Multiplexed catalytic reporter molecule deposition

Abstract

A method for testing the presence of one or more targets in a sample is provided that includes multiplexed catalytic reporter molecule deposition (CARD).

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201980031776.0, whose application date is June 6, 2019 and invention name is “Multiplexed Catalytic Reporter Molecules Deposition”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 682,765, filed on June 8, 2018, and U.S. Provisional Application No. 62 / 760,450, filed on November 13, 2018, the contents of all applications are incorporated herein by reference in their entirety for any purpose. Technical Field

[0004] The present application relates generally to the field of analyte (eg, target) detection, and in particular to detection methods using catalyzed reporter deposition (CARD). Background Art

[0005] For research and medical applications, it may be necessary to detect multiple biologically relevant molecules in a single cell or tissue sample. Immunological methods are generally used for this purpose. Such methods generally involve combining antibodies with target molecules in samples, generating detectable signals associated with antibodies so that each target molecule is distinguished from each other, and using analytical methods to detect signals. Catalytic reporter molecule deposition (CARD) has been used to produce enhanced signals associated with antibodies in such immunological methods. The typical workflow of CARD is that target molecule-specific antibodies are applied to samples, and then the secondary antibodies identifying primary antibodies are applied. Secondary antibodies are connected to enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase), which convert substrates into reactants (such as tyrosine) that are combined with nearby phenolic residues. Because phenolic residues are very abundant in cell samples, the result is that there are dense labels near the primary antibodies.

[0006] One technical problem with CARD is the difficulty in removing the secondary antibody from the sample. This problem arises when additional primary antibodies are needed to detect additional target molecules in the same sample. Heating or microwaves can be used to remove the secondary antibody, but this method can damage the sample, thereby reducing the quality of subsequent tests. In addition, this process is often time-consuming.

[0007] Therefore, a method that avoids harsh sample processing and allows the detection of multiple target molecules in a single sample would be a useful CARD methodology improvement. Summary of the invention

[0008] According to the specification, a method for testing the presence of one or more targets in a sample is provided to multiplex catalyzed reporter deposition (CARD) and achieve a higher level of multiplexed detection.

[0009] In one embodiment, a method for testing a sample for the presence of one or more targets comprises:

[0010] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0011] (2) optionally removing unbound target-specific binding partner;

[0012] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0013] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0014] (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker;

[0015] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0016] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked using a third releasable linker;

[0017] (6) optionally removing unbound substrate conjugates;

[0018] (7) optionally releasing the bound enzyme;

[0019] (8) optionally imaging the sample to detect the bound detectable label; and

[0020] (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to the imaging step (8).

[0021] In some embodiments, the method further comprises repeating steps (3)-(7) prior to the imaging step (8).

[0022] In some embodiments, in step (5), the substrate conjugate comprising the detectably labeled substrate is reacted with the enzyme to form an activated substrate conjugate, and the activated substrate conjugate binds to a receptor of the activated substrate conjugate. In addition, the receptor of the activated substrate conjugate may be present in the sample, which is immobilized on a solid support, thereby causing the detectable label to be deposited.

[0023] In another embodiment, the method may further comprise (10) optionally releasing the bound detectable label by releasing the third releasable linker after step (8), and optionally repeating steps (1)-(10) or any subset thereof.

[0024] In some embodiments, the method may further include amplifying the nucleic acid strand or a portion thereof that is attached to the target-specific binding partner of 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 comprises:

[0026] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0027] (2) optionally removing unbound target-specific binding partner;

[0028] (3) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0029] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0030] (ii) the first member of the binding pair is linked to the complementary nucleic acid strand using a second releasable linker;

[0031] (4) optionally removing unbound first members of the binding pair that are attached to the complementary nucleic acid strand;

[0032] (5) contacting the sample from step (3) or optionally step (4) with a second member of the binding pair optionally linked to an enzyme using a third releasable linker, and optionally removing unbound second member of the binding pair linked to the enzyme;

[0033] (6) contacting the sample from step (5) with a substrate conjugate comprising a labeled substrate, wherein the substrate and the label are optionally linked using a fourth releasable linker;

[0034] (7) optionally removing unbound substrate conjugates;

[0035] (8) optionally releasing the bound first member or second member of the binding pair;

[0036] (9) optionally imaging the sample to detect the bound detectable label; and

[0037] (10) Optionally repeating steps (1)-(9) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof prior to the imaging step (9).

[0038] In some embodiments, the method may further include (11) optionally releasing the bound detectable label by cleaving the third releasable linker between the label and the substrate after step (9), and optionally repeating steps (1)-(11) or any subset thereof.

[0039] In some embodiments, the method may further include amplifying the nucleic acid strand or a portion thereof that is attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0040] In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0041] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0042] (2) optionally removing unbound target-specific binding partner;

[0043] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0044] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0045] (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker;

[0046] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0047] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a nucleic acid strand bound to a substrate, wherein the substrate and the nucleic acid strand are optionally linked using a third releasable linker;

[0048] (6) optionally removing unbound substrate conjugates;

[0049] (7) optionally releasing the bound enzyme;

[0050] (8) contacting the sample from step (5) or optionally steps (6)-(7) with a detectable label bound to a nucleic acid strand, the detectable label optionally being attached using a fourth releasable linker, wherein the nucleic acid strand is a specific binding pair member of the nucleic acid strand in step (5);

[0051] (9) optionally imaging the sample to detect the bound label;

[0052] (10) optionally releasing the bound label from step (8); and

[0053] (11) Optionally repeating steps (1)-(10) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof before step (9).

[0054] In some embodiments, a releasable joint is used to release the combined label. In some embodiments, in step (10), the combined label is released by cleaving step (5) or optionally step (8) or optionally step (5) and step (8). In some embodiments, a releasable joint is not used to release the combined label. In some embodiments, the combined label is released by dehybridizing the nucleic acid chain. In some embodiments, the combined label is released by dehybridizing the nucleic acid chain from step (8) that is combined with the nucleic acid chain from step (5).

[0055] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0056] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) of the nucleic acid chain binding substrate of step (5). In some embodiments, the amplification step is performed after step (5) or optional step (6) or optional step (7).

[0057] In some embodiments, the method may further include first amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1) and secondly amplifying the nucleic acid strand of the nucleic acid strand-bound substrate of 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 optional step (6) or optional step (7).

[0058] In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0059] (1) contacting a sample being tested for the presence of one or more targets with a target-specific binding partner, wherein the target-specific binding partner is attached to a nucleic acid strand,

[0060] (2) optionally removing unbound target-specific binding partner,

[0061] (3) contacting the sample with an HRP-bound nucleic acid strand that is complementary to the nucleic acid strand that is linked to the target-specific binding partner,

[0062] Where (i) or (ii) or both of the following are satisfied:

[0063] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0064] (ii) the HRP is linked to the complementary nucleic acid strand using a second releasable linker;

[0065] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0066] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled phenolic substrate, wherein the substrate and the detectable label are optionally linked using a third releasable linker;

[0067] (6) optionally removing unbound substrate;

[0068] (7) optionally imaging the sample to detect a detectable label;

[0069] (8) optionally releasing the bound label; and

[0070] (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(6) or any subset thereof prior to the imaging step (7).

[0071] In some embodiments, after step (5), the phenolic moiety is enzymatically converted to an activated state, thereby resulting in deposition of the label.

[0072] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0073] In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0074] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0075] (2) optionally removing unbound target-specific binding partner;

[0076] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner;

[0077] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0078] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked using a releasable linker;

[0079] (6) optionally removing unbound substrate conjugates;

[0080] (7) optionally inactivating the bound enzyme;

[0081] (8) optionally imaging the sample to detect the bound detectable label; and

[0082] (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to the imaging step (8).

[0083] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0084] In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0085] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0086] (2) optionally removing unbound target-specific binding partner;

[0087] (3) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner;

[0088] (4) optionally removing unbound first members of the binding pair that are attached to the complementary nucleic acid strand;

[0089] (5) contacting the sample from step (3) or optionally step (4) with the second member of the binding pair linked to an enzyme, and optionally removing unbound second member of the binding pair linked to the enzyme;

[0090] (6) contacting the sample from step (5) with a substrate conjugate comprising a labeled substrate, wherein the substrate and the label are optionally linked using a releasable linker;

[0091] (7) optionally removing unbound substrate conjugates;

[0092] (8) optionally inactivating the bound enzyme;

[0093] (9) optionally imaging the sample to detect the bound detectable label; and

[0094] (10) Optionally repeating steps (1)-(9) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof prior to the imaging step (9).

[0095] In some embodiments, the method further comprises (11) optionally releasing the bound detectable label by cleaving the releasable linker between the label and the substrate after step (9), and optionally repeating any one of steps (1)-(9) and (11).

[0096] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0097] In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0098] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0099] (2) optionally removing unbound target-specific binding partner;

[0100] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner

[0101] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0102] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a nucleic acid strand bound to a substrate, wherein the substrate and the nucleic acid strand are optionally linked using a first releasable linker;

[0103] (6) optionally removing unbound substrate conjugates;

[0104] (7) optionally inactivating the bound enzyme;

[0105] (8) contacting the sample from step (5) or optionally steps (6)-(7) with a detectable label bound to a nucleic acid strand, the detectable label optionally being attached using a second releasable linker, wherein the nucleic acid strand is a specific binding pair member of the nucleic acid strand in step (5);

[0106] (9) optionally imaging the sample to detect the bound label;

[0107] (10) optionally releasing the bound label from step (8); and

[0108] (11) Optionally repeating steps (1)-(10) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof prior to the imaging step (9).

[0109] In some embodiments, a releasable joint is used to release the combined label. In some embodiments, in step (10), the combined label is released by cleaving step (5) or optionally step (8) or optionally step (5) and step (8). In some embodiments, a releasable joint is not used to release the combined label. In some embodiments, the combined label is released by dehybridizing the nucleic acid chain. In some embodiments, the combined label is released by dehybridizing the nucleic acid chain from step (8) that is combined with the nucleic acid chain from step (5).

[0110] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0111] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) of the nucleic acid chain binding substrate of step (5). In some embodiments, the amplification step is performed after step (5) or optional step (6) or optional step (7).

[0112] In some embodiments, the method may further include first amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1) and secondly amplifying the nucleic acid strand of the nucleic acid strand-bound substrate of 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 optional step (6) or optional step (7).

[0113] In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0114] (1) contacting a sample being tested for the presence of one or more targets with a target-specific binding partner, wherein the target-specific binding partner is attached to a nucleic acid strand;

[0115] (2) optionally removing unbound target-specific binding partner,

[0116] (3) contacting the sample with an HRP-bound nucleic acid strand that is complementary to the nucleic acid strand linked to the target-specific binding partner;

[0117] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0118] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled phenolic substrate, wherein the substrate and the detectable label are optionally linked using a releasable linker;

[0119] (6) optionally removing unbound substrate;

[0120] (7) optionally inactivating the HRP;

[0121] (8) optionally imaging the sample to detect the bound detectable label; and

[0122] (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or a subset thereof before step (8).

[0123] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of (1). In some embodiments, the amplification step is performed before step (3).

[0124] In some embodiments, the enzyme is inactivated using an enzyme inactivating agent, wherein the enzyme inactivating agent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, peroxide, cyanide, fluoride, or azide.

[0125] In some embodiments, the step (7) of deactivating the bound enzyme is performed for less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, or less than or equal to 1 minute. In some embodiments, the step (7) of deactivating the bound enzyme is performed for less than 20 minutes. In some embodiments, the step (7) of deactivating the bound enzyme is performed for less than 10 minutes. In some embodiments, the step (7) of deactivating the bound enzyme is performed for less than 5 minutes. In some embodiments, the step (7) of deactivating the bound enzyme is performed for less than 2 minutes. In some embodiments, the step (7) of deactivating the bound enzyme is performed for less than or equal to 1 minute.

[0126] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. Said objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0127] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0128] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiments and, together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] The patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0130] Figure 1A-1B Two schematics of multiplexed catalytic reporter deposition (CARD) are shown. Figure 1A Provides schematics that do not involve stacking. Figure 1B Provide a schematic diagram involving stacking.

[0131] Figure 2 A schematic diagram of multiplexed catalytic reporter deposition (CARD) using a binding pair is provided, wherein the binding pair is a nucleic acid strand (which is or comprises a barcode) and a complementary nucleic acid strand.

[0132] Figure 3 An exemplary embodiment is shown in which two target molecules are detected using the multiplexing method described herein.

[0133] Figure 4A Exemplary embodiments are shown wherein the enzyme is cleaved.

[0134] Figure 4B Exemplary embodiments are shown wherein the enzyme is inactivated.

[0135] Figure 5 An exemplary embodiment is shown in which a substrate barcode is used.

[0136] Figure 6 A scheme for synthesizing exemplary substrate barcodes is shown.

[0137] Figure 7A quadruple image of CD8, CD68, PD-L1, and CK targets obtained using sequential deposition of fluorescent tyramide reagents according to Example 1 is shown (nuclear counterstain not shown). CD8 is shown in green; CD68 is shown in red; PD-L1 is shown in cyan; CK is shown in magenta. The nuclear counterstain is not shown.

[0138] Figure 8A-8B The inactivation of HRP enzyme using TCEP treatment according to Example 2 is shown ( Fig. 8A ) compared with the control without TCEP treatment ( Figure 8B ) comparison.

[0139] Fig. 9 A quadruple image of CD8, CD68, PD-L1, and CK targets obtained using sequential deposition of nucleic acid barcodes according to Example 3 is shown. CD8 is shown in green; CD68 is shown in red; PD-L1 is shown in cyan; CK is shown in magenta. The nuclear counterstain is not shown. DETAILED DESCRIPTION

[0140] I. Definitions

[0141] The term "deposition" refers to the directed binding of the activated substrate conjugate to the receptor resulting from the interaction forming a specific binding pair as described below.

[0142] The term "receptor" is intended to refer to a site to which an activated substrate conjugate will bind through interaction forming a specific binding pair as described below.

[0143] The term "activated substrate conjugate" means that the substrate conjugate has been primed by the reporter enzyme to bind to the receptor.

[0144] The term "detectably labeled" substrate conjugate means that the substrate can be coupled to a detectable label (the term "reporter molecule" can be used interchangeably) or an unlabeled first member of a specific binding pair to facilitate detection after deposition. When the substrate is coupled to an unlabeled member of a specific binding pair, after deposition, the substrate-specific binding pair complex reacts with a second member of the binding pair coupled to a reporter molecule (or label). Alternatively, the substrate-specific binding pair complex can be pre-reacted with a detectably labeled second member of the specific binding pair prior to deposition.

[0145] As described herein, the term "amplification" refers to increasing the number of copies of a nucleic acid sequence (such as a nucleic acid chain or a portion thereof (such as a barcode)) so that multiple copies of the nucleic acid sequence are connected to the corresponding target-specific binding partner. Various amplification methods known in the art can be used to increase the number of copies of nucleic acid sequences. Examples of nucleic acid amplification methods include hybridization chain reaction (HCR) (Dirks et al., 2014, PMID: 15492210, 24712299), dendron polymerization reaction (HDR) based on DNA hairpins (Yin et al., 2008, PMID 18202654), rolling circle amplification (RCA), primer exchange reaction (PER) and other nucleic acid amplification methods, for example, as described in WO 2018 / 107054; WO 2017 / 143006; WO 2018 / 132392A2, the contents of each of which are incorporated herein by reference. This amplification is different from the signal amplification generated by CARD.

[0146] II. Methods for Multiplexed Catalytic Reporter Deposition

[0147] The present application discloses a method for multiplexing catalytic reporter deposition (CARD). Figure 1A , Figure 1B , Figure 2 and Figure 3 A schematic diagram of multiplexed catalytic reporter molecule deposition is provided in .

[0148] According to the description, in some embodiments, a method for testing the presence of one or more targets in a sample comprises:

[0149] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, optionally using a releasable linker, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0150] (2) optionally removing unbound target-specific binding partner,

[0151] (3) contacting the sample with an enzyme or a member of a specific binding pair for subsequent enzymatic labeling, the enzyme or member being attached to a nucleic acid strand complementary to a strand on a target-specific binding partner, wherein if a releasable linker is not used to attach the nucleic acid strand bound to the target-specific binding partner, a releasable linker is used to attach the enzyme or the member of the specific binding pair to the complementary strand of step (3), and if a releasable linker is used to attach the nucleic acid strand bound to the target-specific binding partner, a releasable linker is optionally used to attach the enzyme or the member of the specific binding pair to the complementary strand of step (3),

[0152] (4) optionally removing unbound enzyme or specific binding pair member attached to the complementary nucleic acid strand;

[0153] (5) if a member of a specific binding pair was utilized in step (3), contacting the sample with a second member of the specific binding pair optionally linked to an enzyme using a releasable linker, and optionally removing unbound enzyme-linked second member of the specific binding pair;

[0154] (6) contacting the sample with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally linked using a releasable linker;

[0155] (7) optionally removing unbound substrate conjugates,

[0156] (8) optionally releasing the bound enzyme, first specific binding pair member, or second specific binding pair member,

[0157] (9) optionally detecting a bound detectable label,

[0158] (10) Optionally repeating steps (1)-(9) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof before the detecting step (9).

[0159] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0160] A. Multiplexing CARD using enzymes bound to probe strands

[0161] In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0162] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0163] (2) optionally removing unbound target-specific binding partner;

[0164] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0165] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0166] (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker;

[0167] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0168] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally linked using a third releasable linker;

[0169] (6) optionally removing unbound substrate conjugates;

[0170] (7) optionally releasing the bound enzyme;

[0171] (8) optionally imaging the sample to detect the bound detectable label; and

[0172] (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to the imaging step (8).

[0173] In some embodiments, in step (5), the substrate conjugate comprising the detectably labeled substrate is reacted with the enzyme to form an activated substrate conjugate, and the activated substrate conjugate binds to a receptor for the activated substrate conjugate.

[0174] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0175] In some embodiments, the receptor for the activated substrate conjugate is present in the sample, resulting in deposition of the detectable label.

[0176] In some embodiments, all labels are removed and the above labeling step is started again ("stacking") to achieve another round of multiplexing. In some embodiments, the method further comprises (10) releasing the bound detectable label after the imaging step (8), and optionally repeating steps (1)-(10) or any subset thereof. In some embodiments, a releasable joint is used to release the bound label. In some embodiments, in step (10), the bound label is released by cleaving a third releasable joint. In some embodiments, a releasable joint is not used to release the bound label.

[0177] Figure 1A-1B It shows that there is no accumulation involved ( Figure 1A ) and involves accumulation ( Figure 1B ) is an implementation plan of a method for multiplexing CARDs.

[0178] In such Figure 1A In the illustrated stack-free multiplexing CARD, a sample (tissue, 100) bound to a target-specific binding partner (antibody, 101) is first provided, wherein the target-specific binding partner is connected to a nucleic acid strand comprising a barcode (barcode, 102), and the sample is contacted with an enzyme (103) connected to a nucleic acid strand (probe, 104), the nucleic acid strand being complementary to the nucleic acid strand comprising the barcode (barcode, 102) connected to the antibody (101). The nucleic acid strand comprising the barcode (barcode, 102) is connected to the antibody (101) using a first releasable joint (105). Alternatively, the enzyme (103) is connected to a complementary nucleic acid strand using a second releasable joint (106), the complementary nucleic acid strand comprising a sequence complementary to the barcode (probe, 104). Optionally, unbound enzymes connected to the complementary nucleic acid strand are removed.

[0179] Then, the sample (100) is contacted with a substrate conjugate (labeled substrate, 107) containing a detectable label (108). The detectable label is reacted with the enzyme to form an activated substrate conjugate, and the activated substrate conjugate binds to the receptor of the activated substrate conjugate present in the sample, thereby causing the detectable label to be deposited on the sample (108). Unbound substrate conjugate (107) is optionally removed by a washing step. Then, the enzyme is released by cleaving the releasable joint using a cleaving agent. Depending on the use of the first releasable joint (105) or the second releasable joint (106), after the releasable joint is cleaved, the enzyme (103) connected to the complementary nucleic acid chain (probe, 104) is removed (lower left) or the nucleic acid chain is cleaved (lower right), so that the enzyme, the nucleic acid chain containing the barcode (barcode, 102) connected to the antibody (101) and the complementary nucleic acid chain (probe, 104) connected to the enzyme are removed. For each target, the above deposition of the detectable label can be repeated. The sample is then imaged to detect bound detectable labels of the different targets.

[0180] In such Figure 1B In the multiplexed CARD using stacking shown, a third releasable linker (109) can be used to connect the substrate (107) and the label (108); and after the imaging step, all bound labels are removed by cleaving the third releasable linker (109), and the steps described above are repeated to deposit labels for detecting more targets ("stacking").

[0181] Figure 3 Also illustrated is an exemplary embodiment, wherein two target molecules are detected using the methods described herein. In a tissue sample, two targets 1 and 2 are combined with corresponding antibodies, each antibody being connected to different nucleic acid chains (step A). ​​The enzyme connected to a nucleic acid chain (enzyme probe 1) (wherein the nucleic acid chain is complementary to the nucleic acid chain of the antibody connected to target 1) is combined with the nucleic acid chain of the antibody connected to target 1, and the unbound enzyme probe is optionally washed (step B). Then, the substrate of the label is added to the sample (step C), and it is reacted with the enzyme to form an activated substrate conjugate. The activated substrate conjugate is combined with the receptor of the activated substrate conjugate present in the sample, thereby causing detectable label deposition (step D). Enzyme probe 1 is released by using a cleavage agent to cleave a releasable joint (step E) or by an enzyme inhibitor (or enzyme deactivator) to inactivate the enzyme (step F). In step 3, according to the position of the releasable joint, the enzyme is removed, or the barcode and the probe connected to the enzyme are also removed together with the enzyme (step G). For target 2, the above deposition (steps H and I) of repeatable labeling can be repeated. The sample is then imaged to detect the combined detectable labels of the two targets. Optionally, the signal from the label can be reduced if necessary by using methods well known in the art (eg, chemical bleaching, photobleaching and / or photochemical bleaching).

[0182] B. Multiplexing CARDs using binding pairs

[0183] In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0184] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0185] (2) optionally removing unbound target-specific binding partner;

[0186] (3) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0187] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0188] (ii) the first member of the binding pair is linked to the complementary nucleic acid strand using a second releasable linker;

[0189] (4) optionally removing unbound first members of the binding pair that are attached to the complementary nucleic acid strand;

[0190] (5) contacting the sample from step (3) or optionally step (4) with a second member of the binding pair optionally linked to an enzyme using a third releasable linker, and optionally removing unbound second member of the binding pair linked to the enzyme;

[0191] (6) contacting the sample from step (5) with a substrate conjugate comprising a labeled substrate, wherein the substrate and the label are optionally linked using a fourth releasable linker;

[0192] (7) optionally removing unbound substrate conjugates;

[0193] (8) optionally releasing the bound first member or second member of the binding pair;

[0194] (9) optionally imaging the sample to detect the bound detectable label; and

[0195] (10) Optionally repeating steps (1)-(9) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof prior to the imaging step (9).

[0196] In some embodiments, the method may further include amplifying the nucleic acid strand attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0197] In some embodiments, for stacking, the method further comprises (11) optionally releasing the bound detectable label. In some embodiments, the bound label is released by releasing a third releasable linker between the label and the substrate. In some embodiments, no releasable linker is used to release the bound label.

[0198] In some embodiments, the method further comprises (11) optionally releasing the bound detectable label by releasing the third releasable linker after the imaging step (9), and further comprises optionally repeating steps (1)-(11) or any subset thereof. In some embodiments, a method for testing a sample for the presence of one or more targets comprises:

[0199] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0200] (2) optionally removing unbound target-specific binding partner;

[0201] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0202] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0203] (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker;

[0204] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0205] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a nucleic acid strand bound to a substrate, wherein the substrate and the nucleic acid strand are optionally linked using a third releasable linker;

[0206] (6) optionally removing unbound substrate conjugates;

[0207] (7) optionally releasing the bound enzyme;

[0208] (8) contacting the sample from step (5) or optionally steps (6)-(7) with a detectable label bound to a nucleic acid strand, the detectable label optionally being attached using a fourth releasable linker, wherein the nucleic acid strand is a specific binding pair member of the nucleic acid strand in step (5);

[0209] (9) optionally imaging the sample to detect the bound label;

[0210] (10) optionally releasing the bound label from step (8); and

[0211] (11) Optionally repeating steps (1)-(10) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof prior to the imaging step (9).

[0212] In some embodiments, a releasable joint is used to release the bound label. In some embodiments, the bound label is released by cleaving the releasable joint of step (5) or optionally step (8) or optionally step (5) and step (8). In some embodiments, a releasable joint is not used to release the bound label. In some embodiments, the bound label is released by dehybridizing the nucleic acid chains bound to each other. In some embodiments, the bound label is released by dehybridizing the nucleic acid chain in step (8) bound to the nucleic acid chain in step (5). Dehybridization of nucleic acids can be achieved by conventional methods known in the art, including heating, adding chaotropic agents (such as urea, formamide and guanidine chloride) and low ionic strength. Increasing the temperature or reducing the ionic strength of the medium containing the nucleic acid chain destroys the binding affinity between the nucleic acid chains. Heating, one or more chaotropic agents and low ionic strength can each be used independently to dehybridize nucleic acids; or a combination of one or more of these conditions can be used. As used herein, "low ionic strength" means a medium ionic strength of less than 300 mM as measured by the amount of salt concentration in the medium. In one embodiment, the salt concentration is less than the concentration required to make the Tm below ambient temperature, for example, less than 70 mM. The hybridization rate decreases as the salt concentration decreases. Salt (e.g., Na+) can be reduced by conventional methods, such as by adding water. + ) concentration to achieve dehybridization at low ionic strength.

[0213] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1). In some embodiments, the amplification step is performed before step (3).

[0214] In some embodiments, the method may further include amplifying the nucleic acid chain (or a portion thereof) of the nucleic acid chain binding substrate of step (5). In some embodiments, the amplification step is performed after step (5) or optional step (6) or optional step (7).

[0215] In some embodiments, the method may further include first amplifying the nucleic acid strand (or a portion thereof) attached to the target-specific binding partner of step (1) and secondly amplifying the nucleic acid strand of the nucleic acid strand-bound substrate of 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 optional step (6) or optional step (7).

[0216] Figure 2Embodiments of methods of multiplexed catalytic reporter deposition (CARD) using binding pairs are shown, where the binding pairs are a nucleic acid strand (which is or comprises a barcode) and a complementary nucleic acid strand.

[0217] First, a sample (tissue, 200) is provided that is bound to a target-specific binding partner (antibody, 201), wherein the target-specific binding partner is connected to a nucleic acid strand comprising a barcode (barcode, 202), and the sample is contacted with an enzyme (203) connected to a nucleic acid strand (probe, 204), which is complementary to the nucleic acid strand (barcode, 202) connected to the antibody (201). The nucleic acid strand (barcode, 202) is connected to the antibody (201) using a first releasable linker (205). Alternatively, the enzyme (203) is connected to the complementary nucleic acid strand (probe, 204) using a second releasable linker (206). Optionally, unbound enzyme (203) connected to the complementary nucleic acid strand (202) is removed.

[0218] The sample (200) is then contacted with a nucleic acid strand-bound substrate (217), wherein the substrate (217) and nucleic acid strand (227) are deposited on the tissue. Unbound substrate conjugates and / or bound enzymes are then removed by cleaving the releasable linker using a cleavage agent (such as TCEP, DTT, or periodate). The above deposition of nucleic acid-bound substrates can be repeated for each target. For the last round of deposition, a washing step is optional.

[0219] Then, the tissue sample (200) is contacted with a detectable label (208) combined with a nucleic acid chain, and the detectable label is optionally connected using a fourth releasable joint (209), wherein the nucleic acid chain (probe, 208) is a specific binding pair member of the nucleic acid chain complementary to the probe (227). If four primary antibodies combined with four different targets are provided, four different label probes (208) are provided. Then, the tissue sample (200) is imaged to detect the detectable labels combined with different targets. In order to detect additional targets, after the imaging step, all combined labels are removed by a washing step, and the third releasable joint (209) between the label and the probe (208) is cracked using a cleavage agent, and the steps described above are repeated to deposit ("pile up") the labels for detecting more targets. Repeat the steps described above or any subset thereof as needed for detecting more targets.

[0220] C. Embodiments using phenolic substrates

[0221] In some embodiments, the multiplexing CARD method is a multiplexed tyramide signal amplification (TSA) method. TSA amplification methods can also be used, in which fluorophores can be easily bleached. For example, many cyanine fluorophores and Alexa fluorophores are easily bleached by hydrogen peroxide under acidic or alkaline conditions (PMID: 26399630). Alternatively, TSA dyes containing cleavable bonds between tyramide and fluorophores can be synthesized. In this case, the fluorophore can be inactivated by cleaving this bond and washing.

[0222] In some embodiments, a method for testing a sample for the presence of one or more targets comprises: (1) contacting the sample being tested for the presence of one or more targets with a target-specific binding partner, wherein the target-specific binding partner is attached to a nucleic acid strand, (2) optionally removing unbound target-specific binding partner, (3) contacting the sample with an HRP-bound nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied: (i) the nucleic acid strand is attached to the target-specific binding partner using a first releasable linker; and (ii) the HRP is attached to the target-specific binding partner using a second releasable linker. said complementary nucleic acid strand; (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled phenolic substrate, wherein the substrate and the detectable label are optionally attached using a third releasable linker; (6) optionally removing unbound substrate; (7) optionally imaging the sample to detect the sample; (8) optionally releasing the bound label; and (9) optionally repeating steps (1)-(8) or any subset thereof, and optionally repeating steps (1)-(6) or any subset thereof before the imaging step (7) if more than one target-specific binding partner is used.

[0223] In some embodiments, after step (5), the phenolic substrate is enzymatically converted to an activated state, thereby resulting in the deposition of the label. In some embodiments, the phenolic substrate is tyramide.

[0224] In some embodiments, the method may further include amplifying the nucleic acid strand (or a portion thereof) connected to the target-specific binding partner. In some embodiments, the amplification step is performed prior to the step (3) of contacting the sample with a nucleic acid strand bound to HRP, the nucleic acid strand being complementary to the nucleic acid strand connected to the target-specific binding partner. Catalytic reporter deposition is a signal amplification method, and it utilizes an enzyme coupled directly or indirectly to a target-specific binding partner, the enzyme catalyzing the conversion of a substrate conjugate containing a detectably labeled substrate into an activated substrate conjugate (binding to a receptor of the activated substrate conjugate), thereby resulting in detectable label deposition. Previous examples of catalytic reporter deposition are described in, for example, U.S. Patent Nos. 5,196,306, 5,583,001, and 5,731,158.

[0225] Catalytic reporter deposition has been exploited to enhance signals in immunohistochemistry, immunocytochemistry, in situ hybridization, ELISA, flow cytometry, electron microscopy, and other applications.

[0226] However, there are several limitations in previous catalytic reporter deposition. One of the limitations is the ability to multiplex (i.e., detect multiple assay targets in the same assay). The limitations are exacerbated when antibodies from the same species are utilized in the assay. Detection of two targets using the same species of antibody has been accomplished by using catalytic reporter deposition to detect one target and standard detection of the second target (Shindler J. Histochem Cytochem 1996 Vol. 44 No. 11 1331-1335).

[0227] In previous attempts to multiplex immunohistochemical assays using catalytic reporter deposition, tissue sections were microwaved between each round of reporter deposition (Toth J. Histochem Cytochem 2007 Vol. 55 (6) 545-554). Microwaves remove the antibody and reporter enzyme, while the detectable label remains intact. Microwaves add many steps because each antibody and reporter molecule needs to be added sequentially and then removed by microwaves. As an example of the use of microwaves, Opal TM 7 Solid Tumor Immunoassay Kit (PerkinElmer) requires more than 90 minutes per cycle, and the entire process takes two days to complete. Microwaves can also alter or damage the sample being imaged.

[0228] In another previous attempt, heat inactivation was used to detect a 5-plex assay (Zhang, W., Hubbard, A., Jones, T., et al., J. Immunotherapy Cancer (2015) 3(Suppl 2): ​​P111). Four cycles of heat inactivation resulted in a 12%-50% reduction in fluorescence of the five fluorescent markers used. The assay took nine hours to complete. Other variations of heating have been described in U.S. Pat. Nos. 9,689,875 and 9,874,571, which are incorporated herein by reference for teaching variations of heating. However, heat inactivation may alter or damage the sample being imaged.

[0229] With microwave or heat inactivation between cycles, the number of detectable targets is still limited by the number of labels that the reader and software can resolve. In the field of immuno-oncology, the number of targets that need to be detected is already in the range of 12 to 20, and this is far beyond the limits of readers and software.

[0230] The method described above according to the present disclosure provides a signal amplification method that can be multiplexed in a simpler and faster manner, thereby avoiding harsh sample treatments such as heating and microwaves, and has the ability to be multiplexed at a higher level.

[0231] D. Test kit

[0232] In some embodiments, the kit includes a composition comprising (1) one or more target-specific binding partners, each binding partner being bound to a nucleic acid strand, (2) an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand to which the one or more target-specific binding partners are bound, wherein the following (i) or (ii) or both are satisfied: (i) the nucleic acid strand is attached to each target-specific binding partner using a first releasable linker; and (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker; and (3) a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally attached using a third releasable linker.

[0233] In some embodiments, the kit includes a composition comprising: (1) one or more target-specific binding partners, each binding partner being bound to a nucleic acid strand, (2) a binding pair comprising: a first member attached to a nucleic acid strand complementary to the nucleic acid strand bound to each target-specific binding partner, and a second member attached to an enzyme, wherein the following (i) or (ii) or both are satisfied: (i) the nucleic acid strand is attached to each target-specific binding partner using a first releasable linker; and (ii) the first member of the binding pair is attached to the complementary nucleic acid strand using a second releasable linker; and (3) a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally attached using a third releasable linker, and wherein the second member of the binding pair is attached to the enzyme using a fourth releasable linker.

[0234] In some embodiments, the kit further comprises a fluidic system for performing the fluid exchange step, software for controlling the fluidic system and timing and / or synchronizing the fluidic step with the imaging step. In some embodiments, the kit further comprises an imaging chamber that is attached to a target sample using at least one optically transparent surface to allow imaging of the sample.

[0235] III. Components of the Method

[0236] A. Enzymes for CARD

[0237] Enzymes suitable for direct or indirect coupling to a target-specific binding partner include hydrolases, lyases, oxidoreductases, transferase isomerases, ligases, peroxidases, oxidases, phosphatases, esterases, and glycosidases. Specific examples include alkaline phosphatase, lipase, beta-galactosidase, and horseradish peroxidase (HRP). If desired, more than one enzyme may be used simultaneously in combination with a unique substrate conjugate for each enzyme.

[0238] B. Specific binding pairs

[0239] The members of the specific binding pairs suitable for practicing the present invention can be of immune type or non-immune type. Examples of immunospecific binding pairs are antigen / antibody systems or hapten / anti-hapten systems. The antibody members of the binding pair, whether polyclonal antibodies, monoclonal antibodies or immunoreactive fragments thereof, can be produced by conventional methods familiar to those skilled in the art. The term immunoreactive antibody fragment or immunoreactive fragment means a fragment containing the binding region of an antibody. Such fragments can be Fab-type fragments defined as fragments without Fc parts, such as Fab, Fab' and F(ab')2 fragments, which are obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components of the complete antibody. The fragment can also be a single domain antibody derived from a heavy chain or light chain antibody, and can include nano antibodies. If the antigen member of the specific binding pair is not immunogenic, such as a hapten, it can be covalently coupled to a carrier protein to make it immunogenic.

[0240] Non-immune binding pairs include systems where two components share natural affinity with each other but are not antibodies. Exemplary non-immune binding pairs are biotin-avidin or biotin-streptavidin, complementary probe nucleic acid folic acid-folate binding protein, etc. Also included are non-immune binding pairs that form covalent bonds with each other. Exemplary covalent binding pairs include sulfhydryl reactive groups (such as maleimide and haloacetyl derivatives) and amine reactive groups (such as isothiocyanates, succinimide esters, sulfonyl halides and click chemistry) and coupler dyes (such as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethyl-amino) benzoic acid (DMAB)), etc.

[0241] Complementary probe nucleic acids can be used to increase the signal by increasing the number of binding sites for 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 reaction (WO 2017 / 143006 Al).

[0242] C. Target-specific binding partners

[0243] Target-specific binding partners suitable for implementing the present invention can be of immune type or non-immune type. An example of an immunospecific binding pair is an antigen / antibody system. The antibody member of the binding pair, whether a polyclonal antibody, a monoclonal antibody or an immunoreactive fragment thereof, can be produced by conventional methods familiar to those skilled in the art. The term immunoreactive antibody fragment or immunoreactive fragment means a fragment containing the binding region of an antibody. Such fragments can be Fab-type fragments defined as fragments without an Fc portion, such as Fab, Fab' and F(ab')2 fragments, which are obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components of the complete antibody. The fragment can also be a single domain antibody derived from a heavy chain or light chain antibody, and can include nano antibodies. If the antigen member of the specific binding pair is not immunogenic, such as a carbohydrate or a phosphorylated amino acid, it can be covalently coupled to a carrier protein to make it immunogenic.

[0244] Table 1 provides a representative list of targets and corresponding target recognition moieties.

[0245]

[0246] A list of additional targets is provided in Table 2. Antibodies and other known binding partners for these targets can be used as target recognition moieties.

[0247]

[0248]

[0249] Non-immune binding pairs include systems in which the two components share a natural affinity for each other but are not antibodies.

[0250] An exemplary non-immunological binding pair includes a complementary probe nucleic acid and an aptamer.The complementary probe nucleic acid is a suitable target-specific binding partner for a nucleic acid (eg, DNA or RNA) target.

[0251] 1. Nucleic acid strands used in binding pairs

[0252] In some embodiments, the nucleic acid used in the binding pair is a single-stranded nucleic acid, such as single-stranded DNA, RNA or nucleic acid analogs. Nucleic acid analogs (also referred to as non-natural nucleic acids) can include a changed phosphate backbone, a changed pentose and / or a changed core base. Nucleic acid analogs can include but are not limited to 2'-O-methyl ribonucleic acid, 2'-fluoro ribonucleic acid, peptide nucleic acid, morpholino and locked nucleic acid, glycol nucleic acid and threose nucleic acid.

[0253] In some embodiments, the nucleic acid chain comprises a single-stranded nucleic acid and can be about 5 to 20 nucleotides long, about 8 to 15 nucleotides long, or about 10 to 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.

[0254] The nucleic acid strand may be an independent element, or may be part of the target recognition moiety.

[0255] The nucleic acid strands may be provided in a liquid medium or a buffered solution.

[0256] The target-specific binding partner may be provided in a liquid medium or a buffered solution.

[0257] D. Binding of activated substrate conjugate to receptor

[0258] The directed binding of the activated substrate conjugate to the receptor is caused by the interaction that forms a specific binding pair. The members of the specific binding pair suitable for binding the activated substrate to the receptor can be of immune type or non-immune type. Examples of immunospecific binding pairs are antigen / antibody systems or hapten / anti-hapten systems. The antibody members of the binding pair, whether polyclonal antibodies, monoclonal antibodies or immunoreactive fragments thereof, can be produced by conventional methods familiar to those skilled in the art. The term immunoreactive antibody fragment or immunoreactive fragment means a fragment containing the binding region of an antibody. Such fragments can be Fab-type fragments defined as fragments without an Fc portion, such as Fab, Fab' and F(ab')2 fragments, which are obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components of the intact antibody.

[0259] Non-immune binding pairs include systems where two components share natural affinity with each other but are not antibodies. Exemplary non-immune binding pairs are pairs that form covalent bonds with each other. Exemplary covalent binding pairs include but are not limited to dimerization of phenolic moieties (e.g., tyramine and tyrosine), sulfhydryl reactive groups (such as maleimide and haloacetyl derivatives) and amine reactive groups (such as isothiocyanates, succinimide esters, sulfonyl halides and click chemistry) and coupler dyes (such as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethyl-amino)benzoic acid (DMAB)).

[0260] E. Reporter molecules or markers

[0261] A variety of reporter molecules (or labels) can be used to couple to a substrate to produce a substrate conjugate or to couple to a member of a specific binding pair. The reporter molecule (or label) can be, but is not limited to, an enzyme, a fluorescent material, a colorimetric material, a chemiluminescent material, a mass tag, a magnetic material, a plasmonic material, or an electrochemical material.

[0262] In some embodiments, fluorescent molecules such as small organic molecules can be used, including but not limited to fluorophores such as but not limited to fluorescein, rhodamine, cyanine dyes, Alexa dyes, DyLight dyes, Atto dyes, etc.

[0263] Chromogenic, fluorescent and chemiluminescent materials may include, but are not limited to, 3,3'-diaminobenzidine (DAB), nitro blue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-Gal).

[0264] In some embodiments, organic polymers such as p-dots can be used. In some embodiments, the observable portion can be a biomolecule, including but not limited to fluorescent proteins or fluorescent nucleic acids (including fluorescent RNA, including Spinach and its derivatives). In some embodiments, the observable portion can be an inorganic portion comprising a Q-dot. In some embodiments, the observable portion can be a portion that functions by scattering of elastic or inelastic scattering, such as nanoparticles and surface enhanced Raman spectroscopy (SERS) reporter molecules (e.g., 4-mercaptobenzoic acid, 2,7-mercapto-4-methylcoumarin). In some embodiments, the observable portion can be a chemiluminescent / electrochemiluminescent emitter, such as a ruthenium complex and luciferase. The observable portion can generate an optical signal, an electromagnetic signal (across the entire electromagnetic spectrum), an atomic / molecular mass (e.g., detectable by mass spectrometry), a tangible mass (e.g., detectable by atomic force microscopy), a current, or a voltage.

[0265] F. Substrate

[0266] The substrate for substrate conjugate is specific to the selected enzyme and is known to those skilled in the art. Many phenolic substrates used with horseradish peroxidase have been described (U.S. Patent Nos. 5,196,306, 5,583,001, 5,573,1158, 5,863,748 and 6,355,443), each of which is incorporated by reference for teaching phenolic substrates. The substrate of hydrolase has also been described (U.S. Patent Nos. 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.

[0267] 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.

[0268] G.Releasable joint

[0269] Throughout this disclosure, the first, second or third releasable joints may be mentioned, but in many embodiments, some or all of them are optional. Only mentioning the second or third releasable joint does not require the presence of two or three releasable joints. On the contrary, this numbering is used throughout the application to name the joints and to distinguish them from each other. Therefore, the embodiment comprising the second releasable joint does not have to have two releasable joints, etc. This language only specifies which joint is present, and does not specify how many joints are present.

[0270] Releasable linkers are those containing functional groups that can 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 microwaves. Exemplary bonds that can be cleaved are 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 alkaline conditions), and photocleavable linkers (such as linkers containing 2-nitrobenzyl groups).

[0271] An alternative method using a releasable joint is to completely remove the enzyme or specific binding pair member bound to the nucleic acid strand. Removal can be promoted by destroying the binding affinity between the nucleic acid strands or by the unique nucleic acid sequence of enzymatic cleavage. Exemplary removal methods include USER enzymes (New England Biolabs), reducing the ionic strength of the medium, raising the temperature and / or using a chaotropic agent such as guanidine, ethylene carbonate and / or formamide.

[0272] In some embodiments, the releasable joint includes a photocleavable joint that can be photochemically cleaved (e.g., by UV exposure, visible light, infrared, near infrared, X-rays, microwaves, radio waves, or gamma rays). In some embodiments, the releasable joint includes a part that can be cleaved by an enzyme. Examples of such enzymatic cleavable parts include, but are not limited to, ribonucleotides, which can be cleaved by a variety of RNA enzymes; deoxyuridine, which can be cleaved by enzyme combinations, such as USER (New England Biolabs); and restriction sites, which can be cleaved by sequence-specific nicking enzymes or restriction enzymes. In some embodiments, the releasable joint includes deoxyuridine, wherein the uracil group can be cleaved by uracil-DNA glycosidase. In some embodiments, the releasable joint includes an abasic site, which can be cleaved by an endonuclease.

[0273] 1. Nucleic acid degrading enzyme

[0274] Many enzymes can destroy the covalent bonds in nucleic acid molecules. For example, some glycosidases can remove bases from the sugar moiety of nucleotides, endonucleases can cut the bonds in the phosphodiester bridges inside nucleic acid molecules, and exonucleases can similarly destroy the phosphodiester bridges at the 5' or 3' ends of nucleic acid molecules in a continuous manner. Another example includes DNA enzymes or deoxyribozymes, oligonucleotides with catalytic activity that can crack the phosphodiester bonds in nucleic acid molecules. All these types of enzymes can be engineered to release releasable joints and constitute enzymatic cleavage, modification or degradation of releasable joints.

[0275] Glycosidase. If glycosidase can specifically remove the bases involved in base pairing, it can reduce the interaction strength between the two chains. For example, deoxyuridine (dU) can be used to replace deoxythymidine (dT) in the releasable joint. dU can be paired with dA like dT, but it can be specifically removed by uracil-DNA glycosidase (UDG, commercially available from New England Biolabs, catalog #M0280S). This reaction will produce an abasic site connected to the nucleic acid chain of the detectable label. Such abasic site can be further cleaved by endonuclease VIII. This will further promote the dissociation between the residues of the binding pair. Enzyme blends comprising both UDG and endonuclease VIII are also commercially available (for example, from New England Biolabs, trade name USER, catalog #M5505S). About 1 to 20, 1 to 15, 1 to 10 or 1 to 5 dU nucleotides can be placed in the nucleic acid chain connected to the detectable label. Using USER, dU can be placed in the following manner: after removing U, the residue is short enough (e.g., less than or equal to about 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides) to dissociate spontaneously and rapidly. If only UDG is used (i.e., without using endonuclease VIII), the removal of the dU unit can make the chain unstable enough to facilitate removal. After removing dU, the total number of base pairs between the binding pairs can be less than or equal to 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides. Therefore, in some embodiments, the releasable joint may include at least one U that can be cleaved by USER.

[0276] The sequence and temperature of residue will affect the time length of spontaneous dissociation of residue.For example, the sequence with high GC content may have a greater binding affinity than another sequence with longer length on shorter length.Therefore, in some cases, 9 aggressiveness may be enough to stably bind, and in other cases, 9 aggressiveness may be enough to dissociate.One of ordinary skill in the art can assess sequence, temperature and affinity in the cracking of non-natural nucleotides discussed herein and hereinafter.

[0277] Restriction endonucleases and nicking endonucleases. Restriction sites in releasable joints can be engineered. This allows the use of corresponding restriction endonucleases to cut such restriction sites, which destroys the releasable joints. For example, Cas9 (CRISPR-associated protein 9) is an RNA-guided endonuclease that can be used to specifically cleave releasable joints by engineering specific recognition sites in the corresponding sequence. This causes both chains to be cleaved, thereby preventing one chain from interrogating the corresponding target again. In order to solve this problem, a nicking endonuclease that only cuts one chain can be used. For example, Cas9 nickase is a Cas9 enzyme that has been engineered to contain only one active cleavage site, resulting in single-stranded cleavage while retaining the high specificity of Cas9. Other examples of endonucleases with site-specific activity include, but are not limited to, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and deoxyribozymes.

[0278] RNase. Some or all of the nucleotides may be made in releasable linker RNA nucleotides (also called ribonucleotides) rather than DNA nucleotides (also called deoxynucleotides). Such RNA nucleotides may be removed by RNase.

[0279] Polymerases. Releasable linkers can also be removed by using a polymerase with strand displacement activity or 5' to 3' exonuclease activity.

[0280] Cleavage of non-natural nucleotides. Non-natural nucleotides that serve as substrates for specific enzymes can be used. For example, 8-oxo-bridged guanine can be cleaved by DNA glycosidase OGG1. Abasic sites can also be incorporated into DNA chains, such as DNA chains connected to detectable labels, which can be cleaved by endonucleases. For example, 1', 2'-dideoxyribose, dSpacer, apurinic / apyrimidinic, tetrahydrofuran or abasic furan can be cleaved by endonucleases VIII. Therefore, in some embodiments, the nucleic acid chain connected to a detectable label or an intermediate chain can include at least one abasic site that can be cleaved by endonucleases VIII. In some embodiments, the nucleic acid chain connected to a detectable label or an intermediate chain can include at least one deoxyuridine and at least one abasic site that can be cleaved by USER, UDG or endonucleases VIII. Photocleavable spacers or RNA abasic sites can also be used, such as ribose spacers (rSpacer) or abasic II modifications. Other non-natural nucleotide pairs and their paired enzymes can be used.

[0281] H. Enzyme inactivation

[0282] An alternative method to use a releasable linker or to remove an enzyme or specific binding pair member bound to a nucleic acid strand is to inactivate the enzyme. Exemplary enzyme inactivators include, but are not limited to, peroxides, reducing agents (such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP)) and reduced glutathione, diisopropylfluorophosphate (DFP), α-difluoromethylornithine, fluoride salts, cyanide salts, azides, and specific binding pair members (such as antibodies and aptamers).

[0283] In some embodiments, the enzyme is inactivated using an enzyme inactivating agent, wherein the enzyme inactivating agent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, peroxide, cyanide, fluoride, or azide.

[0284] In some embodiments, inactivation of the enzyme is performed for less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, or less than or equal to 1 minute.

[0285] In some embodiments, inactivation of the enzyme is performed by contacting the sample with the enzyme inactivating agent for less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, or less than or equal to 1 minute.

[0286] I. Intermediate part / amplification / preamplifier

[0287] In some cases, the nucleic acid strand attached to the target-specific binding partner is bound to the nucleic acid strand attached to the enzyme via an intermediate portion.

[0288] In some embodiments, the middle part is an intermediate chain comprising nucleic acid. In some embodiments, the nucleic acid is a single-stranded nucleic acid, such as single-stranded DNA, RNA or nucleic acid analogs. Nucleic acid analogs (also referred to as non-natural nucleic acids) can include a changed phosphate backbone, a changed pentose and / or a changed core base. Nucleic acid analogs can include but are not limited to 2'-O-methyl ribonucleic acid, 2'-fluoro ribonucleic acid, peptide nucleic acid, morpholino and locked nucleic acid, diol nucleic acid and threose nucleic acid.

[0289] In some embodiments, the intermediate strand has a first region complementary to the nucleic acid strand that is attached to the target-specific binding partner and a second region complementary to the nucleic acid strand that is attached to the enzyme. In such embodiments, it is not necessary that the nucleic acid strand that is attached to the target-specific binding partner is complementary to the nucleic acid strand that is attached to the enzyme.

[0290] In some embodiments, the intermediate strand and the nucleic acid strand attached to the target-specific binding partner are not added in discrete steps. In some cases, the intermediate strand and the nucleic acid strand attached to the target-specific binding partner are hybridized together before being added in a single step.

[0291] In some embodiments, the intermediate strand and the nucleic acid strand connected to the enzyme are not added in discrete steps. In some cases, the intermediate strand and the nucleic acid strand connected to the enzyme are hybridized together before being added in a single step.

[0292] In some embodiments, the intermediate strand is a pre-prepared amplicon comprising repetitive sequences, each sequence being capable of binding to a nucleic acid strand attached to an enzyme. In some embodiments, the intermediate strand is a first region complementary to a corresponding region of a nucleic acid strand attached to a target-specific binding partner and a second region comprising repetitive sequences, each sequence specifically binding directly or indirectly to a corresponding sequence of a nucleic acid strand attached to an enzyme.

[0293] In some embodiments, the methods described herein further comprise amplifying the nucleic acid strand (or a portion thereof) that is directly or indirectly linked to the target-specific binding partner of step (1).

[0294] In some embodiments, the methods described herein further comprise amplifying the intermediate portion bound to the nucleic acid strand attached to the target-specific binding partner of step (1) by a primer exchange reaction (PER) as further described below. In some embodiments, labeled nucleotides are used in the amplification reaction.

[0295] In some embodiments, the method described herein further comprises adding a middle portion comprising a repetitive sequence. In some embodiments, a primer exchange reaction (PER) as described herein is used to produce repetitive sequences. In some embodiments, a rolling circle amplification (RCA) as described herein is used to produce a preamplifier.

[0296] 1. Primer Exchange Reaction (PER)

[0297] PER can be used to prepare a nucleic acid product containing multiple probe binding sites (i.e., the middle portion described herein); this nucleic acid product can then be combined with a nucleic acid (or a barcode domain thereof) attached to a target-specific binding partner; where the nucleic acid product can display the probe binding sites. In such embodiments, the probe binding site may not be complementary to the barcode domain of the target-specific binding partner.

[0298] Various PERs and PER-based signal amplification methods have been described in Saka et al., “Highly multiplexed insitu protein imaging with signal amplification by Immuno-SABER” (2018; available as a preprint at www.biorxiv.org / content / 10.1101 / 507566v1 since June 6, 2019); WO 2017 / 143006; and WO 2018 / 132392A2, the contents of each of which are incorporated herein by reference.

[0299] The PER reaction results in the formation of a concatemer (repeat) sequence. The PER concatemer has a first domain complementary to a nucleic acid strand connected to a target-specific binding partner and a second domain comprising a repetitive sequence. The repetitive sequence may be identical to the nucleic acid strand connected to the target-specific binding partner. The repetitive sequence may be different from the nucleic acid strand connected to the target-specific binding partner.

[0300] IV. Supporting Information on Multiplexed Imaging Methods

[0301] A. Spectral multiplexing and temporal multiplexing

[0302] There are two main methods for creating multiplexing in exchange imaging: spectral multiplexing and temporal multiplexing. Spectral multiplexing refers to the ability to use different labels (such as different fluorophores) in a single round of imaging. Spectral multiplexing does not require extinguishing the signal from the first label before viewing the second label. For example, in the case of fluorophores, different fluorophores can be excited separately using light of different excitation wavelengths. This does not require separate rounds of imaging. Temporal multiplexing refers to the ability to use the same label (such as the same fluorophore) in multiple rounds of imaging by extinguishing the signal from the first round of imaging before the second round of imaging. Spectral multiplexing and temporal multiplexing can be used alone or in combination with each other. However, using more than one multiplexing technique can significantly increase the number of targets that a user can visualize during a particular experiment.

[0303] In some embodiments, multiple rounds of imaging are performed using at least some of the same fluorophores. For example, in the first round of imaging, target A can be imaged using label X, target B can be imaged using label Y, and target C can be imaged using label Z. As a next step, the signals from these labels can be extinguished. Then, in the second round of imaging, target D can be imaged using label X, target E can be imaged using label Y, and target F can be imaged using label Z. Again, in some embodiments, at least two targets are imaged using at least two labels, the signal is extinguished, and then at least one identical label is used to image at least one more target, wherein the imaging steps can be performed in any order. This means that the order of the steps can be reversed, so the first imaging step includes imaging at least one target, extinguishing the signal, and the second imaging step includes imaging at least two targets.

[0304] Combining both spectral and temporal multiplexing can increase the overall ease of imaging for the user and reduce disruption to the sample being imaged.

[0305] B. Control Experiments and Background Subtraction

[0306] Control experiments and background subtraction can be used to further improve the results of methods for testing the presence of one or more targets in a sample. Neither aspect is required for useful experiments; however, both can improve the quality of multiplexing and can be used alone or in combination with each other.

[0307] 1. Control experiment

[0308] Control measurements can be added at multiple time points during the multiplexing process by contacting the sample with a binding pair having a nucleotide sequence that is not complementary to the nucleic acid strand that binds to the target-specific binding partner (or a portion thereof, such as a barcode) by performing a control step.

[0309] 2. Background Subtraction

[0310] In any embodiment discussed throughout the application, the method can use background subtraction. In some embodiments, the method includes imaging the sample to detect and / or measure background signal, and subtracting background signal from the image of the sample to detect the combined label. Such background signal can include autofluorescence and / or residual fluorescence associated with the signal of the incomplete extinguishing of the combined label. In some embodiments, the background signal is measured before the sample is imaged to detect the combined label. In other embodiments, the background signal is measured after the sample is imaged to detect the combined label.

[0311] C. Description of Samples

[0312] 1. Sample type

[0313] These methods can be used to image various types of samples. In some embodiments, the sample is a fixed sample. In some embodiments, the sample is a cell, a cell lysate, a tissue, a tissue lysate, and or a whole organism. In some embodiments, the sample is a cell or tissue sample, a cell or tissue lysate, or a body fluid. In some embodiments, the sample is a tissue, and the imaging includes intra-tissue multiplexing for immunostaining.

[0314] The sample may be provided in a liquid medium or a buffered solution.

[0315] 2. Antigen Retrieval

[0316] In some embodiments, staining a sample with a target-specific binding partner requires specific conditions, and not all target-specific binding partners will bind to their antigen under the same conditions. This may be because their target antigen is not available under the same conditions.

[0317] 3. Target description and use in biomarker identification

[0318] In some embodiments, the method can be used to identify biomarkers. In some cases, samples are imaged and data analysis is performed on those samples. In some embodiments, multiple targets are tested using corresponding target-specific binding partners for each target. In some cases, the relationship between different targets can be assessed; for example, compared with healthy tissue, the user may seek to determine the relationship between multiple markers and disease states, and conclude that the level of A of the disease sample is increased, the level of B is reduced, and the level of C is within a certain range compared with healthy tissue without the biomarker distribution.

[0319] In some embodiments, at least 10, 96, 100, 384, or 500 samples are imaged and data analysis is performed on those samples.

[0320] In some embodiments, at least 5, 10, 15, 25, 30, 50, 75, or 100 or more targets are tested for each target using a corresponding target-specific binding partner.

[0321] D. Equipment and Software

[0322] 1. Imaging chamber, such as a flow cell

[0323] In some embodiments, an imaging chamber can be used. In some cases, the imaging chamber is a fixed chamber with no inlet and no outlet. In some embodiments, the imaging chamber has a single inlet / outlet combination. In other cases, the imaging chamber allows flow and is named a flow cell. The flow cell can include a combination of a first optically transparent support and a second optically transparent material (such as a glass or plastic coverslip) to provide a flow cell with a top and bottom surface and a fluid flow between them. If a first and a second optically transparent material are used, they can be placed parallel to each other. With respect to parallelism, it includes a completely parallel geometric arrangement and a geometric arrangement that deviates from parallelism by up to 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9° or 10°. In some embodiments, the second optically transparent material is very close to the first optically transparent material, such as about 5 microns to 5 mm, 50 microns to 500 microns or 500 microns to 5 mm.

[0324] The imaging chamber may also include a first optically transparent support and a gasket (also referred to as an isolator or spacer). The gasket may be open to air on the top surface, or may be closed and have an optically transparent top surface. The gasket may have a combined inlet / outlet or it may have both an inlet and an outlet. The gasket may also have no outlet. The gasket may be plastic, rubber, adhesive. The gasket may include a CoverWell chamber gasket (Thermo Fisher), an ultra-thin sealed chamber for upright and inverted microscopes (Bioscience Tools), or an incubation chamber (Grace Bio-Labs, including HybriSlip TM Hybrid cover, HybriWell TM Sealing system, CoverWell TM Incubation chamber, Imaging pad, SecureSeal TM Hybridization Chamber, FlexWell TM Incubator, FastWells TM Reagent Barriers and Silicone Isolators TM ).

[0325] In some cases, a gasket may be used with a coverslip that forms the top surface of an imaging chamber or flow cell.

[0326] Imaging chambers, such as but not limited to flow cells, may be reusable or disposable.

[0327] 2. Software for controlling fluidic steps

[0328] In some embodiments, all fluid exchange steps are performed using a fluid system that includes electronic, and / or pneumatic, and / or hydraulic, and / or electrofluidic actuators and systems. In some cases, the fluid system is controlled by software. In some embodiments, wherein the fluid system is automatically controlled by software, the aforementioned steps of the method described in the preceding paragraphs are synchronized with the imaging step.

[0329] In some embodiments, the fluidic system is controlled by software that synchronizes the preceding steps of the method with the imaging steps by communicating with the imaging software, with reference to the step numbers described in the preceding paragraphs.

[0330] In some embodiments, all fluidic steps are performed while the sample is on the imaging device.

[0331] The sample can be mounted in a disposable imaging chamber (such as a flow cell), a reusable imaging chamber (such as a flow cell), a slide, a slide with a coverslip, or any other configuration.

[0332] Example

[0333] Example 1. Deposition of fluorescent reporter molecules by HRP cycling

[0334] Four different targets CD8, CD68, PD-L1 and CK were marked using continuous deposition of the corresponding fluorescent tyramide reagent. In particular, the formalin-fixed paraffin-embedded (FFPE) tissue slides were baked and then loaded onto the BOND RX automatic stainer (Leica Biosystems, Nussloch GmbH) for all subsequent steps. The slides were dewaxed and the antigens were repaired using epitope repair solution 2BOND (Leica Biosystems). Endogenous peroxidase activity was quenched with 3% H2O2 and then blocked with antibody diluent (Ultivue, Cambridge, MA) for 15 minutes at room temperature. A mixture of four different primary antibodies (anti-CD8, anti-CD68, anti-PD-L1 and anti-CK) conjugated to nucleic acid barcodes and diluted in antibody diluent was incubated with tissue samples at room temperature for 30 min. The samples were washed with BOND washing solution (Leica Biosystems) and then incubated with the pre-amplification mixture (Ultivue) at room temperature for 10 min, followed by an additional washing step. The samples were incubated with amplification solution (Ultivue) for 15-20 min and then washed. The first set of nucleic acid probe strands complementary to the anti-CD8 antibody conjugate and linked to the HRP enzyme via a disulfide bond were added to the samples and allowed to hybridize with their partner strands for 15 min. A wash step was used to remove unbound HRP-coupled probes. Alexa Fluor TM488 Tyramide Reagent (Invitrogen TM ) was applied to the tissue sample to label the CD8 target. The HRP enzyme attached to the first set of nucleic acid probe chains was inactivated from the sample by adding a solution of TCEP in Tris buffer for 1 minute. After inactivating the HRP enzyme associated with the first round of CARD, a second set of nucleic acid probe chains complementary to the anti-CD68 antibody conjugate, attached to the HRP enzyme via a disulfide bond, was added to the sample and allowed to hybridize with its partner chain. A washing step was used to remove unbound HRP-coupled probes. Alexa Fluor TM A working solution of 555 tyramide reagent (Invitrogen) was applied to the tissue sample to label the CD68 target. The HRP enzyme linked to the second set of nucleic acid probe chains was inactivated from the sample by adding a solution of TCEP in Tris buffer for 1 minute. After inactivating the HRP enzyme associated with the second round of CARD, a third set of nucleic acid probe chains complementary to the anti-PD-L1 antibody conjugate and linked to the HRP enzyme via a disulfide bond were added to the sample and allowed to hybridize with their partner chains. A washing step was used to remove unbound HRP-coupled probes. AlexaFluor TM A working solution of 647 tyramide reagent (Invitrogen) was applied to the tissue sample to label the PD-L1 target. The HRP enzyme linked to the third set of nucleic acid probe chains was inactivated from the sample by adding a solution of TCEP in Tris buffer for 1 minute. After inactivating the HRP enzyme associated with the third round of CARD, a fourth set of nucleic acid probe chains complementary to the anti-CK antibody conjugate and linked to the HRP enzyme via a disulfide bond were added to the sample and allowed to hybridize with their partner chains. A washing step was used to remove unbound HRP-coupled probes. Alexa Fluor TM A working solution of 750 tyramide reagent (Invitrogen) was applied to tissue samples to label CK targets. A nuclear counterstain was applied to slides, and samples were coverslipped and imaged using a fluorescence microscope.

[0335] Figure 7 The four targets CD8, CD68, PD-L1 and CK were successfully labeled using sequential deposition of the corresponding fluorescent reporter molecules. Box A shows an exemplary location of labeled CD8; box B is labeled CD68; box C is labeled PD-L1; box D is labeled CK.

[0336] Example 2. Inactivation of HRP for cyclic deposition

[0337] In order to achieve cyclic deposition of labels using an HRP-based enzymatic reaction, the HRP enzyme needs to be removed or inactivated between successive deposition rounds. The HRP enzyme can be inactivated by heat- or pH-based denaturation or by cleavage from the sample by an unstable linker ( Figure 4A In this example, a reducing agent was used to inactivate the HRP enzyme in the absence of any unstable linker ( Figure 4B ).

[0338] FFPE tissue slides were baked at 60°C for 30 minutes, then dewaxed and antigens were repaired on a BOND RX automatic stainer (Leica Biosystems) using epitope repair solution 2BOND (Leica Biosystems). The slides were removed from the BOND RX for subsequent steps. Endogenous peroxidase activity was quenched with 3% H2O2 for 20 min, washed in PBS, and then blocked with antibody diluent (Ultivue, Cambridge, MA) for 15 min at room temperature. The primary antibody conjugated to the nucleic acid barcode (from Example 1) was diluted in antibody diluent and incubated with the tissue sample for 1 hour at room temperature. The samples were washed and incubated with the pre-amplification mixture (Ultivue) at room temperature for 25 min, followed by an additional washing step. The samples were incubated with the amplification solution (Ultivue) at 30°C for 15 min, then washed in PBS. A first set of nucleic acid probe strands complementary to one of the antibody-conjugated nucleic acid barcodes, linked to the HRP enzyme (without disulfide bonds), were added to the sample and allowed to hybridize with their partner strands. A wash step was used to remove unbound HRP-coupled probes. Tissue slides were exposed to TCEP solution for 5 minutes to inactivate HRP, or to Tris buffer as a control. AlexaFluor TM A working solution of 647 tyramide (Invitrogen) was added to all slides, incubated at room temperature for 10 min, and washed in PBS. Finally, the slides were stained with a nuclear counterstain, washed, and coverslipped. The slides were imaged on a fluorescence microscope.

[0339] Figure 8A-8B It was shown that samples treated with TCEP solution prior to depositing the tyramide reagent did not result in tyramide deposition, whereas the tyramide reagent was successfully deposited in control samples not exposed to TCEP. This surprising result indicates that the TCEP solution inactivates the HRP enzyme.

[0340] Example 3. Deposition of binding pairs by HRP cycling

[0341] FFPE tissue slides are baked and then loaded onto a BOND RX automatic stainer (Leica Biosystems) for all subsequent steps. The slides are dewaxed and antigens are repaired using epitope repair solution 2BOND (Leica Biosystems). Endogenous peroxidase activity is quenched using 3% H2O2, and then blocked with antibody diluent (Ultivue, Cambridge, MA) for 15 minutes at room temperature. A mixture of four different primary antibodies (anti-CD8, anti-CD68, anti-PD-L1, and anti-CK) conjugated to nucleic acid barcodes and diluted in antibody diluent is incubated with tissue samples at room temperature for 30 min. The samples are washed and then incubated with a pre-amplification mixture (Ultivue) at room temperature for 10 min, followed by an additional washing step. The samples are incubated with an amplification solution (Ultivue) for 15-25 min, then washed. The first group of nucleic acid probe chains complementary to the anti-CD8 antibody conjugate and connected to the HRP enzyme by a disulfide bond are added to the sample, and allowed to hybridize with its partner chain for 15 min. A wash step was used to remove unbound HRP-coupled probe. The nucleic acid barcode ( Figure 6 ) is applied to the tissue sample to deposit additional nucleic acid barcodes at the sites of the CD8 target (e.g., depicted in Figure 5). The HRP enzyme connected to the first group of nucleic acid probe chains is inactivated from the sample by adding a TCEP solution in a Tris buffer for 1 minute. After inactivating the HRP enzyme associated with the first round of CARD, a second group of nucleic acid probe chains complementary to the anti-CD68 antibody conjugate and connected to the HRP enzyme by a disulfide bond are added to the sample and allowed to hybridize with its partner chain. Unbound HRP-coupled probes are removed using a washing step. A working solution of a nucleic acid barcode connected to a phenol HRP-reactive substrate is applied to a tissue sample to deposit additional nucleic acid barcodes at the site of the CD68 target. The HRP enzyme connected to the second group of nucleic acid probe chains is inactivated from the sample by adding a TCEP solution in a Tris buffer for 1 minute. After inactivating the HRP enzyme associated with the second round of CARD, a third group of nucleic acid probe chains complementary to the anti-PD-L1 antibody conjugate and connected to the HRP enzyme by a disulfide bond are added to the sample and allowed to hybridize with its partner chain. Unbound HRP-coupled probes are removed using a washing step. A working solution of nucleic acid barcodes connected to a phenol HRP reactive substrate is applied to a tissue sample to deposit additional nucleic acid barcodes at the site of the PD-L1 target. The HRP enzyme connected to the third set of nucleic acid probe chains is inactivated from the sample by adding a TCEP solution in Tris buffer for 1 minute. After inactivating the HRP enzyme associated with the third round of CARD, a fourth set of nucleic acid probe chains complementary to the anti-CK antibody conjugate and connected to the HRP enzyme by a disulfide bond are added to the sample and allowed to hybridize with its partner chain. A washing step is used to remove unbound HRP-coupled probes. A working solution of nucleic acid barcodes connected to a phenol HRP reactive substrate is applied to a tissue sample to deposit additional nucleic acid barcodes at the site of the CK target. The sample is washed and a mixture of fluorescently labeled probe chains complementary to the nucleic acid barcodes is allowed to hybridize with the sample. Unbound fluorescently labeled probe chains are removed and a nuclear counterstain is applied to the slide. The sample is covered with a coverslip and imaged using a fluorescence microscope.

[0342] Fig. 9 The four targets CD8, CD68, PD-L1 and CK were successfully labeled using sequential deposition of the corresponding fluorescent reporter molecules. Box A shows an exemplary location of labeled CD8; box B is labeled CD68; box C is labeled PD-L1; box D is labeled CK.

[0343] Example 4. Additional embodiments

[0344] The following numbered clauses provide additional support and description for implementations of this document.

[0345] Clause 1. A method for testing a sample for the presence of one or more targets, comprising:

[0346] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0347] (2) optionally removing unbound target-specific binding partner;

[0348] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0349] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0350] (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker;

[0351] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0352] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked using a third releasable linker;

[0353] (6) optionally removing unbound substrate conjugates;

[0354] (7) optionally releasing the bound enzyme;

[0355] (8) imaging the sample to detect the bound detectable label; and

[0356] (9) Optionally repeat steps (1)-(8) or any subset thereof.

[0357] Item 2. The method of Item 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 of the activated substrate conjugate.

[0358] Clause 3. The method of Clause 2, wherein the receptor of the activated substrate conjugate is immobilized on a solid support, thereby resulting in the deposition of the detectable label.

[0359] Item 4. The method of Item 1, further comprising (10) optionally releasing the bound detectable label by releasing the third releasable linker after step (8), and optionally repeating steps (1)-(10) or any subset thereof.

[0360] Clause 5. A method for testing a sample for the presence of one or more targets, comprising:

[0361] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0362] (2) optionally removing unbound target-specific binding partner;

[0363] (3) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0364] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0365] (ii) the first member of the binding pair is linked to the complementary nucleic acid strand using a second releasable linker;

[0366] (4) optionally removing unbound first members of the binding pair that are attached to the complementary nucleic acid strand;

[0367] (5) contacting the sample from step (3) or optionally step (4) with a second member of the binding pair optionally linked to an enzyme using a third releasable linker, and optionally removing unbound second member of the binding pair linked to the enzyme;

[0368] (6) contacting the sample from step (5) with a substrate conjugate comprising a labeled substrate, wherein the substrate and the label are optionally linked using a fourth releasable linker;

[0369] (7) optionally removing unbound substrate conjugates;

[0370] (8) optionally releasing the bound first member or second member of the binding pair;

[0371] (9) imaging the sample to detect the bound detectable label; and

[0372] (10) Optionally repeat steps (1)-(9) or any subset thereof.

[0373] Item 6. A method as described in Item 5, which also includes (11) optionally releasing the bound detectable label by cleaving the third releasable linker between the label and the substrate after step (9), and optionally repeating steps (1)-(11) or any subset thereof.

[0374] Clause 7. A method for testing a sample for the presence of one or more targets, comprising:

[0375] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0376] (2) optionally removing unbound target-specific binding partner;

[0377] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied:

[0378] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0379] (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker;

[0380] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0381] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a nucleic acid strand bound to a substrate, wherein the substrate and the nucleic acid strand are optionally linked using a third releasable linker;

[0382] (6) optionally removing unbound substrate conjugates;

[0383] (7) optionally releasing the bound enzyme;

[0384] (8) contacting the sample from step (5) or optionally steps (6)-(7) with a detectable label bound to a nucleic acid strand, the detectable label optionally being attached using a fourth releasable linker, wherein the nucleic acid strand is a specific binding pair member of the nucleic acid strand in step (5);

[0385] (9) optionally imaging the sample to detect the bound label;

[0386] (10) optionally releasing the bound label from step (8) by releasing the releasable linker of step (5) or optionally step (8) or optionally steps (5) and (8); and

[0387] (11) Optionally repeat steps (1)-(10) or any subset thereof.

[0388] Clause 8. A method for testing a sample for the presence of one or more targets, comprising:

[0389] (1) contacting a sample being tested for the presence of one or more targets with a target-specific binding partner, wherein the target-specific binding partner is attached to a nucleic acid strand,

[0390] (2) optionally removing unbound target-specific binding partner,

[0391] (3) contacting the sample with a nucleic acid strand conjugated to horseradish peroxidase (HRP), the nucleic acid strand being complementary to the nucleic acid strand linked to the target-specific binding partner,

[0392] Where (i) or (ii) or both of the following are satisfied:

[0393] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0394] (ii) the HRP is linked to the complementary nucleic acid strand using a second releasable linker;

[0395] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0396] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled phenolic substrate, wherein the substrate and the detectable label are optionally linked using a third releasable linker;

[0397] (6) optionally removing unbound substrate;

[0398] (7) imaging the sample to detect the bound detectable label;

[0399] (8) optionally releasing the bound detectable label; and

[0400] (9) Optionally repeat steps (1)-(8) or any subset thereof.

[0401] Item 9. The method of Item 8, wherein after step (5), the phenolic moiety is enzymatically converted to an activated state, thereby resulting in deposition of the label.

[0402] Clause 10. The method of any one of Clauses 1-9, wherein the releasable linker comprises at least one of a disulfide bond, an ester, a vicinal diol, a sulfone, and a photocleavable linker.

[0403] Clause 11. The method of any one of Clauses 1-10, wherein the enzyme is selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases, and ligases.

[0404] Clause 12. The method of any one of Clauses 1-11, wherein the enzyme is selected from the group consisting of peroxidase, oxidase, phosphatase, esterase and glycosidase.

[0405] Item 13. The method of Item 12, wherein the enzyme is selected from horseradish peroxidase, glucose oxidase, alkaline phosphatase and β-galactosidase.

[0406] Item 14. The method of Item 13, wherein the enzyme is horseradish peroxidase.

[0407] Clause 15. The method of any one of clauses 1 to 14, wherein the binding pair is of an immune type or a non-immune type.

[0408] Clause 16. The method of Clause 15, wherein the immunotypic binding pair is selected from antigen-antibody and hapten-antihapten.

[0409] Clause 17. The method of Clause 16, wherein the antibody member of the binding pair is a polyclonal antibody, a monoclonal antibody, or an immunoreactive fragment thereof.

[0410] Clause 18. The method of Clause 15, wherein the non-immune type binding pair is selected from the group consisting of biotin-avidin, biotin-streptavidin, and complementary probe nucleic acid folate-folate binding protein.

[0411] Item 19. A method as described in Item 18, wherein the non-immune type binding pair forms a covalent bond through a sulfhydryl reactive group (e.g., maleimide and haloacetyl derivatives), an amine reactive group (e.g., isothiocyanate, succinimide ester, sulfonyl halide, click chemistry), and a coupler dye (e.g., 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethyl-amino)benzoic acid (DMAB)).

[0412] Clause 20. The method of any one of Clauses 1-19, wherein the detectable label is selected from an enzyme, a radioisotope, a fluorescent material, a chemiluminescent material, and an electrochemical material.

[0413] Clause 21. The method of any one of Clauses 1-20, wherein the enzyme is horseradish peroxidase (HRP) and the substrate is a phenolic substrate.

[0414] Clause 22. The method of any one of Clauses 1-21, wherein the enzyme is a hydrolase and the substrate is an ester, amide or glycoside substrate.

[0415] Clause 23. A composition comprising:

[0416] (1) a sample bound to more than one target-specific binding partner, each binding partner being bound to a nucleic acid strand,

[0417] (2) an enzyme that produces a nucleic acid strand complementary to the nucleic acid strand bound to the target-specific binding partner,

[0418] Where (i) or (ii) or both of the following are satisfied:

[0419] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0420] (ii) the enzyme is linked to the complementary nucleic acid strand using a second releasable linker; and

[0421] (3) A substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally linked using a third releasable linker.

[0422] Clause 24. A composition comprising:

[0423] (1) a sample bound to more than one target-specific binding partner, each binding partner being bound to a nucleic acid strand,

[0424] (2) A binding pair comprising:

[0425] (i) a first member that is attached to a nucleic acid strand that is complementary to the nucleic acid strand that is bound to the target-specific binding partner, and

[0426] (ii) a second member attached to the enzyme,

[0427] Where (i) or (ii) or both of the following are satisfied:

[0428] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0429] (ii) the first member of the binding pair is linked to the complementary nucleic acid strand using a second releasable linker; and

[0430] (3) a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally linked using a third releasable linker,

[0431] wherein the second member of the binding pair is attached to the enzyme using a fourth releasable linker.

[0432] Clause 25. A composition comprising:

[0433] (1) a sample bound to more than one target-specific binding partner, each binding partner being bound to a nucleic acid strand,

[0434] (2) an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand bound to the target-specific binding partner,

[0435] Where (i) or (ii) or both of the following are satisfied:

[0436] (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and

[0437] (ii) the enzyme is linked to the complementary nucleic acid strand using a second releasable linker; and

[0438] (3) a substrate conjugate comprising a substrate bound to a nucleic acid strand, wherein the substrate and the nucleic acid strand are optionally linked using a third releasable linker;

[0439] (4) a detectable label associated with a nucleic acid strand, optionally linked using a fourth releasable linker, wherein the nucleic acid strand of (4) is a specific binding pair member of the nucleic acid strand of (3).

[0440] Clause 26. A method for testing a sample for the presence of one or more targets, comprising:

[0441] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0442] (2) optionally removing unbound target-specific binding partner;

[0443] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner;

[0444] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0445] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked using a releasable linker;

[0446] (6) optionally removing unbound substrate conjugates;

[0447] (7) optionally inactivating the bound enzyme;

[0448] (8) optionally imaging the sample to detect the bound detectable label; and

[0449] (9) Optionally repeat steps (1)-(8) or any subset thereof.

[0450] Clause 27. A method for testing a sample for the presence of one or more targets, comprising:

[0451] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0452] (2) optionally removing unbound target-specific binding partner;

[0453] (3) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner;

[0454] (4) optionally removing unbound first members of the binding pair that are attached to the complementary nucleic acid strand;

[0455] (5) contacting the sample from step (3) or optionally step (4) with the second member of the binding pair linked to an enzyme, and optionally removing unbound second member of the binding pair linked to the enzyme;

[0456] (6) contacting the sample from step (5) with a substrate conjugate comprising a labeled substrate, wherein the substrate and the label are optionally linked using a releasable linker;

[0457] (7) optionally removing unbound substrate conjugates;

[0458] (8) optionally inactivating the bound enzyme;

[0459] (9) optionally imaging the sample to detect the bound detectable label; and

[0460] (10) Optionally repeat steps (1)-(9) or any subset thereof.

[0461] Item 28. A method as described in Item 27, which also includes (11) optionally releasing the bound detectable label by cleaving the releasable linker between the label and the substrate after step (9), and optionally repeating any one of steps (1)-(9) and (11).

[0462] Clause 29. A method for testing a sample for the presence of one or more targets, comprising:

[0463] (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands;

[0464] (2) optionally removing unbound target-specific binding partner;

[0465] (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner

[0466] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0467] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a nucleic acid strand bound to a substrate, wherein the substrate and the nucleic acid strand are optionally linked using a first releasable linker;

[0468] (6) optionally removing unbound substrate conjugates;

[0469] (7) optionally inactivating the bound enzyme;

[0470] (8) contacting the sample from step (5) or optionally steps (6)-(7) with a detectable label bound to a nucleic acid strand, the detectable label optionally being attached using a second releasable linker, wherein the nucleic acid strand is a specific binding pair member of the nucleic acid strand in step (5);

[0471] (9) optionally imaging the sample to detect the bound label;

[0472] (10) optionally releasing the bound label from step (8) by releasing the linker of step (5) or optionally step (8) or optionally steps (5) and (8); and

[0473] (11) Optionally repeat steps (1)-(10) or any subset thereof.

[0474] Clause 30. A method for testing a sample for the presence of one or more targets, comprising:

[0475] (1) contacting a sample being tested for the presence of one or more targets with a target-specific binding partner, wherein the target-specific binding partner is attached to a nucleic acid strand;

[0476] (2) optionally removing unbound target-specific binding partner,

[0477] (3) contacting the sample with an HRP-bound nucleic acid strand that is complementary to the nucleic acid strand linked to the target-specific binding partner;

[0478] (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand;

[0479] (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled phenolic substrate, wherein the substrate and the detectable label are optionally linked using a releasable linker;

[0480] (6) optionally removing unbound substrate;

[0481] (7) optionally imaging the sample to detect the bound detectable label;

[0482] (8) optionally inactivating the HRP; and

[0483] (9) Optionally repeat a subset of steps (1)-(8).

[0484] Clause 31. The method of any one of Clauses 26-30, wherein the enzyme is inactivated using dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, peroxide, cyanide, fluoride or azide.

[0485] Equivalent

[0486] The foregoing written description is considered sufficient to enable those skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and describe the best mode contemplated by the inventors. However, it should be understood that no matter how detailed the foregoing appears in the text, the embodiments can be practiced in many ways and should be interpreted according to the appended claims and any equivalents thereof.

[0487] As used herein, whether or not explicitly stated, the term approximately refers to a numerical value, including, for example, integers, fractions, and percentages. The term approximately generally refers to a numerical range (e.g., + / - 5-10% of the listed range) that one of ordinary skill in the art considers to be equivalent to the listed value (e.g., having the same function or result). When a term (such as at least and approximately) precedes a list of numerical values ​​or ranges, the term modifies all values ​​or ranges provided in the list. In some cases, the term approximately can include a numerical value rounded to the nearest significant figure.

Claims

1. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands; (2) contacting the sample with (a) an enzyme linked to a nucleic acid strand and (b) an intermediate portion, the intermediate portion comprising a first domain capable of specifically binding to the nucleic acid strand linked to the target-specific binding partner; and a second domain capable of specifically binding to the nucleic acid strand linked to the enzyme, in one or two steps; (3) contacting the sample from step (2) with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked using a releasable linker; (4) optionally inactivating the bound enzyme; (5) optionally detecting a signal from the bound detectable label; as well as (6) Optionally repeating steps (1)-(5) or a subset thereof, and optionally repeating steps (3)-(4) or any subset thereof prior to the imaging step (5).

2. The method of claim 1, wherein the second region of the middle portion comprises repeated sequences, each of which specifically binds directly or indirectly to a corresponding sequence of the nucleic acid strand attached to the enzyme.

3. The method of claim 1, wherein the method further comprises amplifying the intermediate portion bound to the nucleic acid chain connected to the target-specific binding partner of step (1) by a nucleic acid amplification reaction to form an amplicon of a repeated sequence, each of which specifically binds directly or indirectly to a corresponding sequence of the nucleic acid chain connected to the enzyme.

4. The method of claim 3, wherein the nucleic acid amplification reaction comprises a hairpin-based tandem polymerization reaction or a hairpin-based dendron polymerization 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, comprising: (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partner; (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner; (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked using a releasable linker; (6) optionally removing unbound substrate conjugates; (7) optionally inactivating the bound enzyme; (8) optionally imaging the sample to detect the bound detectable label; and (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to the imaging step (8).

6. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partner; (3) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner; (4) optionally removing unbound first members of the binding pair that are attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with the second member of the binding pair linked to an enzyme, and optionally removing unbound second member of the binding pair linked to the enzyme; (6) contacting the sample from step (5) with a substrate conjugate comprising a labeled substrate, wherein the substrate and the label are optionally linked using a releasable linker; (7) optionally removing unbound substrate conjugates; (8) optionally inactivating the bound enzyme; (9) optionally imaging the sample to detect the bound detectable label; and (10) Optionally repeating steps (1)-(9) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof prior to the imaging step (9).

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

8. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partner; (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a nucleic acid strand bound to a substrate, wherein the substrate and the nucleic acid strand are optionally linked using a first releasable linker; (6) optionally removing unbound substrate conjugates; (7) optionally inactivating the bound enzyme; (8) contacting the sample from step (5) or optionally steps (6)-(7) with a detectable label bound to a nucleic acid strand, the detectable label optionally being attached using a second releasable linker, wherein the nucleic acid strand is a specific binding pair member of the nucleic acid strand in step (5); (9) optionally imaging the sample to detect the bound label; (10) optionally releasing the bound label from step (8) by releasing the linker of step (5) or optionally step (8) or optionally steps (5) and (8); and (11) Optionally repeating steps (1)-(10) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof prior to the imaging step (9).

9. A method for testing a sample for the presence of one or more targets, the method comprising: (1) contacting a sample being tested for the presence of one or more targets with a target-specific binding partner, wherein the target-specific binding partner is attached to a nucleic acid strand; (2) optionally removing unbound target-specific binding partner; (3) contacting the sample with a nucleic acid strand conjugated to horseradish peroxidase (HRP), the nucleic acid strand being complementary to the nucleic acid strand linked to the target-specific binding partner; (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled phenolic substrate, wherein the substrate and the detectable label are optionally linked using a releasable linker; (6) optionally removing unbound substrate; (7) optionally inactivating the HRP; (8) optionally imaging the sample to detect the bound detectable label; as well as (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or a subset thereof before step (8).

10. The method of claim 1, wherein the enzyme is inactivated using an enzyme inactivating agent, wherein the enzyme inactivating agent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), reduced glutathione, peroxide, cyanide, fluoride or azide.

11. The method of claim 1, wherein the step (4) of inactivating the bound enzyme is performed for less than 20 minutes.

12. The method of claim 1, wherein step (4) of inactivating the bound enzyme is performed for less than 10 minutes.

13. The method of claim 1, wherein the step (4) of inactivating the bound enzyme is performed for less than 2 minutes.

14. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partner; (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied: (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker; (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the detectable label are optionally linked using a third releasable linker; (6) optionally removing unbound substrate conjugates; (7) optionally releasing the bound enzyme; (8) optionally imaging the sample to detect the bound detectable label; and (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to the imaging step (8).

15. The method of 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 of the activated substrate conjugate.

16. The method of claim 15, wherein the receptor for the activated substrate conjugate is present in the sample, thereby resulting in deposition of the detectable label.

17. The method of claim 5, further comprising (10) optionally releasing the bound detectable label by releasing the third releasable linker after step (8), and optionally repeating steps (1)-(10) or any subset thereof.

18. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partner; (3) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied: (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and (ii) the first member of the binding pair is linked to the complementary nucleic acid strand using a second releasable linker; (4) optionally removing unbound first members of the binding pair that are attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with a second member of the binding pair optionally linked to an enzyme using a third releasable linker, and optionally removing unbound second member of the binding pair linked to the enzyme; (6) contacting the sample from step (5) with a substrate conjugate comprising a labeled substrate, wherein the substrate and the label are optionally linked using a fourth releasable linker; (7) optionally removing unbound substrate conjugates; (8) optionally releasing the bound first member or second member of the binding pair; (9) optionally imaging the sample to detect the bound detectable label; and (10) Optionally repeating steps (1)-(9) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof prior to the imaging step (9).

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

20. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is attached to a nucleic acid strand, and wherein target-specific binding partners with different specificities are attached to different nucleic acid strands; (2) optionally removing unbound target-specific binding partner; (3) contacting the sample from step (1) or optionally step (2) with an enzyme attached to a nucleic acid strand that is complementary to the nucleic acid strand attached to the target-specific binding partner, wherein the following (i) or (ii) or both are satisfied: (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and (ii) the enzyme is attached to the complementary nucleic acid strand using a second releasable linker; (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a nucleic acid strand bound to a substrate, wherein the substrate and the nucleic acid strand are optionally linked using a third releasable linker; (6) optionally removing unbound substrate conjugates; (7) optionally releasing the bound enzyme; (8) contacting the sample from step (5) or optionally steps (6)-(7) with a detectable label bound to a nucleic acid strand, the detectable label optionally being attached using a fourth releasable linker, wherein the nucleic acid strand is a specific binding pair member of the nucleic acid strand in step (5); (9) optionally imaging the sample to detect the bound label; (10) optionally releasing the bound label from step (8) by releasing the releasable linker of step (5) or optionally step (8) or optionally steps (5) and (8); and (11) Optionally repeating steps (1)-(10) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(8) or any subset thereof before step (9).

21. A method for testing a sample for the presence of one or more targets, comprising: (1) contacting a sample being tested for the presence of one or more targets with a target-specific binding partner, wherein the target-specific binding partner is attached to a nucleic acid strand, (2) optionally removing unbound target-specific binding partner, (3) contacting the sample with a nucleic acid strand conjugated to horseradish peroxidase (HRP), the nucleic acid strand being complementary to the nucleic acid strand linked to the target-specific binding partner, Where (i) or (ii) or both of the following are satisfied: (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and (ii) the HRP is linked to the complementary nucleic acid strand using a second releasable linker; (4) optionally removing unbound enzyme attached to the complementary nucleic acid strand; (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprising a detectably labeled phenolic substrate, wherein the substrate and the detectable label are optionally linked using a third releasable linker; (6) optionally removing unbound substrate; (7) optionally imaging the sample to detect the bound detectable label; (8) optionally releasing the bound detectable label; and (9) Optionally repeating steps (1)-(8) or any subset thereof, and optionally if more than one target-specific binding partner is used, repeating steps (1)-(6) or any subset thereof prior to the imaging step (7).

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

23. The method of claim 1, wherein the bound label is releasable with or without a releasable linker.

24. The method of claim 23, wherein the bound label is released by cleaving the releasable linker between the label and the substrate or by cleaving the releasable linker between the substrate and the nucleic acid.

25. The method of claim 8, wherein the bound label is released by dehybridizing the nucleic acid strand from step (8) that is bound to the nucleic acid strand from step (5).

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

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

28. The method of claim 8, wherein the method further comprises: First, the nucleic acid chain or a portion thereof attached to the target-specific binding partner of step (1) is amplified, optionally wherein the amplification step is performed before step (3); and secondly, the nucleic acid chain or a portion thereof of the substrate to which the nucleic acid chain of step (5) is bound is amplified, optionally wherein the second amplification step is performed after step (5) or optional step (6) or optional step (7).

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

30. The method of claim 1, wherein the enzyme is selected from the group consisting of an oxidoreductase, a hydrolase, a lyase, a transferase, an isomerase, and a ligase.

31. The method of claim 1, wherein the enzyme is selected from the group consisting of peroxidase, oxidase, phosphatase, esterase and glycosidase.

32. The method of claim 31, wherein the enzyme is selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and beta-galactosidase.

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

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

35. The method of claim 34, wherein the immunotypic binding pair is selected from the group consisting of antigen-antibody and hapten-antihapten.

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

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

38. The method of claim 33, wherein the non-immune type binding pair forms a covalent bond through a sulfhydryl reactive group, an amine reactive group, and a coupler dye optionally selected from the group consisting of maleimide and haloacetyl derivatives, isothiocyanates, succinimidyl esters, sulfonyl halides, click chemistry, 3-methyl-2-benzothiazolinone hydrazone (MBTH), and 3-(dimethyl-amino)benzoic acid (DMAB).

39. The method of claim 1, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorescent material, a chemiluminescent material, and an electrochemical material.

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

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

42. A composition comprising: (1) a sample bound to more than one target-specific binding partner, each binding partner being bound to a nucleic acid strand, (2) an enzyme that produces a nucleic acid strand complementary to the nucleic acid strand bound to the target-specific binding partner, Where (i) or (ii) or both of the following are satisfied: (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and (ii) the enzyme is linked to the complementary nucleic acid strand using a second releasable linker; and (3) A substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally linked using a third releasable linker.

43. A composition comprising: (1) a sample bound to more than one target-specific binding partner, each binding partner being bound to a nucleic acid strand, (2) A binding pair comprising: (i) a first member that is attached to a nucleic acid strand that is complementary to the nucleic acid strand that is bound to the target-specific binding partner, and (ii) a second member attached to the enzyme, Where (i) or (ii) or both of the following are satisfied: (i) the nucleic acid strand is linked to the target-specific binding partner using a first releasable linker; and (ii) the first member of the binding pair is linked to the complementary nucleic acid strand using a second releasable linker; and (3) a substrate conjugate comprising a detectably labeled substrate, wherein the substrate and the label are optionally linked using a third releasable linker, wherein the second member of the binding pair is attached to the enzyme using a fourth releasable linker.

44. A method of depositing a detectable label on a sample, comprising: The detectable label is reacted with an enzyme to form an activated substrate conjugate, and the activated substrate conjugate is allowed to bind to a receptor for the activated substrate conjugate present in the sample, thereby resulting in deposition of the detectable label on the sample.

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