The method according to claim 3, wherein the nucleic acid amplification reaction comprises a hairpin-based concatemerization reaction or a hairpin-based dendritic reaction, and optionally the amplification reaction is a primer exchange reaction.

5. A method for testing a sample for the presence of one or more targets, (1) The method includes contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains, and further, the method (2) Optionally, remove unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) optionally remove unbound enzymes linked to complementary nucleic acid chains, and (5) The method further comprises bringing the sample from step (3) or optionally from step (4) into contact with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked by an evacuation linker, and the method further comprises (6) Optionally, remove any unbound substrate conjugates. (7) Optionally, inactivate the bound enzyme. (8) Optionally, imaging the sample to detect the conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (7) or any subset thereof before imaging step (8). The method, including the method described above.

6. A method for testing a sample for the presence of one or more targets, (1) The method includes contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains, and further, the method (2) Optionally, remove unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with a first member of a binding pair that is linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) Optionally, remove the unbound first member of a binding pair linked to a complementary nucleic acid chain. (5) Contacting the sample from step (3) or optionally from step (4) with the second member of the binding pair linked to the enzyme, and optionally removing the unbound second member of the binding pair linked to the enzyme, (6) The method further includes bringing the sample from step (5) into contact with a substrate conjugate consisting of a labeled substrate, wherein the substrate and label are optionally linked by an evacuation linker, (7) Optionally, remove any unbound substrate conjugates. (8) Optionally, inactivate the aforementioned bound enzyme. (9) Optionally, imaging the sample to detect the conjugated detectable label, and (10) Optionally, repeat steps (1) to (9) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before imaging step (9). The method, including the method described above.

7. The method according to claim 6, further comprising (11) optionally releasing the bound-type detectable label after step (9) by cleaving the release-type linker between the label and the substrate, and optionally repeating any of steps (1) to (9) and (11).

8. A method for testing a sample for the presence of one or more targets, (1) The method includes contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains, and further, the method (2) Optionally, remove unbound target-specific binding partners. (3) Contact the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner. (4) optionally remove unbound enzymes linked to complementary nucleic acid chains, and (5) The method further comprises contacting the sample from step (3) or optionally from step (4) with a substrate conjugate comprising a nucleic acid chain-binding substrate, wherein the substrate and nucleic acid chain are optionally linked by a first release-type linker, and the method further comprises (6) Optionally, remove the unbound substrate conjugate. (7) Optionally, inactivating the aforementioned bound enzyme, and (8) The method further comprises bringing the sample from step (5) or optionally from steps (6) to (7) into contact with a nucleic acid chain-bound detectable label, optionally linked by a second release linker, wherein the nucleic acid chain is a specific binding pair member to the nucleic acid chain from step (5), and the method further comprises (9) Optionally, imaging the sample in order to detect the conjugated label. (10) optionally releasing the linked label in step (8) by optionally releasing the linker in step (5) or step (8) or steps (5) and (8), and (11) Optionally, repeat steps (1) to (10) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before imaging step (9). The method, including the method described above.

9. A method for testing a sample for the presence of one or more targets, wherein the method is (1) The method comprises bringing a sample to be tested for the presence of one or more targets into contact with a target-specific binding partner, wherein the target-specific binding partner is linked to a nucleic acid chain, and further, (2) Optionally, remove unbound target-specific binding partners. (3) Contacting the sample with a horseradish peroxidase (HRP)-bound nucleic acid chain that is complementary to the nucleic acid chain linked to the target-specific binding partner, (4) optionally remove unbound enzymes linked to complementary nucleic acid chains, and (5) The method further comprises contacting the sample from step (3) or optionally from step (4) with a substrate conjugate comprising a detectably labeled phenol-containing substrate, wherein the substrate and the detectably labeled substrate are optionally linked by an evacuation linker, and the method further comprises (6) Optionally, remove unbound substrates. (7) Optionally, inactivate the HRP. (8) Optionally, imaging the sample to detect the conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (7) or a subset thereof before step (8). The method, including the method described above.

10. The method according to claim 1, wherein the enzyme is inactivated using an enzyme inactivator, the enzyme inactivator comprising dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, peroxide, cyanide, fluoride, or azide.

11. The method according to claim 1, wherein the step (4) of inactivating the bound enzyme is carried out in less than 20 minutes.

12. The method according to claim 1, wherein the step (4) of inactivating the bound enzyme is carried out in less than 10 minutes.

13. The method according to claim 1, wherein the step (4) of inactivating the bound enzyme is performed in less than two minutes.

14. A method for testing a sample for the presence of one or more targets, (1) The method includes contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains, and further, the method (2) Optionally, remove unbound target-specific binding partners, and (3) The process includes contacting the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner, provided that (i) or (ii) or both of the following are satisfied: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release-type linker, and (ii) The enzyme is linked to the complementary nucleic acid chain by a second release linker, further the method (4) optionally remove unbound enzymes linked to complementary nucleic acid chains, and (5) The method further comprises bringing the sample from step (3) or optionally from step (4) into contact with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectably labeled substrate are optionally linked by a third release linker, (6) Optionally, remove any unbound substrate conjugates. (7) Optionally, release the bound enzyme, (8) Optionally, imaging the sample to detect the conjugated detectable label, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (7) or any subset thereof before imaging step (8). The method, including the method described above.

15. The method according to claim 1, wherein in step (3), the substrate conjugate comprising the detectably labeled substrate reacts with the enzyme to form an activated substrate conjugate, and the activated substrate conjugate binds to a receptor for the activated substrate conjugate.

16. The method according to claim 15, wherein the receptor for the activating substrate conjugate is present in the sample, and the deposition of the detectable label occurs.

17. The method according to claim 5, further comprising (10) optionally releasing the third release linker to release the coupled detectable marker after step (8), and optionally repeating steps (1) to (10) or any subset thereof.

18. A method for testing a sample for the presence of one or more targets, (1) The method includes contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains, and further, the method (2) Optionally, remove unbound target-specific binding partners, and (3) The process includes bringing the sample from step (1) or optionally from step (2) into contact with a first member of a binding pair linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner, provided that (i) or (ii) or both of the following are satisfied: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release-type linker, and (ii) The first member of the bond pair is linked to the complementary nucleic acid chain by a second release linker, further the method (4) Optionally, remove the unbound first member of a binding pair linked to a complementary nucleic acid chain. (5) optionally bringing the sample from step (3) or step (4) into contact with the second member of the binding pair linked to the enzyme by a third release linker, and optionally removing the unbound second member of the binding pair linked to the enzyme, (6) The method further comprises bringing the sample from step (5) into contact with a substrate conjugate comprising a labeled substrate, wherein the substrate and label are optionally linked by a fourth release linker. (7) Optionally, remove any unbound substrate conjugates. (8) Optionally, release the first or second member of the coupling type of the coupling pair. (9) Optionally, imaging the sample to detect the conjugated detectable label, and (10) Optionally, repeat steps (1) to (9) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before imaging step (9). The method, including the method described above.

19. The method according to claim 18, further comprising (11) optionally releasing the bound-type detectable label after step (9) by cleaving the third release-type linker between the label and the substrate, and optionally repeating steps (1) to (11) or any subset thereof.

20. A method for testing a sample for the presence of one or more targets, (1) The method includes contacting a sample to be tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid chain, and target-specific binding partners with different specificities are linked to different nucleic acid chains, and further, the method (2) Optionally, remove unbound target-specific binding partners. (3) The process includes contacting the sample from step (1) or optionally from step (2) with an enzyme linked to a nucleic acid chain complementary to the nucleic acid chain linked to the target-specific binding partner, provided that (i) or (ii) or both of the following are satisfied: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release-type linker, and (ii) The enzyme is linked to the complementary nucleic acid chain by a second release linker, further the method (4) optionally remove unbound enzymes linked to complementary nucleic acid chains, and (5) The method further comprises contacting the sample from step (3) or optionally from step (4) with a substrate conjugate comprising a nucleic acid chain-binding substrate, wherein the substrate and nucleic acid chain are optionally linked by a third release-type linker, and the method further comprises (6) Optionally, remove any unbound substrate conjugates. (7) Optionally, release the bound enzyme, (8) The method further comprises bringing the sample from step (5) or optionally from steps (6) to (7) into contact with a nucleic acid chain-bound detectable label, optionally linked by a fourth release linker, wherein the nucleic acid chain is a specific binding pair member to the nucleic acid chain from step (5), and the method further comprises (9) Optionally, imaging the sample in order to detect the conjugated label. (10) optionally releasing the bonded label of step (8) by optionally releasing the release-type linker of step (5) or optionally step (8) or optionally steps (5) and (8), and (11) Optionally, repeat steps (1) to (10) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (8) or any subset thereof before step (9). The method, including the method described above.

21. A method for testing a sample for the presence of one or more targets, (1) The method comprises bringing a sample to be tested for the presence of one or more targets into contact with a target-specific binding partner, wherein the target-specific binding partner is linked to a nucleic acid chain, and further, (2) Optionally, remove unbound target-specific binding partners, and (3) The method includes contacting the sample with a horseradish peroxidase (HRP)-bound nucleic acid chain that is complementary to the nucleic acid chain linked to the target-specific binding partner. The following conditions (i) or (ii) or both must be met: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release-type linker, and (ii) The HRP is linked to the complementary nucleic acid chain by a second release linker, further the method (4) Optionally, remove any unbound enzymes linked to complementary nucleic acid chains. (5) The method further comprises contacting the sample from step (3) or optionally from step (4) with a substrate conjugate comprising a detectably labeled phenol-containing substrate, wherein the substrate and the detectably labeled substrate are optionally linked by a third release-type linker, and the method further comprises (6) Optionally, remove unbound substrates. (7) Optionally, imaging the sample to detect the conjugated detectable label. (8) optionally releasing the concatenated detectable marker, and (9) Optionally, repeat steps (1) to (8) or any subset thereof, and optionally, if multiple target-specific binding partners are used, repeat steps (1) to (6) or any subset thereof before imaging step (7). The method, including the method described above.

22. The method according to claim 9, wherein, after step (5), the phenol portion is enzymatically converted to an activated state, resulting in the deposition of the label.

23. The method according to claim 1, wherein the bonded label is released using or without an emission linker.

24. The method according to claim 23, wherein the conjugated label is released by cleaving the release linker between the label and the substrate, or by cleaving the release linker between the substrate and the nucleic acid.

25. The method according to claim 8, wherein the conjugated label is released by dehybridization of the nucleic acid chain in step (8) that is bound to the nucleic acid chain in step (5).

26. The method according to claim 5, wherein the method further comprises amplifying the nucleic acid chain or a portion thereof that is linked to the target-specific binding partner of step (1), and optionally the amplification step is performed before step (3).

27. The method according to claim 8, wherein the method further comprises amplifying the nucleic acid chain or a portion thereof of the nucleic acid chain-bound substrate of step (5), and optionally the amplification step is performed after step (5) or any step (6) or any step (7).

28. The method according to claim 8, wherein the method further comprises a first amplification of the nucleic acid chain or a portion thereof linked to the target-specific binding partner of step (1), optionally the amplification step being performed before step (3), and further the method comprises a second amplification of the nucleic acid chain or a portion thereof of the nucleic acid chain-bound substrate of step (5), optionally the second amplification step being performed after step (5) or any step (6) or any step (7).

29. The method according to claim 1, wherein the release-type linker comprises at least one of a disulfide bond, an ester, a vicinal diol, a sulfone, and a photocleavable linker.

30. The method according to claim 1, wherein the enzyme is selected from oxidoreductase, hydrolase, lyase, transferase, isomerase, and ligase.

31. The method according to claim 1, wherein the enzyme is selected from peroxidase, oxidase, phosphatase, esterase, and glycosidase.

32. The method according to claim 31, wherein the enzyme is selected from horseradish peroxidase, glucose oxidase, alkaline phosphatase, and β-galactosidase.

33. The method according to claim 32, wherein the enzyme is horseradish peroxidase.

34. The method according to claim 1, wherein the binding pair is immunotype or non-immune type.

35. The method according to claim 34, wherein the immunotype binding pair is selected from antigen-antibody and hapten-antihapten.

36. The method according to claim 35, wherein the antibody member of the binding pair is polyclonal, monoclonal, or an immunoreactive fragment thereof.

37. The method according to claim 36, wherein the non-immune binding pair is selected from biotin-avidin, biotin-streptavidin, and complementary probe nucleic acid folate-folate binding protein.

38. The method according to claim 33, wherein the non-immunotype bond pair optionally forms a covalent bond via a sulfhydryl reactive group, an amine reactive group, and a coupler dye selected from maleimide and haloacetyl derivative isothiocyanates, succinimidyl esters, sulfonyl halides, click chemistry, 3-methyl-2-benzothiazolinone hydrazone (MBTH), and 3-(dimethylamino)benzoic acid (DMAB).

39. The method according to claim 1, wherein the detectable label is selected from enzymes, radioisotopes, fluorescence, chemiluminescence, and electrochemical substances.

40. The method according to claim 1, wherein the enzyme is horseradish peroxidase (HRP) and the substrate is a phenolic substrate.

41. The method according to claim 1, wherein the enzyme is a hydrolase and the substrate is an ester, amide, or glycoside substrate.

42. A composition, (1) A sample comprising a sample bound to multiple target-specific binding partners, wherein each binding partner is bound to a nucleic acid chain, and the composition is (2) an enzyme for a nucleic acid chain complementary to the nucleic acid chain bound to the target-specific binding partner, The following conditions (i) or (ii) or both must be met: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release-type linker, and (ii) The enzyme is linked to the complementary nucleic acid chain by a second release linker, and the composition further comprises (3) The composition comprising a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally linked by a third release linker.

43. A composition, (1) The composition comprises a sample bound to multiple target-specific binding partners, each binding partner being bound to a nucleic acid chain, and the composition is (2) (i) A first member linked to a nucleic acid chain complementary to the nucleic acid chain bound to the target-specific binding partner, and (ii) The second member linked to the enzyme, Includes bond pairs containing, The following conditions (i) or (ii) or both must be met: (i) The nucleic acid chain is linked to the target-specific binding partner by a first release-type linker, and (ii) The composition wherein the first member of the bond pair is linked to the complementary nucleic acid chain by a second release linker, (3) comprising a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and label are optionally linked by a third release linker, The composition wherein the second member of the bond pair is linked to the enzyme by a fourth release-type linker.

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