Ultra-sensitive molecular detection by hybridization chain reaction

Abstract

This application relates to hybridization chain reactions (HCRs). In particular, compositions and methods for ultra-sensitive molecular detection using HCR signal amplification are presented. Several embodiments and methods involve coordinated probe junctions, reporter-labeled probes, and nonlinear HCR signal amplification.

Description

[Technical Field]

[0001] References to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 457,043, filed on April 4, 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0002] Statement on federally supported research and development This invention relates to the National Institutes of Health (NHI). This invention was supported by government funding from the National Institutes of Health (NIH) under grant numbers R01EB006192 and R44GM140796. The U.S. government has certain rights to this invention.

[0003] Reference to the sequence listing file. This application incorporates the XML array list document presented herein, titled CALTE.163WOseqlist.xml, created on April 1, 2024, and having a size of 4,755 bytes. [Background technology]

[0004] This application relates to hybridization chain reactions (HCRs). In particular, compositions and methods for ultra-high-sensitivity molecular detection using HCR signal amplification are provided. [Overview of the project]

[0005] Compositions and methods for ultra-high sensitivity molecular detection using HCR signal amplification are provided. Several embodiments and methods include one, two, or three of the following embodiments: 1) a coordinated probe junction, 2) a reporter-labeled probe, and 3) nonlinear HCR signal amplification. In some embodiments relating to coordinated probe junctions, a target is detected using a probe set comprising one or more probe units, each containing two or more fragmentary initiator probes, wherein the fragmentary initiator probes include a target binding region, a linker region, and a fragmentary initiator, the target binding region on the probe within the probe unit is configured to bind to overlapping or non-overlapping binding sites on the target, the linker regions on the probes within the probe unit are configured to bind to each other as needed, the fragmentary initiator on the probe within each probe unit is configured to bind to overlapping or non-overlapping binding sites on the HCR hairpin, and when the probes within the probe unit bind to the cognitive binding sites on the target and the cognitive binding sites on the HCR hairpin, a coordinated probe junction is formed, mutually promoting target binding and co-localization of the complete HCR initiator, thereby efficiently triggering HCR signal amplification. In some embodiments relating to reporter-labeled probes, the target is detected by an anti-target reporter-labeled probe (e.g., an anti-target hapten-labeled primary antibody), which is then detected by an anti-reporter initiator-labeled probe (e.g., an anti-hapten initiator-labeled primary antibody) or one or more fragmentary initiator probes (e.g., one or more anti-hapten fragmentary initiator primary antibodies), eliminating the need to conjugate the initiator to the target-binding probe and the need to use a secondary antibody. In some situations, conjugating the initiator to the target-binding probe may interfere with probe / target binding, and in some situations, the use of a secondary antibody may interfere with multiplexing. In some embodiments relating to nonlinear HCR signal amplification, ultra-high sensitivity detection is achieved by using multiple rounds of HCR signal amplification to grow a branched HCR amplification polymer.In some embodiments, during the linear amplification phase, an anti-target initiator-labeled signal probe triggers a reporter-labeled HCR hairpin, which is linked to the target and contains reporter-modified first H. A CR-amplified polymer is grown. In some embodiments, an anti-reporter initiator-labeled bridging probe binds to a reporter that modifies a first HCR-amplified polymer, thereby modifying the first HCR-amplified polymer with the HCR initiator. In some embodiments, during the nonlinear amplification stage, each HCR initiator modifying the first HCR-amplified polymer triggers an auxiliary reporter-labeled HCR hairpin, causing the growth of an auxiliary reporter-modified HCR-amplified polymer branching off from the first HCR-amplified polymer bonded to the target. In some embodiments, multiple rounds of bridging and nonlinear HCR signal amplification occur, growing multiple generations of HCR-amplified polymers bonded to each other and to the target. In some embodiments, the reporter and / or auxiliary reporter on the HCR-amplified polymer directly or indirectly mediate catalytic reporter deposition (CARD) near the target.

[0006] According to several embodiments, a method for HCR including a coordinated probe junction is provided. In some embodiments, the method includes: a) providing a sample which may contain a target; b) contacting the sample with a probe set which includes one or more probe units, each containing two or more fragmentary initiator probes; c) contacting the sample with an HCR amplifier labeled with a reporter; and d) detecting a signal directly or indirectly from the reporter. According to some embodiments, each of the two or more fragmentary initiator probes includes a target binding region, a linker region, and a fragmentary initiator. According to some embodiments, the target binding region on the probe in each probe unit is configured to bind to different binding sites on the target. According to some embodiments, the linker regions on the two or more fragmentary initiator probes are configured to bind to each other. According to some embodiments, the fragmentary initiators on the two or more fragmentary initiator probes are configured to bind to different binding sites on an HCR hairpin. In some embodiments, the probes within each probe unit are configured to form a cooperative probe junction when they bind to the target and to the HCR hairpins. In some embodiments, one HCR amplifier includes two or more HCR hairpins. In some embodiments, one HCR hairpin includes an input domain comprising a single-stranded toehold and a stem section, and an output domain comprising a single-stranded loop and a complement of the stem section. In some embodiments, at least one HCR hairpin further includes a reporter. In some embodiments, a washing step is performed between any of steps (b) to (d). In some embodiments, each of the two or more fragmentary initiator probes further includes a proximity domain. In some embodiments, the proximity domain is configured to bind to the proximity probe. In some embodiments, the sample is brought into contact with one or more proximity probes after step (b) and before step (c). In some embodiments, after providing one or more proximity probes, a washing step is performed to remove any unbound proximity probes.In some embodiments, the coordinating probe junction further includes the one or more adjacent probes. In some embodiments, one or more auxiliary reporter-labeled readout probes are provided after step (c). In some embodiments, a washing step is performed after providing the one or more auxiliary reporter-labeled readout probes to remove any unbound auxiliary reporter-labeled readout probes. In some embodiments, a signal is detected from the auxiliary reporter. In some embodiments, the method further includes step (e) after step (d) in which the signal is removed. In some embodiments, the method further includes repeating any of the steps of the method to detect another target in the sample, wherein the target-binding domain is for a different target. In some embodiments, the target-binding regions on the two or more fragmentary initiator probes are configured to bind to overlapping binding sites on the target. In some embodiments, the target-binding regions on the two or more fragmentary initiator probes are configured to bind to non-overlapping binding sites on the target. According to some implementations, the fragment initiators on the two or more fragment initiator probes bind to the overlapping binding sites on the HCR hairpin. The device is configured such that the fragment initiators on the two or more fragment initiator probes bind to non-overlapping binding sites on the HCR hairpin. In some embodiments, the target includes RNA molecules, DNA molecules, proteins, small molecules, chemicals, biological molecules, pathogens, molecular complexes, or sets of adjacent molecules. In some embodiments, the reporter and the auxiliary reporter may be the same or different, and each may contain a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, magnetic carbon nanotube, gold nanoparticle (AuNP), gold nanoshell, gold nanorod, silver-shelled gold nanoparticle, latex, magnetic nanoparticle, silica nanoparticle, fluorophore-supported nanoparticle, dye-supported nanoparticle, hapten, ligand, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzyme, combination thereof, or a molecule that directly or indirectly mediates signal generation.

[0007] In some embodiments, a composition is provided for detecting a target in a sample, the composition comprising: a) an anti-target reporter-labeled signal probe configured to bind to the target; b) an anti-reporter initiator-labeled signal probe configured to bind to a reporter; and c) an auxiliary reporter-labeled HCR amplifier configured to be triggered by the initiator to grow an auxiliary reporter-modified HCR amplification polymer linked to the target, the auxiliary reporter directly or indirectly mediating the generation of a signal. In some embodiments, the composition further comprises an anti-auxiliary reporter tertiary reporter-labeled readout probe configured to bind to the auxiliary reporter-modified amplification polymer, the tertiary reporter directly or indirectly mediating the generation of a signal. In some embodiments, the target comprises an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules. According to some implementations, the reporter, the auxiliary reporter, and the tertiary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon This includes atoms, chemical linkers, magnetic molecules, carbon black (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

[0008] According to several implementations, a composition is provided for detecting N targets (N is a positive integer) in a sample, the composition comprising: a) a j-th reporter and a j-th anti-target signal probe (j=1, ..., N, j is a positive integer) configured to bind to the j-th target; b) a j-th HCR initiator and a j-th anti-reporter signal probe configured to bind to the j-th reporter; and c) a j-th auxiliary reporter and a j-th HCR amplifier configured to grow a j-th auxiliary reporter-modified HCR amplification polymer triggered by the j-th HCR initiator and linked to the j-th target, wherein the j-th auxiliary reporter is the j-th It directly or indirectly mediates the generation of a signal to a target of the eye. In some embodiments, the composition further comprises a j-th anti-auxiliary reporter readout probe configured to bind to the j-th auxiliary reporter modified amplification polymer, comprising a j-th tertiary reporter. In some embodiments, the j-th tertiary reporter directly or indirectly mediates the generation of a signal to the j-th target. In some embodiments, the j-th target comprises an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules. In some embodiments, the j-th reporter, the j-th auxiliary reporter, and the j-th tertiary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent, FRET pair, member of a FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, compound The compound includes composite substrates, carbon atoms, chemical linkers, magnetic molecules, carbon black (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

[0009] According to some implementations, an HCR method using a reporter-labeled probe is provided. In some implementations, the method includes: providing a sample containing a target; contacting the sample with at least one reporter-labeled signal probe including a target-binding region and at least one reporter; contacting the sample with at least one anti-reporter-initiator-labeled signal probe including a reporter-binding region and at least one initiator; contacting the sample with a first HCR hairpin including a first input domain including a first toehold and a first stem section, and a first output domain including a first hairpin loop and a complement of the first stem section; contacting the sample with a second HCR hairpin including a second input domain including a second toehold and a second stem section, and a second output domain including a second hairpin loop and a complement of the second stem section; at least one of the first HCR hairpin and the second HCR hairpin further including an auxiliary reporter, and detecting a signal directly or indirectly from the reporter and / or the auxiliary reporter. In some embodiments, a washing step is performed during any of the above steps. In some embodiments, the signal is removed after detection. In some embodiments, the first HCR hairpin includes a first auxiliary reporter. In some embodiments, the first auxiliary reporter directly or indirectly mediates the generation of the signal. In some embodiments, the second HCR hairpin includes a second auxiliary reporter. In some embodiments, the second auxiliary reporter directly or indirectly mediates the generation of the signal. In some embodiments, (a) the reporter-labeled signal probe includes an anti-target primary antibody or nanobody, (b) the reporter includes a hapten, and (c) the anti-reporter initiator-labeled signal probe includes an anti-hapten primary antibody or nanobody.In some embodiments, the method further includes: binding the first HCR hairpin to the at least one initiator; binding the second HCR hairpin to the first HCR hairpin; contacting the sample with an anti-co-reporter readout probe containing a CARD-mediating enzyme; contacting the sample with one or more CARD substrates; and measuring the signal from one or more deposited CARD reporters generated from the CARD substrates by the CARD-mediating enzyme. In some embodiments, the anti-co-reporter readout probe is a primary antibody that binds to the co-reporter or The sample comprises a nanobody, and further comprises a secondary antibody or nanobody (labeled with one or more enzymes mediating CARD) that binds to the primary antibody or nanobody. According to some embodiments, the method further comprises repeating any of the steps of the method to detect another target in the sample. According to some embodiments, the target comprises an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules. In some embodiments, the reporter and the auxiliary reporter may be the same or different, and each may contain a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, magnetic carbon nanotube, gold nanoparticle (AuNP), gold nanoshell, gold nanorod, silver-shelled gold nanoparticle, latex, magnetic nanoparticle, silica nanoparticle, fluorophore-supported nanoparticle, dye-supported nanoparticle, hapten, ligand, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzyme, combination thereof, or a molecule that directly or indirectly mediates signal generation.

[0010] According to several embodiments, a composition for nonlinear HCR signal amplification is provided, comprising: a) a first HCR initiator; b) a first HCR amplifier comprising two or more HCR hairpins, at least one of which comprises a reporter; c) an anti-reporter bridging probe comprising a reporter-binding domain and a second HCR initiator; and d) a second HCR amplifier comprising two or more HCR hairpins, at least one of which comprises an auxiliary reporter. According to several embodiments, the first HCR initiator is configured to trigger the HCR hairpins constituting the first HCR amplifier to grow a reporter-modified first HCR amplification polymer linked to the first HCR initiator. According to several embodiments, the anti-reporter bridging probe is configured to bind to a reporter that modifies the first HCR amplification polymer and to modify it with the second HCR initiator. In some embodiments, the second HCR initiator is configured to trigger an HCR hairpin constituting the second HCR amplifier to grow a second HCR amplifying polymer modified with an auxiliary reporter linked to the first HCR amplifying polymer. In some embodiments, the reporter and / or auxiliary reporter is configured to directly or indirectly mediate the generation of an amplified signal. In some embodiments, the first HCR initiator is bound to a signal probe configured to directly or indirectly bind to a target. In some embodiments, the first HCR initiator is a colocalized complete first HCR initiator formed when two or more fragmentary initiator probes specifically bind to their cognitive binding sites on a target. In some embodiments, the first HCR initiator is a colocalized complete first HCR initiator formed when two or more fragmentary initiator probes are compounded or specifically bind to their cognitive binding sites on two or more adjacent targets. According to some implementations, the system additionally includes one or more neighboring probes configured to couple to the two or more fragmentary initiator probes.In some embodiments, (a) the auxiliary reporter is the same as the reporter, (b) the first HCR initiator has the same sequence as the second HCR initiator, the first HCR amplifier has the same sequence as the second HCR amplifier, and / or (c) the reporter and the auxiliary reporter may be the same or different, each comprising a hapten, a fluorophore, a chromophore, or a rare earth element or compound. In some embodiments, a) the auxiliary reporter is configured to mediate catalytic reporter deposition (CARD), and b) the composition comprises an anti-auxiliary reporter. The present invention further comprises a reporter readout probe, wherein c) the tertiary reporter comprises an enzyme, and / or d) the enzyme is configured to act on a CARD substrate near the target to catalytically deposit a CARD reporter, either directly or indirectly, to produce a fluorescent or chromogenic signal. In some embodiments, the target comprises an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules. In some embodiments, the reporter, the auxiliary reporter, and the tertiary reporter may be the same or different, each comprising a fluorophore, a chromophore, a luminescent phore, a phosphorescent body, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a nucleotide, an amino acid, an oligonucleotide, DNA, RNA, 2'OMe-RNA, a chemically modified nucleic acid, a synthetic nucleic acid analog, a chemically modified protein, a synthetic protein analog, a peptide, a binding substrate, or a carbon This includes atoms, chemical linkers, magnetic molecules, carbon black (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

[0011] According to several implementations, a composition is provided for detecting N targets (N is a positive integer) in a sample using nonlinear HCR signal amplification, the composition comprising (a) a j-th signal probe set (j=1, ..., N, j is a positive integer) configured to bind to the j-th target, i) a j-th initiator-labeled signal probe containing the j-th first HCR initiator, or ii) a j-th fragmentary initiator signal probe set configured to colocalize the j-th complete first HCR initiator when the probe in the probe unit specifically binds to a cognitive binding site on the j-th target, or iii) the probe in the probe unit specifically binds to the j-th target complex or a set of j-th nearby targets, and one or more j-th nearby probes The present invention provides a set of j-th fragmentary initiator signal probes configured to colocalize the j-th complete first HCR initiator when bound, or iv) a set of j-th signal probes comprising a j-th anti-target reporter-labeled primary signal probe containing the j-th reporter and a j-th anti-reporter initiator-labeled secondary signal probe containing the j-th first HCR initiator; (b) a j-th first HCR amplifier comprising two or more HCR hairpins, at least one of which comprises the j-th auxiliary reporter; (c) a j-th anti-auxiliary reporter bridging probe comprising the j-th auxiliary reporter-binding domain and the j-th second HCR initiator; and (d) a j-th second HCR amplifier comprising two or more HCR hairpins, at least one of which comprises the j-th tertiary reporter. In some embodiments, the j-th first HCR initiator is configured to trigger the HCR hairpin constituting the j-th first HCR amplifier to grow a j-th auxiliary reporter-modified first HCR amplifying polymer linked to the j-th target. In some embodiments, the j-th anti-auxiliary reporter bridging probe is configured to bind to the j-th auxiliary reporter that modifies the j-th first HCR amplifying polymer, thereby modifying the j-th first HCR amplifying polymer with the j-th second HCR initiator.In some embodiments, the j-th second HCR initiator modifying the j-th first HCR amplification polymer is configured to trigger the HCR hairpin constituting the j-th second HCR amplifier to grow a second HCR amplification polymer modified by a j-th tertiary reporter linked to the j-th first HCR amplification polymer. In some embodiments, the j-th reporter, the j-th auxiliary reporter, and / or the j-th tertiary reporter directly generate the j-th amplified signal at the site of the j-th target. It is configured to mediate indirectly. In some embodiments, the j-th target includes RNA molecules, DNA molecules, proteins, small molecules, chemicals, biological molecules, pathogens, molecular complexes, or sets of adjacent molecules. In some embodiments, the j-th reporter, the j-th auxiliary reporter, and the j-th tertiary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent, FRET pair, member of a FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acids, synthetic nucleic acid analogs, chemically modified proteins, synthetic protein analogs, peptides, etc. The compound includes composite substrates, carbon atoms, chemical linkers, magnetic molecules, carbon black (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

[0012] According to several embodiments, a composition for nonlinear HCR signal amplification is provided, comprising: a) a first HCR initiator; b) a self-bridging first HCR amplifier comprising two or more HCR hairpins, each comprising one split initiator tail; and c) a second HCR amplifier comprising two or more HCR hairpins, at least one comprising a reporter. According to several embodiments, the first HCR initiator is configured to trigger the HCR hairpins constituting the first HCR amplifier to grow a first HCR amplification polymer coupled to the first HCR initiator, wherein the split initiator tails on the two or more self-bridging HCR hairpins constituting the first HCR amplification polymer are configured to colocalize a complete second HCR initiator within the polymer. In some embodiments, each of the colocalized complete second HCR initiators within the first HCR amplification polymer is configured to trigger the HCR hairpins constituting the second HCR amplifier, thereby growing a reporter-modified second HCR amplification polymer linked to the first HCR amplification polymer. In some embodiments, the reporter is configured to directly or indirectly mediate the generation of an amplified signal. In some embodiments, the target includes RNA molecules, DNA molecules, proteins, small molecules, chemicals, biological molecules, pathogens, molecular complexes, or sets of adjacent molecules.According to some embodiments, the reporter comprises a fluorophore, a chromophore, a luminophore, a phosphor, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a nucleotide, an amino acid, an oligonucleotide, DNA, RNA, 2’OMe-RNA, a chemically modified nucleic acid, a synthetic nucleic acid analog, a chemically modified protein, a synthetic protein analog, a peptide, a binding substrate, a carbon atom, a chemical linker, a magnetic molecule, carbon black (CB), a carbon nanotube, a magnetic carbon nanotube, a gold nanoparticle (AuNP), a gold nanoshell, a gold nanorod, a silver-shelled gold nanoparticle, a latex, a magnetic nanoparticle, a silica nanoparticle, a fluorophore-bearing nanoparticle, a dye-bearing nanoparticle, a hapten, a ligand, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, an enzyme, a combination thereof, or a molecule that directly or indirectly mediates signal generation.

[0013] According to some embodiments, a method for target detection by non-linear HCR signal amplification is provided. According to some embodiments, the method comprises: a) providing a sample comprising one or more targets; b) contacting the sample with a probe set comprising one or more initiator-labeled probes, or one or more probe units each comprising two or more fragmentary initiator probes; c) contacting the sample with a first HCR amplifier labeled with a reporter and The method comprises: (a) contacting the sample to initiate a linear amplification step; (d) contacting the sample with an anti-reporter initiator-labeled bridging probe containing one or more HCR initiators; (e) contacting the sample with a second HCR amplifier labeled with an auxiliary reporter to initiate a nonlinear HCR amplification step; and (f) detecting one or more signals from the reporter and / or the auxiliary reporter. In some embodiments, the probe set comprises one or more initiator-labeled probes, each of which comprises a target binding region and one or more HCR initiators. In some embodiments, the probe set comprises one or more probe units, each comprising two or more fragmentary initiator probes, each of which comprises a target binding region and fragmentary initiators. In some embodiments, the method comprises an additional HCR signal amplification round, each additional round comprising repeating steps (c) to (d). In some embodiments, the reporter comprises a hapten, and the bridging probe comprises an anti-hapten primary antibody or nanobody. In some embodiments, the auxiliary reporter is a fluorophore, chromophore, or rare earth element or compound. In some embodiments, the anti-reporter bridging probe comprises a primary antibody or nanobody that binds to the reporter, and further comprises an initiator-labeled secondary antibody or nanobody that binds to the primary antibody or nanobody. In some embodiments, the first HCR amplifier and the second HCR amplifier have the same sequence. In some embodiments, the reporter and the auxiliary reporter are the same. In some embodiments, the method further comprises contacting the sample with an anti-auxiliary reporter readout probe comprising a CARD-mediated enzyme; contacting the sample with one or more CARD substrates; and measuring the signal from one or more catalytically deposited CARD reporters. In some embodiments, the method further comprises performing a washing step during any of steps (b) to (f). According to some implementations, the method further includes a step (g) in which the signal is removed after step (f).In some embodiments, one HCR amplifier comprises two or more HCR hairpins. In some embodiments, one HCR hairpin comprises an input domain comprising a single-stranded toehold and a stem section. In some embodiments, one HCR hairpin further comprises an output domain comprising a single-stranded loop and complements of the stem section. In some embodiments, at least one HCR hairpin of the first HCR amplifier further comprises one or more reporters, and at least one HCR hairpin of the second HCR amplifier further comprises one or more auxiliary reporters. In some embodiments, the method further comprises repeating any of the steps of the method to detect another target in the sample. In some embodiments, the target comprises an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules. In some embodiments, the reporter and the auxiliary reporter may be the same or different, and each may contain a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, magnetic carbon nanotube, gold nanoparticle (AuNP), gold nanoshell, gold nanorod, silver-shelled gold nanoparticle, latex, magnetic nanoparticle, silica nanoparticle, fluorophore-supported nanoparticle, dye-supported nanoparticle, hapten, ligand, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzyme, combination thereof, or a molecule that directly or indirectly mediates signal generation.

[0014] According to some implementations, a method for amplifying a nonlinear HCR signal is provided. According to some implementations, the method involves a) a sample being subjected to a first HCR initiator or a complete first HCR a) Contacting the sample with two or more fragmentary initiators that together constitute an R initiator; b) Contacting the sample with an HCR amplifier labeled with a first reporter and incubating it to initiate the growth of a first reporter-modified HCR amplifying polymer linked to the first HCR initiator; c) Contacting the sample with an anti-first reporter bridging probe containing a second HCR initiator and incubating it so that the first HCR amplifying polymer is modified with the second HCR initiator; d) Contacting the sample with a second reporter-labeled HCR amplifier, the e) incubating to initiate growth of a second reporter-modified HCR amplification polymer linked to each second HCR initiator that modifies the first HCR amplification polymer linked to one HCR initiator; and e) detecting a signal directly or indirectly generated by the first reporter and / or the second reporter, wherein at least one HCR hairpin of the first reporter-labeled HCR amplification agent comprises one or more first reporters, and at least one HCR hairpin of the second reporter-labeled HCR amplification agent comprises one or more second reporters. According to some embodiments, the method further comprises preparing an anti-second reporter bridging probe comprising a third HCR initiator and incubating so that the second HCR amplification polymer is modified with the third HCR initiator. In some embodiments, the method further comprises preparing a third reporter-labeled HCR amplifier and incubating it to initiate the growth of a third reporter-modified HCR amplifier linked to each third HCR initiator, modifying the first HCR amplifier polymer linked to the first HCR initiator, modifying the second HCR amplifier polymer linked to the second HCR initiator, and at least one HCR hairpin of the third reporter-labeled HCR amplifier comprises one or more third reporters. In some embodiments, the third reporters directly or indirectly mediate the generation of a signal.In some embodiments, (a) some or all of the first reporter, second reporter, and third reporter are identical; (b) some or all of the first reporter-modified HCR amplifier, the second reporter-modified HCR amplifier, and the third reporter-modified HCR amplifier have the same sequence; and / or (c) the first, second, and third reporters may be the same or different, each comprising a hapten, fluorophore, chromophore, or rare earth element or compound. In some embodiments, the third reporter is used to mediate CARD signal amplification. In some embodiments, the anti-reporter initiator-labeled bridging probe used to bridge between different rounds of HCR signal amplification is the same. In some embodiments, the method further includes performing a washing step during any of steps (a) to (e). In some embodiments, the method further includes removing the signal after step (e). In some embodiments, the method further includes repeating any of steps (a) to (f). In some embodiments, one HCR amplifier comprises two or more HCR hairpins. In some embodiments, one HCR hairpin comprises an input domain comprising a single-stranded toehold and a stem section. In some embodiments, one HCR hairpin further comprises an output domain comprising a single-stranded loop and complement of the stem section. In some embodiments, the target comprises an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.According to some embodiments, the first reporter, the second reporter, and the third reporter may be the same or different, and each may be a fluorophore, chromophore, luminophore, phosphor, FRET pair, member of a FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2’OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, magnetic carbon nanotube, gold nanoparticle (AuNP), gold nanoshell, gold nanorod, silver-shelled gold nanoparticle, latex, magnetic nanoparticle, silica nanoparticle, fluorophore-bearing nanoparticle, dye-bearing nanoparticle, hapten, ligand, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzyme, a combination thereof, or a molecule that directly or indirectly mediates signal generation. including particles, silica nanoparticles, fluorophore-bearing nanoparticles, dye-bearing nanoparticles, hapten, ligand, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzyme, a combination thereof, or a molecule that directly or indirectly mediates signal generation.

Brief Description of the Drawings

[0015] [Figure 1A] Figures 1A to 1C are diagrams showing some embodiments of HCR RNA-FISH using an initiator-labeled probe. [Figure 1B] Figures 1A to 1C are diagrams showing some embodiments of HCR RNA-FISH using an initiator-labeled probe. [Figure 1C] Figures 1A to 1C are diagrams showing some embodiments of HCR RNA-FISH using an initiator-labeled probe. [Figure 2A] Figures 2A to 2B are diagrams showing some embodiments of HCR RNA-FISH using a fragmented initiator probe. [Figure 2B] Figures 2A to 2B are diagrams showing some embodiments of HCR RNA-FISH using a fragmented initiator probe. [Figure 3A] Figure 3A shows several embodiments of a fragmented initiator probe co-localized to a target. Figures 3B–3C show test tube data demonstrating the use of a fragmented initiator probe co-localized to a target to trigger HCR. [Figure 3B] Figure 3A shows several embodiments of a fragmented initiator probe co-localized to a target. Figures 3B–3C show test tube data demonstrating the use of a fragmented initiator probe co-localized to a target to trigger HCR. [Figure 3C] Figure 3A shows several embodiments of a fragmented initiator probe co-localized to a target. Figures 3B–3C show test tube data demonstrating the use of a fragmented initiator probe co-localized to a target to trigger HCR. [Figure 4A] Figures 4A to 4D show the background and signal-to-background ratio using initiator-labeled probes and fragmented initiator probes. [Figure 4B] Figures 4A to 4D show the background and signal-to-background ratio using initiator-labeled probes and fragmented initiator probes. [Figure 4C] Figures 4A to 4D show the background and signal-to-background ratio using initiator-labeled probes and fragmented initiator probes. [Figure 4D] Figures 4A to 4D show the background and signal-to-background ratio using initiator-labeled probes and fragmented initiator probes. [Figure 5A] Figures 5A to 5D show multiplex imaging of high-signal versus background mRNA expression in fixed whole-mount chicken embryos using fragmentary initiator probes without probe set optimization. [Figure 5B]Figures 5A to 5D show multiplex imaging of high-signal versus background mRNA expression in fixed whole-mount chicken embryos using fragmentary initiator probes without probe set optimization. [Figure 5C] Figures 5A to 5D show multiplex imaging of high-signal versus background mRNA expression in fixed whole-mount chicken embryos using fragmentary initiator probes without probe set optimization. [Figure 5D] Figures 5A to 5D show multiplex imaging of high-signal versus background mRNA expression in fixed whole-mount chicken embryos using fragmentary initiator probes without probe set optimization. [Figure 6A] Figures 6A and 6B show quantitative imaging of mRNA expression at intracellular resolution in fixed whole-mount chicken embryos using a fragmentary initiator probe. [Figure 6B] Figures 6A and 6B show quantitative imaging of mRNA expression at intracellular resolution in fixed whole-mount chicken embryos using a fragmentary initiator probe. [Figure 7] Figure 7 shows single-molecule mRNA imaging in fixed whole-mount chicken embryos using a fragmentary initiator probe. [Figure 8A] Figures 8A to 8D illustrate several embodiments of HCR IF using initiator-labeled probes. Figures 8E to 8F illustrate several embodiments of HCR RNA-FISH / IF using fragmentary initiator probes for RNA targeting and initiator-labeled probes for protein targeting. [Figure 8B]Figures 8A to 8D illustrate several embodiments of HCR IF using initiator-labeled probes. Figures 8E to 8F illustrate several embodiments of HCR RNA-FISH / IF using fragmentary initiator probes for RNA targeting and initiator-labeled probes for protein targeting. [Figure 8C] Figures 8A to 8D illustrate several embodiments of HCR IF using initiator-labeled probes. Figures 8E to 8F illustrate several embodiments of HCR RNA-FISH / IF using fragmentary initiator probes for RNA targeting and initiator-labeled probes for protein targeting. [Figure 8D] Figures 8A to 8D illustrate several embodiments of HCR IF using initiator-labeled probes. Figures 8E to 8F illustrate several embodiments of HCR RNA-FISH / IF using fragmentary initiator probes for RNA targeting and initiator-labeled probes for protein targeting. [Figure 8E] Figures 8A to 8D illustrate several embodiments of HCR IF using initiator-labeled probes. Figures 8E to 8F illustrate several embodiments of HCR RNA-FISH / IF using fragmentary initiator probes for RNA targeting and initiator-labeled probes for protein targeting. [Figure 8F] Figures 8A to 8D illustrate several embodiments of HCR IF using initiator-labeled probes. Figures 8E to 8F illustrate several embodiments of HCR RNA-FISH / IF using fragmentary initiator probes for RNA targeting and initiator-labeled probes for protein targeting. [Figure 9A] Figures 9A and 9B show HCR IF for multiplex imaging of high signal-to-background ratio protein expression in FFPE mouse brain tissue sections using initiator-labeled primary antibody probes. [Figure 9B]Figures 9A and 9B show HCR IF for multiplex imaging of high signal-to-background ratio protein expression in FFPE mouse brain tissue sections using initiator-labeled primary antibody probes. [Figure 10A] Figures 10A and 10B show HCR IFs for multiplex imaging of high signal-to-background ratio protein expression in FFPE mouse brain tissue sections using primary antibody probes and initiator-labeled secondary antibody probes. [Figure 10B] Figures 10A and 10B show HCR IFs for multiplex imaging of high signal-to-background ratio protein expression in FFPE mouse brain tissue sections using primary antibody probes and initiator-labeled secondary antibody probes. [Figure 11A] Figures 11A–11C show HCR IF for quantitative imaging of protein expression at intracellular resolution in FFPE mouse brain tissue sections. [Figure 11B] Figures 11A–11C show HCR IF for quantitative imaging of protein expression at intracellular resolution in FFPE mouse brain tissue sections. [Figure 11C] Figures 11A–11C show HCR IF for quantitative imaging of protein expression at intracellular resolution in FFPE mouse brain tissue sections. [Figure 12A] Figures 12A and 12B show HCR RNA-FISH / IF for simultaneous multiplex imaging of RNA and protein targets in high-signal versus background conditions in FFPE mouse brain tissue sections. This was achieved by using fragmentary initiator probes for RNA targets and initiator-labeled primary antibody probes for protein targets, and performing HCR signal amplification for all targets simultaneously. [Figure 12B]Figures 12A and 12B show HCR RNA-FISH / IF for simultaneous multiplex imaging of RNA and protein targets in high-signal versus background conditions in FFPE mouse brain tissue sections. This was achieved by using fragmentary initiator probes for RNA targets and initiator-labeled primary antibody probes for protein targets, and performing HCR signal amplification for all targets simultaneously. [Figure 13A] Figures 13A and 13B show HCR RNA-FISH / IF for simultaneous multiplex imaging of RNA and protein targets with a high signal-to-background ratio in FFPE mouse brain tissue sections. This is achieved by using fragmentary initiator probes for RNA target detection and primary antibody probes and initiator-labeled secondary antibody probes for protein target detection, and performing HCR signal amplification simultaneously for all labels. [Figure 13B] Figures 13A and 13B show HCR RNA-FISH / IF for simultaneous multiplex imaging of RNA and protein targets with a high signal-to-background ratio in FFPE mouse brain tissue sections. This is achieved by using fragmentary initiator probes for RNA target detection and primary antibody probes and initiator-labeled secondary antibody probes for protein target detection, and performing HCR signal amplification simultaneously for all labels. [Figure 14] Figure 14 shows several embodiments of HCR amplification using two HCR hairpins. Figure 14 shows a colocalized complete HCR initiator i1 formed by two fragmentary initiator probes colocalized by the target. Only a portion of the fragmentary initiator probes is shown. [Figure 15] Figure 15 shows several embodiments of HCR amplification using two HCR hairpins. [Figure 16A] Figures 16A to 16D illustrate several embodiments of HCR amplification using four HCR hairpins. [Figure 16B]Figures 16A to 16D illustrate several embodiments of HCR amplification using four HCR hairpins. [Figure 16C] Figures 16A to 16D illustrate several embodiments of HCR amplification using four HCR hairpins. [Figure 16D] Figures 16A to 16D illustrate several embodiments of HCR amplification using four HCR hairpins. [Figure 17A] Figures 17A to 17F show several embodiments of the HCR amplifier. [Figure 17B] Figures 17A to 17F show several embodiments of the HCR amplifier. [Figure 17C] Figures 17A to 17F show several embodiments of the HCR amplifier. [Figure 17D] Figures 17A to 17F show several embodiments of the HCR amplifier. [Figure 17E] Figures 17A to 17F show several embodiments of the HCR amplifier. [Figure 17F] Figures 17A to 17F show several embodiments of the HCR amplifier. [Figure 18A] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18B] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18C] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18D] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18E] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18F] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18G] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18H] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18I] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18J] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18K] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18L] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18M] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 18N] Figures 18A to 18N illustrate several embodiments of initiator-labeled probes. [Figure 19A] Figures 19A to 19F illustrate several embodiments of initiator-labeled probes. [Figure 19B] Figures 19A to 19F illustrate several embodiments of initiator-labeled probes. [Figure 19C] Figures 19A to 19F illustrate several embodiments of initiator-labeled probes. [Figure 19D] Figures 19A to 19F illustrate several embodiments of initiator-labeled probes. [Figure 19E] Figures 19A to 19F illustrate several embodiments of initiator-labeled probes. [Figure 19F] Figures 19A to 19F illustrate several embodiments of initiator-labeled probes. [Figure 20A]Figures 20A to 20E illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 20B] Figures 20A to 20E illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 20C] Figures 20A to 20E illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 20D] Figures 20A to 20E illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 20E] Figures 20A to 20E illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 21A] Figures 21A to 21D illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 21B] Figures 21A to 21D illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 21C] Figures 21A to 21D illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 21D] Figures 21A to 21D illustrate several embodiments of fragmentary initiator probes colocalized by labeling. [Figure 22A] Figures 22A to 22E illustrate several embodiments of fragmentary initiator probes colocalized by a labeling complex. [Figure 22B] Figures 22A to 22E illustrate several embodiments of fragmentary initiator probes colocalized by a labeling complex. [Figure 22C] Figures 22A to 22E illustrate several embodiments of fragmentary initiator probes colocalized by a labeling complex. [Figure 22D] Figures 22A to 22E illustrate several embodiments of fragmentary initiator probes colocalized by a labeling complex. [Figure 22E] Figures 22A to 22E illustrate several embodiments of fragmentary initiator probes colocalized by a labeling complex. [Figure 23] Figure 23 shows several embodiments of a fragmentary initiator probe colocalized by labeling. [Figure 24A] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24B] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24C] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24D] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24E] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24F] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24G] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24H] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24I]Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24J] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24K] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24L] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24M] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24N] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24O] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24P] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24Q] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 24R] Figures 24A to 24R illustrate several embodiments of fragmentary initiator probes that are directly or indirectly colocalized by a target. [Figure 25A] Figures 25A to 25E illustrate several embodiments of mediating CARD signal amplification using HCR amplification for various targets and signal probes. [Figure 25B] Figures 25A to 25E illustrate several embodiments of mediating CARD signal amplification using HCR amplification for various targets and signal probes. [Figure 25C] Figures 25A to 25E illustrate several embodiments of mediating CARD signal amplification using HCR amplification for various targets and signal probes. [Figure 25D] Figures 25A to 25E illustrate several embodiments of mediating CARD signal amplification using HCR amplification for various targets and signal probes. [Figure 25E] Figures 25A to 25E illustrate several embodiments of mediating CARD signal amplification using HCR amplification for various targets and signal probes. [Figure 26A] Figures 26A to 26C illustrate several embodiments of mediating CARD signal amplification using HCR signal amplification for commercially available targets and signal probes. [Figure 26B] Figures 26A to 26C illustrate several embodiments of mediating CARD signal amplification using HCR signal amplification for commercially available targets and signal probes. [Figure 26C] Figures 26A to 26C illustrate several embodiments of mediating CARD signal amplification using HCR signal amplification for commercially available targets and signal probes. [Figure 27A] Figures 27A and 27B illustrate several embodiments of mediating CARD signal amplification using reporter-labeled or fragmentary reporter-labeled HCR hairpins. [Figure 27B] Figures 27A and 27B illustrate several embodiments of mediating CARD signal amplification using reporter-labeled or fragmentary reporter-labeled HCR hairpins. [Figure 28A]Figures 28A and 28B illustrate several embodiments of fragmentary initiator probes designed to be complementary to the overlapping region of the HCR hairpin. [Figure 28B] Figures 28A and 28B illustrate several embodiments of fragmentary initiator probes designed to be complementary to the overlapping region of the HCR hairpin. [Figure 29] Figure 29 shows several embodiments of a fragmented initiator probe designed to be complementary to the overlapping region of the target. [Figure 30] Figure 30 shows several embodiments of a fragmentation initiator probe designed to be complementary to the overlapping region of the HCR hairpin and to the overlapping region of the target. [Figure 31A] Figures 31A to 31C illustrate several embodiments of in vitro optimization examples of cooperative probe junctions to enhance fragmented initiator HCR suppression (off state) and conversion (on state). [Figure 31B] Figures 31A to 31C illustrate several embodiments of in vitro optimization examples of cooperative probe junctions to enhance fragmented initiator HCR suppression (off state) and conversion (on state). [Figure 31C] Figures 31A to 31C illustrate several embodiments of in vitro optimization examples of cooperative probe junctions to enhance fragmented initiator HCR suppression (off state) and conversion (on state). [Figure 32A] Figures 32A and 32B illustrate several embodiments of multiplex in situ hybridization in cultured human cells using repeated reporter detection. [Figure 32B] Figures 32A and 32B illustrate several embodiments of multiplex in situ hybridization in cultured human cells using repeated reporter detection. [Figure 33] Figure 33 shows several embodiments of ultra-sensitive HCR RNA-CISH for chromogenic staining of CARD-mediated RNA targets using HCR. [Figure 34A] Figures 34A and 34B show the performance of ultra-high-sensitivity HCR RNA-CISH in FFPE tissue sections compared with weak staining using bDNA CARD and damaged tissue morphology. [Figure 34B] Figures 34A and 34B show the performance of ultra-high-sensitivity HCR RNA-CISH in FFPE tissue sections compared with weak staining using bDNA CARD and damaged tissue morphology. [Figure 35A] Figures 35A and 35B illustrate several embodiments of ultra-sensitive multiplex HCR RNA-CISH. [Figure 35B] Figures 35A and 35B illustrate several embodiments of ultra-sensitive multiplex HCR RNA-CISH. [Figure 36A] Figures 36A and 36B show multiplex imaging of RNA targets in FFPE mouse duodenal tissue sections using ultra-high sensitivity HCR RNA-CISH. [Figure 36B] Figures 36A and 36B show multiplex imaging of RNA targets in FFPE mouse duodenal tissue sections using ultra-high sensitivity HCR RNA-CISH. [Figure 37A] Figures 37A to 37C illustrate several embodiments of probes for detecting short RNA targets, including miRNAs and mRNA splice junctions. [Figure 37B] Figures 37A to 37C illustrate several embodiments of probes for detecting short RNA targets, including miRNAs and mRNA splice junctions. [Figure 37C] Figures 37A to 37C illustrate several embodiments of probes for detecting short RNA targets, including miRNAs and mRNA splice junctions. [Figure 38A]Figures 38A and 38B illustrate several embodiments of nonlinear HCR signal amplification used to mediate CARD signal amplification for ultra-sensitive HCR RNA-CISH. [Figure 38B] Figures 38A and 38B illustrate several embodiments of nonlinear HCR signal amplification used to mediate CARD signal amplification for ultra-sensitive HCR RNA-CISH. [Figure 39A] Figures 39A and 39B illustrate several embodiments of ultra-high sensitivity HCR IHC for chromogenic staining of CARD-mediated protein targets using HCR. [Figure 39B] Figures 39A and 39B illustrate several embodiments of ultra-high sensitivity HCR IHC for chromogenic staining of CARD-mediated protein targets using HCR. [Figure 40A] Figures 40A and 40B illustrate the performance of ultra-high sensitivity HCR IHC for protein imaging in tissue sections and cell pellets compared to weak staining using two competing conventional IHC products. [Figure 40B] Figures 40A and 40B illustrate the performance of ultra-high sensitivity HCR IHC for protein imaging in tissue sections and cell pellets compared to weak staining using two competing conventional IHC products. [Figure 41A] Figures 41A and 41B illustrate several embodiments of an ultra-high-sensitivity multiplex HCR IHC that uses HCR signal amplification to mediate CARD signal amplification. [Figure 41B] Figures 41A and 41B illustrate several embodiments of an ultra-high-sensitivity multiplex HCR IHC that uses HCR signal amplification to mediate CARD signal amplification. [Figure 42] Figure 42 shows multiplex imaging of protein targets in FFPE human tonsil sections mediated by CARD using HCR for ultra-high sensitivity HCR IHC. [Figure 43]Figure 43 shows several embodiments of ultra-sensitive multiplex HCR RNA-CISH / IHC that use HCR signal amplification to mediate CARD signal amplification for simultaneous RNA / protein imaging by chromogenic staining. [Figure 44A] Figures 44A and 44B show simultaneous imaging of RNA and protein targets in FFPE human breast cancer tissue sections using ultra-high sensitivity HCR RNA-CISH (HCR-mediated CARD) in combination with conventional IHC. This demonstrates the advantage that ultra-high sensitivity HCR RNA-CISH does not require protease pretreatment, thus preventing damage to protein targets and allowing them to be used for imaging in the same sample as RNA targets. [Figure 44B] Figures 44A and 44B show simultaneous imaging of RNA and protein targets in FFPE human breast cancer tissue sections using ultra-high sensitivity HCR RNA-CISH (HCR-mediated CARD) in combination with conventional IHC. This demonstrates the advantage that ultra-high sensitivity HCR RNA-CISH does not require protease pretreatment, thus preventing damage to protein targets and allowing them to be used for imaging in the same sample as RNA targets. [Figure 45] Figure 45 shows several embodiments of ultra-sensitive HCR RNA-FISH for fluorescent staining of RNA targets using nonlinear enzyme-free HCR signal amplification. [Figure 46] Figure 46 shows the enhanced sensitivity of HCR RNA-FISH to linear HCR signal amplification using ultra-high sensitivity nonlinear HCR signal amplification (using an anti-reporter 1°Ab bridging probe and an anti-primary initiator labeled 2°Ab bridging probe—see Figure 45). [Figure 47A] Figures 47A and 47B illustrate several embodiments of ultra-sensitive multiplex HCR RNA-FISH using nonlinear enzyme-free HCR signal amplification. [Figure 47B]Figures 47A and 47B illustrate several embodiments of ultra-sensitive multiplex HCR RNA-FISH using nonlinear enzyme-free HCR signal amplification. [Figure 48] Figure 48 shows multiplex imaging of RNA targets in FFPE mouse duodenal sections using ultra-sensitive HCR RNA-FISH with nonlinear enzyme-free HCR signal amplification. [Figure 49A] Figures 49A and 49B illustrate several embodiments of nonlinear enzyme-free HCR signal amplification for ultra-sensitive HCR RNA-FISH. [Figure 49B] Figures 49A and 49B illustrate several embodiments of nonlinear enzyme-free HCR signal amplification for ultra-sensitive HCR RNA-FISH. [Figure 50A] Figures 50A to 50C illustrate several embodiments of imaging target microRNAs and mRNAs in whole-mount zebrafish embryos using initiator-labeled probes. [Figure 50B] Figures 50A to 50C illustrate several embodiments of imaging target microRNAs and mRNAs in whole-mount zebrafish embryos using initiator-labeled probes. [Figure 50C] Figures 50A to 50C illustrate several embodiments of imaging target microRNAs and mRNAs in whole-mount zebrafish embryos using initiator-labeled probes. [Figure 51A] Figures 51A and 51B illustrate several embodiments of ultra-sensitive HCR IF for fluorescent staining of protein targets using nonlinear or linear enzyme-free HCR signal amplification. [Figure 51B] Figures 51A and 51B illustrate several embodiments of ultra-sensitive HCR IF for fluorescent staining of protein targets using nonlinear or linear enzyme-free HCR signal amplification. [Figure 52A]Figures 52A and 52B illustrate several embodiments of ultra-sensitive multiplex HCR IF using nonlinear enzyme-free HCR signal amplification. [Figure 52B] Figures 52A and 52B illustrate several embodiments of ultra-sensitive multiplex HCR IF using nonlinear enzyme-free HCR signal amplification. [Figure 53] Figure 53 shows multiplex protein imaging in FFPE human tonsil tissue using HCR IF with enzyme-free HCR signal amplification. [Figure 54] Figure 54 shows several embodiments of ultra-sensitive multiplex HCR RNA-FISH / IF using nonlinear enzyme-free HCR signal amplification for simultaneous RNA / protein imaging by fluorescence staining. [Figure 55] Figure 55 shows imaging of small RNA targets and protein targets in FFPE human breast cancer tissue using HCR RNA-FISH / IF with enzyme-free HCR signal amplification. [Figure 56A] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56B] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56C] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56D] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56E] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56F] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56G]Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56H] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56I] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56J] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56K] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56L] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56M] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 56N] Figures 56A to 56N illustrate several embodiments of reporter-labeled signal probes. [Figure 57A] Figures 57A and 57B illustrate several embodiments of polyreporter-labeled signal probes. [Figure 57B] Figures 57A and 57B illustrate several embodiments of polyreporter-labeled signal probes. [Figure 58A] Figures 58A to 58C illustrate several embodiments of reporter-labeled signal probes, initiator-labeled signal probes, fragmentary initiator signal probes, and proximity probes. [Figure 58B] Figures 58A to 58C illustrate several embodiments of reporter-labeled signal probes, initiator-labeled signal probes, fragmentary initiator signal probes, and proximity probes. [Figure 58C]Figures 58A to 58C illustrate several embodiments of reporter-labeled signal probes, initiator-labeled signal probes, fragmentary initiator signal probes, and proximity probes. [Figure 59A] Figures 59A to 59F show several embodiments of reporter-labeled signal probes and initiator-labeled signal probes. [Figure 59B] Figures 59A to 59F show several embodiments of reporter-labeled signal probes and initiator-labeled signal probes. [Figure 59C] Figures 59A to 59F show several embodiments of reporter-labeled signal probes and initiator-labeled signal probes. [Figure 59D] Figures 59A to 59F show several embodiments of reporter-labeled signal probes and initiator-labeled signal probes. [Figure 59E] Figures 59A to 59F show several embodiments of reporter-labeled signal probes and initiator-labeled signal probes. [Figure 59F] Figures 59A to 59F show several embodiments of reporter-labeled signal probes and initiator-labeled signal probes. [Figure 60A] Figures 60A to 60D illustrate several embodiments of ultra-high sensitivity multiplex HCR IHC and ultra-high sensitivity multiplex HCR IF using anti-target reporter-labeled signal probes and anti-reporter initiator-labeled signal probes. [Figure 60B] Figures 60A to 60D illustrate several embodiments of ultra-high sensitivity multiplex HCR IHC and ultra-high sensitivity multiplex HCR IF using anti-target reporter-labeled signal probes and anti-reporter initiator-labeled signal probes. [Figure 60C]Figures 60A to 60D illustrate several embodiments of ultra-high sensitivity multiplex HCR IHC and ultra-high sensitivity multiplex HCR IF using anti-target reporter-labeled signal probes and anti-reporter initiator-labeled signal probes. [Figure 60D] Figures 60A to 60D illustrate several embodiments of ultra-high sensitivity multiplex HCR IHC and ultra-high sensitivity multiplex HCR IF using anti-target reporter-labeled signal probes and anti-reporter initiator-labeled signal probes. [Figure 61A] Figures 61A to 61E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 61B] Figures 61A to 61E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 61C] Figures 61A to 61E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 61D] Figures 61A to 61E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 61E] Figures 61A to 61E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 62A] Figures 62A to 62D illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 62B] Figures 62A to 62D illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 62C] Figures 62A to 62D illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 62D]Figures 62A to 62D illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 63A] Figures 63A to 63E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 63B] Figures 63A to 63E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 63C] Figures 63A to 63E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 63D] Figures 63A to 63E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 63E] Figures 63A to 63E illustrate several embodiments of a coordinated probe junction for a fragmented initiator probe. [Figure 64A] Figures 64A to 64D illustrate several embodiments of a probe set, which includes one or more probe units and optionally one or more helper probes. [Figure 64B] Figures 64A to 64D illustrate several embodiments of a probe set, which includes one or more probe units and optionally one or more helper probes. [Figure 64C] Figures 64A to 64D illustrate several embodiments of a probe set, which includes one or more probe units and optionally one or more helper probes. [Figure 64D] Figures 64A to 64D illustrate several embodiments of a probe set, which includes one or more probe units and optionally one or more helper probes. [Figure 65A] Figures 65A to 65C illustrate several embodiments of a probe unit including a fragmentary initiator probe. [Figure 65B]Figures 65A to 65C illustrate several embodiments of a probe unit including a fragmentary initiator probe. [Figure 65C] Figures 65A to 65C illustrate several embodiments of a probe unit including a fragmentary initiator probe. [Figure 66] Figure 66 shows multiplex HCR IF in FFPE human spleen tissue sections, where an anti-target reporter-labeled primary antibody signal probe is combined with an anti-reporter and initiator-labeled primary antibody signal probe to trigger HCR signal amplification. [Figure 67] Figure 67 shows the enhanced sensitivity of HCR RNA-FISH compared to linear HCR signal amplification using ultra-high sensitivity nonlinear HCR signal amplification (using an anti-reporter initiator labeled 1°Ab bridging probe—see Figure 49A). [Figure 68A] Figure 68A illustrates several embodiments for detecting targets within or near a complex using a fragmentary initiator probe and a proximity probe, where the fragmentary initiator probe binds directly to the sample. [Figure 68B] Figure 68B illustrates several embodiments for detecting targets within or near a complex using a fragmentary initiator probe and a proximity probe, where the fragmentary initiator probe binds indirectly to the sample. [Figure 68C] Figure 68C illustrates several embodiments of target detection using a fragmentary initiator probe and a proximity probe. [Figure 68D] Figure 68D illustrates several embodiments for detecting targets within or near a complex using an anti-target reporter labeled primary probe, an anti-reporter fragment initiator secondary probe, and a proximity probe. [Figure 68E] Figures 68E to 68I illustrate several embodiments for detecting targets within or in the vicinity of a complex using a proximity probe and different signal probe compositions for each of two targets within or in the vicinity of the complex. [Figure 68F] Figures 68E to 68I illustrate several embodiments for detecting targets within or in the vicinity of a complex using a proximity probe and different signal probe compositions for each of two targets within or in the vicinity of the complex. [Figure 68G] Figures 68E to 68I illustrate several embodiments for detecting targets within or in the vicinity of a complex using a proximity probe and different signal probe compositions for each of two targets within or in the vicinity of the complex. [Figure 68H] Figures 68E to 68I illustrate several embodiments for detecting targets within or in the vicinity of a complex using a proximity probe and different signal probe compositions for each of two targets within or in the vicinity of the complex. [Figure 68I] Figures 68E to 68I illustrate several embodiments for detecting targets within or in the vicinity of a complex using a proximity probe and different signal probe compositions for each of two targets within or in the vicinity of the complex. [Figure 69A] Figures 69A and 69B illustrate several embodiments of HCR imaging of protein:protein target complexes using primary antibody probes with and without secondary antibody probes. [Figure 69B] Figures 69A and 69B illustrate several embodiments of HCR imaging of protein:protein target complexes using primary antibody probes with and without secondary antibody probes. [Figure 70] Figure 70 illustrates several embodiments of a protocol for simultaneous HCR imaging of protein targets, protein:protein target complexes, and RNA targets in a sample using enzyme-free nonlinear HCR signal amplification for all target classes simultaneously. [Figure 71]Figure 71 illustrates several embodiments of HCR imaging of RNA targets, protein targets, and / or target complexes using enzyme-free nonlinear HCR signal amplification, employing a 4-hairpin amplifier with a split initiator tail in the linear stage and a 2-hairpin fluorophore-labeled amplifier in the nonlinear stage. [Figure 72A] Figures 72A to 72C show the enhanced sensitivity of HCR RNA-FISH using ultra-high sensitivity nonlinear HCR signal amplification (using the approach in Figure 71) compared to linear HCR signal amplification. [Figure 72B] Figures 72A to 72C show the enhanced sensitivity of HCR RNA-FISH using ultra-high sensitivity nonlinear HCR signal amplification (using the approach in Figure 71) compared to linear HCR signal amplification. [Figure 72C] Figures 72A to 72C show the enhanced sensitivity of HCR RNA-FISH using ultra-high sensitivity nonlinear HCR signal amplification (using the approach in Figure 71) compared to linear HCR signal amplification. [Figure 73] Figure 73 illustrates several embodiments of nonlinear HCR signal amplification, including an anti-target signal probe containing a first HCR initiator that triggers a first round of HCR signal amplification, bridging to a second HCR initiator to trigger a second round of HCR signal amplification, and bridging to a third HCR initiator to trigger a third round of HCR signal amplification. [Modes for carrying out the invention]

[0016] Hybridization chain reaction (HCR) 1 Signal amplification based on this mechanism enables the detection of molecular targets within immobilized biological samples such as cells or tissues. 2~8Target molecules within a fixed sample are detected using a probe that triggers a chain reaction in which a reporter-labeled HCR hairpin self-assembles into a reporter-modified HCR-amplified polymer, generating an amplified signal in situ at the location of the target molecule or target complex within the sample (see, for example, Figures 1B and 2A). 2~9 RNA targets, HCR Imaging can be performed within a fixed sample using fluorescent staining by RNA fluorescence in situ hybridization (HCR RNA-FISH) or chromogenic staining by HCR RNA chromogenic in situ hybridization (HCR RNA-CISH). DNA targets can be imaged within a fixed sample using fluorescent staining by HCR DNA fluorescence in situ hybridization (HCR DNA-FISH) or chromogenic staining by HCR DNA chromogenic in situ hybridization (HCR DNA-CISH). Protein targets within a fixed sample can be imaged using fluorescent staining by HCR immunofluorescence (HCR IF) or chromogenic staining by HCR immunohistochemistry (IHC). Target molecules that are bound to each other or in close proximity within a complex can be imaged within a fixed sample using either fluorescent staining or chromogenic staining. In contrast to branched DNA in situ hybridization, which requires the use of long preamplifiers and amplification strands to achieve strong signal amplification, making it difficult for bDNA amplification reagents to penetrate deep into the fixed sample. 10~12 HCR signal amplification is performed using small HCR hairpins that penetrate deep into the fixed sample, and then a long HCR amplification polymer is grown at the target site, enabling strong signal amplification and achieving deep penetration of the sample. 2~8

[0017] Hybridization chain reaction (HCR) signal amplification In some embodiments, the target is detected using a signal probe set that includes one or more initiator-labeled probes, each containing a target-binding domain and an amplification domain containing one or more HCR initiators (e.g., Figures 18A-18N, 19A-19F, 52A, 54, 60C, and 71). In some embodiments, the target is detected using a signal probe set that includes one or more primary probes, each containing a target-binding domain and one or more reporters, and one or more secondary probes, each containing a reporter-binding domain and an amplification domain containing one or more HCR initiators (e.g., Figures 58A-58C, 59A-59F, and 60A-60D). In some embodiments, a signal probe set comprising one or more probe units (see, for example, the probe sets in Figures 2A-2B and 64A-64D) is used to detect a target in a sample, and the probe unit comprises two or more fragmentary initiator probes (see, for example, the probe units in Figures 20A-20E, 21A-21D, 22A-22E, 47A, 65A-65C, and 71), and each fragmentary initiator probe comprises a target-binding domain and an amplification domain containing a fragmentary initiator (see, for example, the fragmentary initiator probes in Figures 23, 47A, 65A-65C, and 71). In some embodiments, when each probe in the probe unit binds to an adjacent cognitive binding site on the target, fragmentary initiators colocalize to form a colocalized complete HCR initiator that can trigger HCR signal amplification (e.g., Figures 2A, 20A-20E, 21A-21D, 22A-22E, 24A-24R, 37C, 38A-38B, 47A, 61A-61). E forms a colocalized complete HCR initiator (see Figures 64A-64D and 71). In some embodiments, each fragmentary initiator probe within the probe unit further comprises a proximity domain (see, for example, Figures 58C, 68A-68I, and 69A-69C). In some embodiments, each fragmentary initiator probe within the probe unit binds to a corresponding cognitive binding site on a proximal target, so that one or more proximity probes bind to the proximity domain within the probe unit, colocalizing the fragmentary initiators and forming a colocalized complete HCR initiator (see, for example, Figures 58C, 68A-68B, 68D-68I, and 69A-69C), which can trigger HCR signal amplification.

[0018] In some embodiments, HCR signal amplification increases the signal intensity by 2, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000 times, or within the range defined by any two of the aforementioned values.

[0019] In some embodiments, the target-binding domain within the probe unit is configured to bind to overlapping or non-overlapping regions of the target (see, for example, Figures 2A-2B, 28, 29, and 30). In some embodiments, the fragmentary initiator within the probe unit is designed to hybridize to overlapping or non-overlapping regions of the HCR hairpin (see, for example, Figures 2A-2B, 28, 29, and 30). In some embodiments, the non-specifically binding individual fragmentary initiator probes do not co-localize with the complete HCR initiator, thereby suppressing the false generation of amplified HCR background.

[0020] In some embodiments, the HCR amplifier comprises two or more HCR hairpins (see, for example, the HCR amplifiers in Figures 1A-1B, 17A-17F, and 16A-16B). In some embodiments, each HCR hairpin (also referred to as HCR monomer, HCR hairpin monomer, or hairpin monomer) comprises an input domain including a single-stranded toehold and stem section, and an output domain including a single-stranded loop and complement of the stem section (see, for example, the HCR hairpins in Figures 1A-1B, 14, 15, 16A-16B, and 17A-17F). In some embodiments, one or more HCR initiators on an initiator-labeled probe each initiate a chain reaction of the polymerization step. In this chain reaction, the initiator hybridizes to the input domain of a first HCR hairpin, opening the first hairpin and exposing its output domain, then hybridizes to the input domain of a second HCR hairpin, opening the second hairpin and exposing its output domain, and so on, until the hairpins polymerize to produce an HCR-amplified polymer linked to a target (see, for example, the amplified polymers in Figures 14, 15, 16A-16B, 25A, 25C-25E, 26A, 39A-39B, 41A, and 60A). In some embodiments, in the absence of a colocalized complete HCR initiator, the HCR hairpins are kinetically captured, do not polymerize, and suppress background noise. However, when a fragmentary initiator probe within the probe unit binds to an adjacent cognitive binding site on the target and colocalizes a complete HCR initiator, the colocalized complete HCR initiator (also referred to as the colocalized complete initiator, complete HCR initiator, or complete initiator) initiates a polymerization chain reaction in which the colocalized complete HCR initiator hybridizes to the input domain of the first HCR hairpin, opening the first hairpin and exposing its output domain. This then hybridizes to the input domain of the second HCR hairpin, opening the second hairpin and exposing its output domain.This hybridizes to the input domain of the second copy of the first HCR hairpin, opening up the second copy of the first hairpin and exposing its output domain. This then hybridizes to the second of the second HCR hairpin. The first and second HCR hairpins hybridize to the input domain of the copy, opening up a second copy of the second hairpin to expose its output domain, and so on, until the first and second HCR hairpins alternately polymerize to produce an HCR amplified polymer linked to the target (see, for example, the amplified polymers in Figures 2A, 17C-17F, 16A-16B, 33, 35A, and 47A).

[0021] In some embodiments, the HCR hairpin further includes zero, one, or more reporters that directly or indirectly lead to the generation of an amplified signal (see, for example, Figures 17A–17F). In some embodiments, the zero, one, or more reporters on the HCR hairpin play a role in mediating an additional layer of signal amplification via HCR (see, for example, Figures 45, 47A–47B, 49A–49B, 51A, 52A–52B, 60C–60D, and 71), catalytic reporter deposition (CARD) (see, for example, Figures 25A–25E, 26A–26C, 33, 35A, 39A–39B, 41A, 43, and 60A–60B), or both HCR and CARD (see, for example, Figures 38A–38B). In some embodiments, the reporter on the HCR hairpin includes a fragmentary reporter such that a readout probe labeled with an auxiliary reporter does not strongly bind to the fragmentary reporter on the individual hairpin, but following HCR polymerization, adjacent hairpins in the HCR amplified polymer colocalize with a complete reporter, and the colocalized complete reporter (also referred to as the complete reporter) strongly binds to the readout probe labeled with the auxiliary reporter (e.g., Figures 17D and 27B). In some embodiments, the readout probe includes one or more auxiliary reporters and further includes a reporter-binding domain configured to bind to a reporter on the HCR amplified polymer, or to bind to a colocalized complete reporter (also referred to as the complete reporter) in the HCR amplified polymer (e.g., see the readout probes in Figures 27A–27B). In some embodiments, the amplified signal is generated by one or more reporters or auxiliary reporters associated with the HCR amplified polymer linked to a target in the sample. In some embodiments, the signal is removed from the sample. In some embodiments, the HCR signal is generated, detected, and removed from the sample one or more times.

[0022] In some embodiments, the HCR amplifier includes (see, for example, Figures 1A and 15): 1) a first HCR hairpin (h1) comprising a first input domain (sequence domain "ab") comprising a first toehold (sequence domain "a") and a first stem section (sequence domain "b"), and a first output domain (sequence domain "c*-b*") comprising a first loop (sequence domain "c*") and a partner of the first stem section (sequence domain "b*") configured to couple to the first stem section; and 2) a second HCR hairpin (h2) comprising a second input domain (sequence domain "bc") comprising a second toehold (sequence domain "c") and a second stem section (sequence domain "b"), and a second output domain (sequence domain "b*-a*") comprising a second loop (sequence domain "a*") and a partner of the second stem section (sequence domain "b*") configured to couple to the second stem section. In some embodiments, a first initiator (i1; sequence domain "b*-a*") includes a partner of the first toehold (sequence domain "a*") and a partner of the first stem section (sequence domain "b*"). In some embodiments, the first initiator is configured to bind to a first input domain. In some embodiments, when the first initiator binds to the first input domain, the first HCR hairpin opens, exposing the first output domain. In some embodiments, the exposed first output domain is configured to bind to a second input domain. In some embodiments, when the exposed first output domain is bound to the second input domain, the second HCR hairpin opens, exposing the second output domain. In some embodiments, the exposed second output domain is configured to bind to the first input domain. When the second output domain is coupled to the first input domain, HCR polymerization occurs, and the first and second HCR hairpins open sequentially and are alternately added to the growing polymer.

[0023] In some embodiments, a second initiator (i2; domain "c*-b*") is configured to bind to a second input domain. In some embodiments, when the second initiator binds to the second input domain, a second HCR hairpin opens, exposing a second output domain. In some embodiments, the exposed second output domain is configured to bind to a first input domain. In some embodiments, when the exposed second output domain binds to the first input domain, a first HCR hairpin opens, exposing a first output domain. In some embodiments, the exposed first output domain is configured to bind to a second input domain. In some embodiments, when the exposed first output domain binds to the second input domain, HCR polymerization occurs, opening the second and first HCR hairpins and alternately adding them to the growing polymer.

[0024] In some embodiments, the first and second HCR hairpins are kinetically captured so as not to polymerize in the absence of the first or second HCR initiator.

[0025] Further information relating to HCR, fragmentary initiators, and their use is contained in U.S. Patent No. 10,450,599, filed June 30, 2017, titled "Fractional Initiator Hybridization Chain Reaction," which is incorporated herein by reference in its entirety. Further details regarding HCR and its use are described in U.S. Patent Application Publication No. 2022 / 0282300 (A1), filed on 22 February 2022, entitled "Analysis of Target Molecules within a Sample via Hybridization Chain Reaction," and in PCT / US2024 / 017915 (International Publication No. 2024 / 017915), filed on 29 February 2024, entitled "Probes for Measuring Molecular Proximity in a Sample," both of which are expressly incorporated by reference in their entirety.

[0026] HCR initiators. In some embodiments, the initiator-labeled probe comprises one or more HCR initiators that can initiate an HCR polymerization cascade leading to the growth of an HCR-amplified polymer linked to the initiators. In some embodiments, the initiator is fully complementary to the input domain of the HCR hairpin, so that it hybridizes with the input domain of the hairpin to open the hairpin and initiate the HCR polymerization cascade. In some embodiments, the initiator is fragmentarily complementary to the input domain of the HCR hairpin, but is sufficiently complementary to hybridize with the input domain of the hairpin to open the hairpin and initiate the HCR polymerization cascade. In some embodiments, the initiator is shorter or longer than the input domain of the HCR hairpin and / or has incomplete complementarity with the input domain of the hairpin, but can still hybridize with the input domain of the hairpin to open the hairpin and initiate the HCR polymerization cascade. In some embodiments, the HCR initiator has 50%, 60%, 70%, 80%, 90%, or 100% (or any intermediate value between these) complementarity to the input domain of the HCR hairpin, hybridizes to the input domain of the hairpin to open the hairpin, and initiates the HCR polymerization cascade. In some situations, initiator-labeled probes containing one or more initiators may cause an increase in background due to the nonspecific binding of the initiators to DNA, RNA, proteins, or other molecules in the sample. In some embodiments, the initiators on the initiator-labeled probe undergo base pairing. This protects the probe from nonspecific binding within the sample (and thus reduces background noise). In some embodiments, the initiator may be protected by a hairpin structure. In some embodiments, the initiator may be protected by one or more auxiliary oligos. In some embodiments, the initiator may be protected by self-complementarity and / or complementarity to one or more auxiliary chains within the oligo containing the initiator.

[0027] Automated background suppression by HCR fragmentary initiator probes. In some embodiments, fragmentary initiator probes automatically suppress background because the HCR initiator is divided into a pair of probes (see, for example, Figures 2–4, 33, 38, 45, 49, 58C, 61–63, and 68A–68I). In some embodiments, if the probe specifically binds to the target at a proximal cognitive binding site, the target colocalizes the two probes within the probe pair to form a colocalized complete HCR initiator (also referred to as a complete HCR initiator, complete initiator, or colocalized complete initiator). In some embodiments, individual fragmentary initiator probes that bind nonspecifically do not trigger HCR because each probe carries only a portion of the HCR initiator. HCR signal amplification is triggered only when a complete HCR initiator is colocalized.

[0028] Automatic background suppression by HCR hairpins. In some embodiments, HCR hairpins are kinetically captured so as not to polymerize in the absence of HCR initiators, thus automatically suppressing background. In some embodiments, if both probes in a fragmentary initiator probe pair specifically bind to a proximal cognitive binding site on the target, the resulting colocalized complete HCR initiator triggers the growth of the linked HCR-amplified polymer (see, e.g., Figure 2A). In some embodiments, individual HCR hairpins that bind nonspecifically are kinetically captured and therefore do not trigger HCR.

[0029] Automated background suppression using HCR fragment initiator probes and HCR hairpins. In some embodiments, a combination of an HCR fragment initiator probe for target detection and an HCR amplification hairpin for signal amplification provides automated background suppression throughout the protocol, ensuring that amplified background is not generated even if the reagent binds nonspecifically.

[0030] HCR with coordinated probe junction Several embodiments of the cooperative probe junction are shown in Figures 28A-28B, 29, 30, 61A-61E, 62A-62D, 63A-63E, 68A-68C, and 69A-69B. In some embodiments, a target is detected using a probe set comprising one or more probe units, each containing two or more fragmentary initiator probes. In some embodiments, a fragmentary initiator probe comprises a target binding region, a linker region, and a fragmentary initiator. In some embodiments, the target binding region on the probe within the probe unit is configured to bind to overlapping or non-overlapping binding sites on the target (Figures 28A-28B, 29, and 30). In some embodiments, the linker regions on the probes within the probe unit are configured to bind to each other (Figures 61A-61E, 62A-62D, 63A-63E). In some embodiments, fragmentary initiators on the probe within each probe unit are configured to bind to overlapping or non-overlapping binding sites on the HCR hairpin (Figures 28A-28B, 29, and 30). In some embodiments, the probe within the probe unit binds to the corresponding cognitive binding site on the target, as needed. The linker regions on the probes within the probe unit bond to each other, forming a cooperative probe junction. This facilitates binding to the target and co-localization of the complete HCR initiator, efficiently triggering HCR signal amplification. In some embodiments, the co-localized complete initiator initiates polymerization of the HCR monomer, thereby generating a signal. In some embodiments, the cooperative probe junction enhances the intensity of HCR signal amplification.

[0031] In some embodiments, the cooperative probe junction includes two or more fragmentary initiator probes bound to the proximity domain by a proximity probe (see, for example, Figures 68A–68I and Figures 69A–69B). In some embodiments, the cooperative probe junction is used to perform proximity measurements within a sample. In some embodiments, the fragmentary initiator probe includes a fragmentary initiator (also referred to as an HCR fragmentary initiator), a proximity domain, and a target-binding domain configured to bind directly to a target (see, for example, Figures 68A, 68E, 68G, and 69A). In some embodiments, the fragmentary initiator probe includes a fragmentary initiator (also referred to as an HCR fragmentary initiator), a proximity domain, and a target-binding domain configured to bind indirectly to a target (see, for example, Figures 68B, 68C–68I, and 69B). In some embodiments, different probe compositions are used for different targets within or adjacent to the complex (e.g., Figures 68E-68I). In some embodiments, the adjacent domain is an array within a fragmentary initiator probe configured to be bound by the adjacent probe. In some embodiments, the fragmentary initiator is an array within a fragmentary initiator probe that cannot trigger HCR signal amplification on its own, but can form a cooperative probe junction that facilitates the formation of a co-localized complete HCR initiator (also referred to as a complete HCR initiator, complete initiator, or co-localized complete initiator) when co-localized with one or more fragmentary initiators from one or more other fragmentary initiator probes, for example, by binding the fragmentary initiator probe to one or more adjacent probes, thereby triggering HCR signal amplification. In some embodiments, the co-localized complete initiator initiates polymerization of HCR monomers, thereby generating a signal. In some embodiments, the fragmentary initiator probe further includes linker regions, and the linker regions on the probes within the probe unit are configured to connect to each other as needed.

[0032] In some embodiments, the first fragmentary initiator signal probe includes a first target-binding domain, a first proximity domain, and a first fragmentary initiator that binds directly or indirectly to a first target; the second fragmentary initiator signal probe includes a second target-binding domain, a second proximity domain, and a second fragmentary initiator that binds directly or indirectly to a second target, and the proximity probe is configured to bind the first proximity domain and the second proximity domain (see, for example, Figures 68A-68I and 69A-69B). In some embodiments, the first fragmentary initiator probe may further include a first linker region, and the second fragmentary initiator probe may further include a second linker region. In some embodiments, the first linker region is configured to bind to the second linker region. In some embodiments, the first linker region is coupled to the second linker region when the first and second linker regions are in close proximity to each other. In some embodiments, when a proximity probe is coupled to the first and second proximity domains, the first and second fragmentary initiators colocalize. In some embodiments, when a proximity probe is coupled to the first and second proximity domains, a cooperative probe junction is formed that facilitates the colocalization of the first and second fragmentary initiators, resulting in a complete HCR initiator (also referred to as a complete initiator) that can trigger HCR signal amplification. (These are colocalized.)

[0033] In some embodiments, the first and second targets are sufficiently close to each other, and when the first fragmentary initiator probe binds to the first target and the second fragmentary initiator probe binds to the second target, the proximity probes can bind to both the first proximity domain of the first fragmentary initiator probe and the second proximity domain of the second fragmentary initiator probe to form a cooperative probe junction. In some embodiments, the first and second targets are bound to each other within the target complex (see, for example, Figures 68A–68B, 68D–68I, and 69A–69B). In some embodiments, the first and second targets do not form a complex with each other but do form a complex with a third target. In some embodiments, the first and second targets are close to each other but are not bound to each other. In some embodiments, the first and second targets are the same molecule (see, for example, Figure 68C).

[0034] In some embodiments, the first fragmentary initiator probe, the second fragmentary initiator probe, and the proximity probe form a coordinated probe junction (see, for example, Figures 69A-69B). In some embodiments, the triggering of HCR occurs as a result of the formation of the coordinated probe junction and the co-localization of the first and second fragmentary initiators via the proximity probe, thus indicating that the first and second targets are in close proximity to each other within the sample.

[0035] HCR using reporter-labeled signal probes Some embodiments of reporter-labeled signal probes are shown in Figures 37B, 56A-56N, 57A-57B, 58A-58B, 59A-59F, and 60A-60D. In some embodiments, the reporter-labeled signal probe binds directly to the target (e.g., Figures 37B, 56A-56B, 56E-56J, 56M-56N, 57A-57B, 58A-58B, 59A-59F, 68D, and 68F-68I). In some embodiments, the target is bound indirectly by the reporter-labeled signal probe (e.g., Figures 56C-56D and 56K-56L). In some embodiments, the reporter-labeled signal probe includes one or more reporters (e.g., Figures 56A-56N, 57A-57B, 58A-58B, 59A-59F, 68D, and 68F-68I). In some embodiments, the reporter-labeled signal probe includes multiple reporters (e.g., Figures 56B, 56D, 56F, 56J, 56N, 57A-57B, and 58B). In some embodiments, the target is detected by an anti-target reporter-labeled signal probe (e.g., Figures 37B, 56A-56N, 57A-57B, 58A-58B, 59A-59F, 60A-60D, 68D, and 68F-68I) (e.g., an anti-target reporter-labeled primary antibody; Figures 56I-56J, 59C-59D, 60A-60D). This is detected by an anti-reporter initiator-labeled signal probe (e.g., Figures 58A-58B, 59A-59F, 60A-60D, 68D, and 68F-68I) (e.g., an anti-reporter initiator-labeled primary antibody; Figures 58A, 59C, and 60A-60D), or by two or more anti-reporter fragment initiator probes (e.g., Figure 58B) (e.g., two or more anti-reporter fragment initiator primary antibodies).

[0036] In some embodiments, using a reporter-labeled signal probe eliminates the need to conjugate one or more initiators to an anti-target signal probe containing a target-binding region, thereby eliminating a potential source of probe / target binding interference. Another strategy to avoid conjugation between initiators and anti-target signal probes is to use an unmodified anti-target primary antibody signal probe, and then use an initiator-labeled secondary antibody to conjugate the primary antibody The goal is to detect the target. However, this approach presents challenges in multiplex imaging of multiple different targets within the same sample. This is because each anti-target primary antibody needs to be of a different isotype or produced in a different host organism to enable specific detection by different secondary antibodies, or because the probe needs to be removed from the sample after imaging each target and before imaging the next target. In some embodiments, using reporter-labeled signal probes avoids these difficulties and allows for easy multiplexing because the anti-target reporter-labeled signal probe can be detected by the anti-reporter initiator-labeled signal probe (e.g., Figures 58A, 59A-59F, 60A-60D). For example, in some embodiments, the target is detected by an anti-target reporter-labeled primary antibody (Figures 56I-56J, 59C-59D, 60A-60D), which is then detected by an anti-reporter initiator-labeled primary antibody (Figures 58A, 59C, and 60A-60D). In particular, in some embodiments, a two-layer primary antibody is used, and no secondary antibody is used. In some embodiments, one of the two layers of the primary antibody can be a primary nanobody (Figures 59B, 59D-59F). Multiplexing with reporter-labeled signal probes allows all targets to be detected simultaneously by different anti-target primary antibodies (or anti-target primary nanobody) that are of the same isotype or produced in the same host organism and labeled with different reporters (Figures 60A-60D). All of these reporters can be detected simultaneously by different anti-reporter initiator-labeled primary antibodies (or anti-reporter initiator-labeled primary nanobody), and the initiator sequence is different for each target (Figures 60A-60D). In some embodiments, the reporter-labeled signal probe enhances probe / target binding and facilitates easy multiplexing.

[0037] In some embodiments, the target is detected using an anti-target reporter labeled primary signal probe (e.g., Figures 58A-58B, 59A-59F, 60A, 60C). In some embodiments, the anti-target reporter labeled primary signal probe includes multiple reporters (e.g., Figures 56B, 56D, 56F, 56J, 56N, 57A-57B, 58B). In some embodiments, the anti-target reporter labeled primary signal probe is detected by an anti-reporter initiator labeled secondary signal probe (e.g., Figures 58A, 59A-59F, 60A, 60C). In some embodiments, the anti-target reporter labeled primary signal probe is detected by an anti-reporter fragment initiator secondary signal probe (e.g., Figure 58B). In some cases, labeling an anti-target signal probe with one or more initiators may inhibit the probe's binding to the target or increase background due to nonspecific binding mediated by one or more initiators. In some embodiments, the target comprises a small RNA or another oligonucleotide, and the anti-target signal probe is a nucleic acid probe labeled with one or more initiators at one or both ends (e.g., Figure 37A), or the anti-target signal probe is a nucleic acid probe labeled with one or more reporters (e.g., Figure 37B), which are detected by an anti-reporter initiator-labeled secondary signal probe. In some embodiments, using an anti-target reporter-labeled primary signal probe and an anti-target initiator-labeled secondary signal probe increases the probe:target binding intensity compared to using an anti-target initiator-labeled signal probe. In some embodiments, using an anti-target reporter-labeled primary signal probe and an anti-reporter initiator-labeled secondary signal probe reduces background compared to using an anti-target initiator-labeled signal probe.

[0038] In some embodiments, reporter-labeled signal probes are used to perform HCR imaging with or without catalytic reporter deposition (CARD) (e.g., Figures 60A and 60C). In some embodiments, the target is detected by an anti-target reporter-labeled signal probe, and the target is detected by an anti-reporter initiator-labeled signal probe. This is done (for example, in Figures 60A and 60C). In some embodiments, HCR signal amplification is performed using an auxiliary reporter-labeled HCR amplifier to produce an auxiliary reporter-modified HCR amplification polymer linked to a target. In some embodiments, HCR signal amplification is linear (for example, in Figure 60A) or nonlinear (for example, in Figures 38A-38B, 60C, and 73). In some embodiments, an anti-auxiliary reporter enzyme-labeled readout probe is used to bind to the auxiliary reporter on the HCR amplification polymer and to mediate CARD signal amplification directly or indirectly (for example, in Figures 38A-38B and 60A). In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, the reporter labeling the anti-target signal probe is the same as the auxiliary reporter labeling the HCR amplifier. In some embodiments, CARD signal amplification produces a fluorescent or chromogenic signal.

[0039] In some embodiments, a reporter-labeled signal probe is used to perform HCR imaging on two targets within or adjacent to a complex, with or without catalytic reporter deposition (CARD). In some embodiments, two targets within or adjacent to a complex (target 1 and target 2) are detected using an anti-target 1 reporter 1-labeled signal probe to bind to target 1 and an anti-target 2 reporter 2-labeled signal probe to bind to target 2 (e.g., Figures 58C and 68D). In some embodiments, reporter 1 is then detected using an anti-reporter 1 signal probe containing a fragmentary initiator 1 and an adjacent domain 1, and reporter 2 is detected using an anti-reporter 2 signal probe containing a fragmentary initiator 2 and an adjacent domain 2 (e.g., Figures 58C and 68D). In some embodiments, proximity domains 1 and 2 are close enough to simultaneously bind to the proximity probe and colocalize a complete HCR initiator i1 capable of triggering HCR signal amplification only when targets 1 and 2 are in or near each other within the complex (see, e.g., Figures 58C and 68D). In some embodiments, HCR signal amplification is performed using an auxiliary reporter-labeled hairpin to generate a linked fluorescent amplification polymer. In some embodiments, HCR signal amplification is linear (e.g., Figure 60A) or nonlinear (e.g., Figures 38A-38B and 73). In some embodiments, the auxiliary reporter is a hapten, fluorophore, chromophore, or rare earth element or compound. In some embodiments, the auxiliary reporter mediates CARD signal amplification. In some embodiments, an anti-auxiliary reporter enzyme-labeled readout probe is used to bind to the auxiliary reporter on the HCR amplification polymer and mediate CARD signal amplification directly or indirectly. In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, a fluorescent signal or a chromogenic signal is generated by CARD signal amplification.

[0040] In some embodiments, with or without catalytic reporter deposition (CARD), HCR imaging is performed for two or more targets within or adjacent to a complex, using one or more proximity probes in combination with optionally different signal probe compositions for each target within or adjacent to the complex. At least one target within or adjacent to the complex is detected using a reporter-labeled signal probe, which is then detected by an anti-reporter signal probe (e.g., Figures 68F-68I). In some embodiments, the targets within or adjacent to the complex may be the same type of molecule or different types of molecules. For example, in the case of three targets within or adjacent to the complex, one target may be a protein, one target may be RNA, and one target may be DNA, or all three targets may be proteins, or two targets may be proteins and one target may be RNA. For example, in the case of two targets within or adjacent to the complex, one target may be a protein and one target may be RNA. It is possible that one target is a protein and the other is DNA, or that one target is RNA and the other is DNA, or that both targets are proteins, or that both targets are RNA targets, or that both targets are DNA targets, or that one target is a protein and the other is a small molecule target, or that one target is a protein target and the other is a non-protein target, and so on.

[0041] In some embodiments, two targets (target 1 and target 2) located within or in proximity to each other are detected using an anti-target 1 signal probe bound to target 1 and an anti-target 2 reporter 2 labeled signal probe bound to target 2 (e.g., Figure 68F). ​​In some embodiments, anti-target 1 is then detected using an anti-anti-target 1 signal probe containing a fragmentary initiator 1 and a proximity domain 1, and reporter 2 is detected using an anti-reporter 2 signal probe containing a fragmentary initiator 2 and a proximity domain 2 (e.g., Figure 68F). ​​In some embodiments, proximity domains 1 and 2 are close enough to simultaneously bind to the proximity probe only when targets 1 and 2 are within or in proximity to each other, so that a complete HCR initiator i1 capable of triggering HCR signal amplification colocalizes (see e.g., Figure 68F). ​​In some embodiments, HCR signal amplification is performed using an auxiliary reporter labeled hairpin to generate a linked fluorescent amplification polymer. In some embodiments, HCR signal amplification is linear (e.g., Figure 60A) or nonlinear (e.g., Figures 38A-38B and Figure 73). In some embodiments, the auxiliary reporter is a hapten, fluorophore, chromophore, or rare earth element or compound. In some embodiments, the auxiliary reporter mediates CARD signal amplification. In some embodiments, an anti-auxiliary reporter enzyme-labeled readout probe is used to bind to the auxiliary reporter on the HCR amplification polymer and to mediate CARD signal amplification directly or indirectly. In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, CARD signal amplification generates a fluorescent or chromogenic signal.

[0042] In some embodiments, two targets (target 1 and target 2) located within or in proximity to the complex are detected using an anti-target 1 signal probe that binds to target 1, comprising a fragmentary initiator 1 and a proximity domain 1, and an anti-target 2 reporter 2 labeled signal probe that binds to target 2 (e.g., Figure 68G). In some embodiments, reporter 2 is detected using an anti-reporter 2 signal probe comprising a fragmentary initiator 2 and a proximity domain 2 (e.g., Figure 68G). In some embodiments, proximity domains 1 and 2 are close enough to simultaneously bind to the proximity probe and colocalize a complete HCR initiator i1 that can trigger HCR signal amplification only when targets 1 and target 2 are within or in proximity to the complex (see e.g., Figure 68G). In some embodiments, HCR signal amplification is performed using an auxiliary reporter labeled hairpin to generate a coupled fluorescent amplification polymer. In some embodiments, HCR signal amplification is linear (e.g., Figure 60A) or nonlinear (e.g., Figures 38A-38B and Figure 73). In some embodiments, the auxiliary reporter is a hapten, fluorophore, chromophore, or rare earth element or compound. In some embodiments, the auxiliary reporter mediates CARD signal amplification. In some embodiments, an anti-auxiliary reporter enzyme-labeled readout probe is used to bind to the auxiliary reporter on the HCR amplification polymer and to directly or indirectly mediate CARD signal amplification. In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, CARD signal amplification generates a fluorescent or chromogenic signal.

[0043] In some embodiments, two targets (target 1 and target 2) located within or in close proximity to each other are The targets are detected using an anti-target 1 signal probe that binds to target 1 and an anti-target 2 reporter 2-labeled signal probe that binds to target 2 (e.g., Figure 68H). In some embodiments, anti-target 1 is then detected using an anti-anti-target 1 signal probe containing a fragmentary initiator 1 and a neighboring domain 1, and reporter 2 is detected using an anti-reporter 2 signal probe (e.g., Figure 68H). In some embodiments, anti-reporter 2 is detected using an anti-anti-reporter 2 signal probe containing a fragmentary initiator 2 and a neighboring domain 2 (e.g., Figure 68H). In some embodiments, neighboring domains 1 and 2 are close enough to bind simultaneously to the neighboring probe only if targets 1 and 2 are in the complex or in proximity, and a complete HCR initiator i1 that can trigger HCR signal amplification colocalizes (see e.g., Figure 68H). In some embodiments, HCR signal amplification is performed using an auxiliary reporter-labeled hairpin to generate a linked fluorescent amplification polymer. In some embodiments, HCR signal amplification is linear (e.g., Figure 60A) or nonlinear (e.g., Figures 38A-38B and Figure 73). In some embodiments, the auxiliary reporter is a hapten, fluorophore, chromophore, or rare earth element or compound. In some embodiments, the auxiliary reporter mediates CARD signal amplification. In some embodiments, an anti-auxiliary reporter enzyme-labeled readout probe is used to bind to the auxiliary reporter on the HCR amplification polymer and to mediate CARD signal amplification directly or indirectly. In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, CARD signal amplification generates a fluorescent or chromogenic signal.

[0044] In some embodiments, two targets (target 1 and target 2) located within or in proximity to the complex are detected using an anti-target 1 reporter 1-labeled signal probe that binds to target 1 and an anti-target 2 reporter 2a-labeled signal probe that binds to target 2 (e.g., Figure 68I). In some embodiments, reporter 1 is then detected using an anti-reporter 1 signal probe containing a fragmentary initiator 1 and a proximity domain 1, and reporter 2a is detected using an anti-reporter 2a reporter 2b-labeled signal probe (e.g., Figure 68I). In some embodiments, reporter 2b is detected using an anti-reporter 2b signal probe containing a fragmentary initiator 2 and a proximity domain 2 (e.g., Figure 68I). In some embodiments, proximity domains 1 and 2 are close enough to simultaneously bind to the proximity probes and colocalize a complete HCR initiator i1 that can trigger HCR signal amplification only when targets 1 and target 2 are within or in proximity to the complex (see e.g., Figure 68I). In some embodiments, HCR signal amplification is performed using an auxiliary reporter-labeled hairpin to generate a linked fluorescent amplification polymer. In some embodiments, HCR signal amplification is linear (e.g., Figure 60A) or nonlinear (e.g., Figures 38A-38B and Figure 73). In some embodiments, the auxiliary reporter is a hapten, fluorophore, chromophore, or rare earth element or compound. In some embodiments, the auxiliary reporter mediates CARD signal amplification. In some embodiments, an anti-auxiliary reporter enzyme-labeled readout probe is used to bind to the auxiliary reporter on the HCR amplification polymer and mediate CARD signal amplification directly or indirectly. In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, CARD signal amplification generates a fluorescent or chromogenic signal.

[0045] In some embodiments, a reporter-labeled signal probe is used for HCR imaging by CARD against N targets in a sample (where N is a positive integer; see, for example, the reagent in Figure 60B). In some embodiments, the j-th target (j=1, ..., N, where j is a positive integer) is detected by the j-th anti-target signal probe containing the j-th reporter (see, for example, Figure 60A). In some embodiments, the j-th reporter is The j-th target is detected by a j-th anti-reporter signal probe containing the j-th HCR initiator (see, e.g., Figure 60A). In some embodiments, signal amplification of the j-th target is provided by a j-th HCR amplifier labeled with a j-th auxiliary reporter (see, e.g., Figure 60A). In some embodiments, HCR signal amplification of the j-th target is linear HCR signal amplification (e.g., Figure 60A) or nonlinear HCR signal amplification (e.g., Figures 38A-38B and Figure 73). In some embodiments, the j-th auxiliary reporter is the j-th fluorophore. In some embodiments, the j-th auxiliary reporter mediates CARD signal amplification for the j-th target. In some embodiments, a j-th anti-auxiliary reporter readout probe containing the j-th enzyme is used to bind to the j-th auxiliary reporter on the j-th HCR amplification polymer and to mediate CARD signal amplification directly or indirectly for the j-th target. In some embodiments, the j-th readout probe acts on the j-th CARD substrate to catalyze the deposition of the j-th CARD reporter in the vicinity of the j-th target. In some embodiments, the same enzyme is used to mediate CARD for all targets. In some embodiments, CARD signal amplification generates a j-th fluorescent signal or j-th chromogenic signal for the j-th target.

[0046] HCR by reporter-labeled probe and nonlinear HCR signal amplification In some embodiments, a reporter-labeled signal probe is used to perform HCR imaging by nonlinear HCR signal amplification (e.g., Figure 60C). In some embodiments, a target is detected by an anti-target signal probe containing a reporter, and then by an anti-reporter signal probe containing a first HCR initiator (e.g., Figure 60C). In some embodiments, in a linear amplification step, the HCR signal is amplified by an auxiliary reporter-labeled first HCR amplifier using the first HCR initiator, triggering the growth of an auxiliary reporter-modified HCR amplification polymer linked to the target (e.g., Figures 38A and 60C). In some embodiments, bridging to a nonlinear amplification step is provided by an anti-auxiliary reporter bridging probe containing a second HCR initiator (e.g., Figures 38A and 60C). In some embodiments, the bridging probe includes a primary bridging probe containing an anti-auxiliary reporter domain and a secondary bridging probe containing a second HCR initiator (e.g., Figures 38A, 45, and 47A). In some embodiments, the bridging probe directly or indirectly bridges the reporter and the second HCR initiator. In some embodiments, in the nonlinear amplification stage, the second HCR initiator is used to amplify the HCR signal by a tertiary reporter-labeled second HCR amplifier, triggering the growth of the tertiary reporter-modified HCR amplifier polymer linked to the HCR amplifier polymer from the linear amplification stage (e.g., Figures 38A and 60C). In some embodiments, the reporter on the signal probe, the auxiliary reporter on the first HCR amplifier, and / or the tertiary reporter on the second HCR amplifier are the same. In some embodiments, the first HCR initiator on the signal probe and the second HCR initiator on the bridging probe are the same. In some embodiments, the first HCR amplifier in the linear stage and the second HCR amplifier in the nonlinear stage are the same.In some embodiments, additional rounds of bridging and nonlinear HCR amplification are performed (e.g., an additional 1 round of bridging and HCR signal amplification, an additional 2 rounds of bridging and HCR signal amplification, or an additional M rounds of bridging and HCR signal amplification; see, for example, Figure 73). In some embodiments, multiple HCR signal amplifications are performed using the same bridging probe and HCR amplifier. In some embodiments, the tertiary reporter is a fluorophore (e.g., Figure 60C). In some embodiments, the tertiary reporter mediates CARD signal amplification (e.g., Figure 38A). In some embodiments, an anti-auxiliary reporter enzyme-labeled readout probe is used to bind to the tertiary reporter on the HCR amplification polymer and to directly or indirectly mediate CARD signal amplification (e.g., (For example, Figures 38A-38B). In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, the anti-target signal probe includes an antibody, nanobody, oligonucleotide, or a molecule containing a target-binding domain. In some embodiments, the anti-reporter signal probe includes an antibody, nanobody, nucleic acid, or a molecule containing a reporter-binding domain. In some embodiments, the target is a protein, and HCR imaging is used to perform HCR immunohistochemistry (IHC) or immunofluorescence (IF) with or without CARD. In some embodiments, the target is a nucleic acid (for example, Figures 37B, 56G-56H, and 59A-59B), and HCR imaging is used to perform HCR in situ hybridization (ISH) with or without CARD. In some embodiments, CARD signal amplification generates a fluorescent or chromogenic signal.

[0047] In some embodiments, N reporter-labeled signal probes are used for HCR imaging of N targets (N is a positive integer) in a sample using nonlinear HCR signal amplification (see reagents in Figure 60D, e.g.). In some embodiments, the j-th target (j=1, ..., N, j is a positive integer) is detected by the j-th anti-target signal probe containing the j-th reporter. In some embodiments, the j-th reporter is detected by the j-th anti-reporter signal probe containing the j-th first HCR initiator. In some embodiments, in the linear amplification stage, signal amplification for the j-th target is provided by the j-th first HCR amplifier containing the j-th auxiliary reporter, and the growth of the j-th auxiliary reporter-modified HCR amplification polymer linked to the j-th target is initiated using the j-th first HCR initiator. In some embodiments, bridging to the nonlinear amplification stage is provided by the j-th anti-auxiliary reporter bridging probe containing the j-th second HCR initiator. In some embodiments, the j-th bridging probe includes a j-th primary bridging probe containing a j-th anti-auxiliary reporter domain and a j-th secondary bridging probe containing a j-th second HCR initiator (e.g., Figures 38A, 45, and 47A). In some embodiments, the j-th bridging probe directly or indirectly bridges the j-th reporter and the j-th second HCR initiator. In some embodiments, the nonlinear amplification step involves HCR signal amplification of the j-th target by the j-th second HCR amplifier containing the j-th tertiary reporter using the j-th second HCR initiator, triggering the growth of the j-th tertiary reporter-modified HCR amplifying polymer linked to the j-th HCR amplifying polymer from the linear amplification step (e.g., Figures 38A and 60C). In some embodiments, the j-th reporter on the j-th signal probe, the j-th auxiliary reporter on the j-th first HCR amplifier, and / or the j-th tertiary reporter on the j-th second HCR amplifier are the same.In some embodiments, the j-th first HCR initiator supported by the j-th signal probe and the j-th second HCR initiator supported by the j-th bridging probe are the same. In some embodiments, the j-th first HCR amplifier in the linear stage and the j-th second HCR amplifier in the nonlinear stage are the same. In some embodiments, additional rounds of bridging and nonlinear HCR signal amplification are performed (e.g., 1 round of additional bridging and HCR signal amplification, 2 rounds of additional bridging and HCR signal amplification, or M rounds of additional bridging and HCR signal amplification). In some embodiments, the same j-th bridging probe and j-th HCR amplifier are used for multiple rounds of HCR signal amplification against the j-th target. In some embodiments, the j-th tertiary reporter is the j-th fluorophore (e.g., Figure 60D). In some embodiments, the j-th tertiary reporter mediates CARD signal amplification against the j-th target (e.g., Figure 38A). In some embodiments, a j-th anti-tertiary reporter readout probe containing the j-th enzyme binds to the j-th tertiary reporter on the j-th HCR amplification polymer, directly or indirectly to the j-th target. It is used to mediate CARD signal amplification. In some embodiments, the j-th readout probe acts on the j-th CARD substrate and catalyzes the deposition of the j-th CARD reporter in the vicinity of the j-th target. In some embodiments, the same enzyme is used to mediate CARD for all targets. In some embodiments, CARD signal amplification generates the j-th fluorescent signal or j-th chromogenic signal for the j-th target. In some embodiments, the j-th anti-target signal probe comprises the j-th antibody, the j-th nanobody, the j-th nucleic acid, or the j-th molecule containing the j-th target binding domain. In some embodiments, the j-th anti-reporter signal probe comprises the j-th antibody, the j-th nanobody, the j-th nucleic acid, or the j-th molecule containing the j-th reporter binding domain. In some embodiments, the j-th target is a protein, and HCR imaging is used to perform HCR immunohistochemistry (IHC) or immunofluorescence (IF) with or without CARD. In some embodiments, the j-th target is nucleic acid (e.g., Figures 37B, 56G-56H, and 59A-59B), and HCR imaging is used to perform HCR in situ hybridization (ISH) with or without CARD.

[0048] Nonlinear HCR signal amplification Several embodiments of nonlinear HCR signal amplification using CARD for HCR RNA-ISH are shown in Figures 38A and 38B. Several embodiments of enzyme-free nonlinear HCR signal amplification for HCR RNA-FISH are shown in Figures 45, 47A-47B, and 49A-49B. Several embodiments of nonlinear HCR signal amplification for HCR IF are shown in Figures 51A, 52A-52B, and 54. In some embodiments, ultra-high sensitivity detection is achieved by using branched HCR amplification polymers (Figures 38A, 45, 47A-47B, 49A-49B, 51A, 52A-52B, and 54), where the probe triggers the self-assembly of reporter-labeled HCR hairpins to form reporter-modified HCR amplification polymers in the linear amplification stage, the anti-reporter initiator-labeled bridging probe binds to the reporter on the linear amplification polymer, and the bridging probe triggers the self-assembly of auxiliary reporter-labeled HCR hairpins to form coupled auxiliary reporter-modified branched amplification polymers in the nonlinear amplification stage. In some embodiments, ultra-high sensitivity detection is achieved by using branched HCR amplification polymers (Figures 38B, 71, and 73), where an initiator-labeled probe containing a first HCR initiator (or a fragmented initiator probe that colocalizes a complete first HCR initiator upon specific binding to the target) triggers the self-assembly of a self-bridging split initiator-tailed HCR hairpin, forming a linked HCR amplification polymer that colocalizes a complete second HCR initiator in the linear amplification stage, and the colocalized complete second HCR initiator triggers the self-assembly of a reporter-labeled HCR hairpin, forming a linked reporter-modified branched amplification polymer in the nonlinear amplification stage. In some embodiments, the split initiator tail on the HCR hairpin contains a fragmented initiator. In some embodiments, polymerization of a reporter-modified HCR amplification polymer linked to a target colocalizes a complete initiator from a split-initiator-tail type HCR hairpin, and from each colocalized fragmentary initiator, amplification of a second set of hairpins in different directions becomes possible.In some embodiments, the auxiliary reporter on the branched amplification polymer mediates catalytic reporter deposition (CARD) directly or indirectly (Figures 38A-38B).

[0049] Nonlinear HCR signal amplification using bridging probes In some embodiments, nonlinear HCR signal amplification is used to detect targets in the sample. In some embodiments, targets are detected by an initiator-labeled signal probe (e.g., Figures 52A and 71) containing a first HCR initiator. In some embodiments, targets are detected by fragments colocalizing with the complete first HCR initiator. The target is detected by a neuter signal probe when it specifically binds to the corresponding cognitive binding site on the target (e.g., Figures 38A-38B, 47A, 49A, and 71). In some embodiments, the target is a target complex (or a set of adjacent target molecules) detected using two or more fragmentary initiator probes and one or more proximity probes to colocalize the complete first HCR initiator (e.g., Figures 58C, 68AB, 68D-68I, and 69AB). In some embodiments, the target is detected by an anti-target signal probe containing a reporter, and then by an anti-reporter signal probe containing the first HCR initiator (e.g., Figure 60C). In some embodiments, the linear amplification step involves HCR signal amplification using an auxiliary reporter-labeled first HCR amplifier, and the growth of the auxiliary reporter-modified HCR amplification polymer linked to the target is triggered using a first HCR initiator (or a colocalized complete first HCR initiator) (e.g., Figures 38A, 47A, 49A, 52A, and 60C). In some embodiments, bridging to the nonlinear amplification step is provided by an anti-auxiliary reporter bridging probe containing a second HCR initiator (e.g., Figures 38A, 49A, 52A, and 60C). In some embodiments, the bridging probe includes a primary bridging probe containing an anti-auxiliary reporter domain and a secondary bridging probe containing a second HCR initiator (e.g., Figures 38A, 45, and 47A). In some embodiments, the bridging probe directly or indirectly bridges the reporter and the second HCR initiator. In some embodiments, during the nonlinear amplification stage, the HCR signal is amplified using the second HCR initiator by a tertiary reporter-labeled second HCR amplifier, triggering the growth of the tertiary reporter-modified HCR amplifier polymer linked to the HCR amplifier polymer from the linear amplification stage (e.g., Figures 38A, 47A, 49A, 52A, and 60C).In some embodiments, the desired reporter on the signal probe, the auxiliary reporter on the first HCR amplifier, and / or the tertiary reporter on the second HCR amplifier are the same. In some embodiments, the first HCR initiator supported by the signal probe and the jth second HCR initiator supported by the bridging probe are the same. In some embodiments, the arrangement of the first HCR amplifier in the linear stage and the arrangement of the second HCR amplifier in the nonlinear stage are the same. In some embodiments, additional rounds of bridging and HCR signal amplification are performed (e.g., an additional 1 round of bridging and HCR signal amplification, an additional 2 rounds of bridging and HCR signal amplification, or an additional M rounds of bridging and HCR signal amplification; see, for example, Figure 73). In some embodiments, multiple HCR signal amplifications are performed using the same bridging probe and HCR amplifier (see, for example, Figure 73). In some embodiments, the tertiary reporter is a fluorophore, chromophore, or rare earth element or compound (e.g., Figures 47A, 49A, 52A, and 60C). In some embodiments, the tertiary reporter mediates CARD signal amplification (e.g., Figure 38A). In some embodiments, an anti-co-reporter enzyme-labeled readout probe is used to bind to the tertiary reporter on the HCR amplification polymer and to mediate CARD signal amplification directly or indirectly (e.g., Figure 38A). In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, the signal probe includes antibodies, nanobodies, nucleic acids, molecules containing a target-binding domain, and / or molecules containing a reporter-binding domain. In some embodiments, the target is a protein, and HCR imaging is used to perform HCR immunohistochemistry (IHC) or immunofluorescence (IF) with or without CARD. In some embodiments, the target is a nucleic acid, and HCR imaging is used to perform HCR in situ hybridization (ISH) with or without CARD.In some embodiments, a fluorescent signal or a chromogenic signal is generated by CARD signal amplification.

[0050] Nonlinear HCR signal amplification through multi-round bridging and HCR signal amplification. In some embodiments, the target is detected using an anti-target signal probe containing a first HCR initiator (or using a fragmented initiator signal probe that colocalizes the complete first HCR initiator when it specifically binds to the corresponding cognitive binding site on the target). In some embodiments, a first round of HCR signal amplification (also referred to as a linear amplification step) is performed by a first reporter-labeled HCR amplifier using the first HCR initiator, triggering the growth of a first reporter-modified HCR amplification polymer linked to the target (see, for example, Figure 73). In some embodiments, bridging to a second round of HCR amplification is provided by an anti-first reporter bridging probe containing a second HCR initiator. In some embodiments, a second round of HCR signal amplification (also referred to as the first nonlinear amplification step or second-order amplification step) is performed by a second reporter-labeled HCR amplifier using a second HCR initiator, triggering the growth of the second reporter-modified HCR amplifier polymer linked to the first reporter-modified HCR amplifier polymer bound to the target (see, for example, Figure 73). In some embodiments, bridging to a third round of HCR amplification is provided by an anti-second reporter-bridging probe containing a third HCR initiator. In some embodiments, a third round of HCR signal amplification (also referred to as the second nonlinear amplification step or third-order amplification step) is performed by a third reporter-labeled HCR amplifier using a third HCR initiator, triggering the growth of the third reporter-modified HCR amplifier polymer linked to the second reporter-modified HCR amplifier polymer linked to the first reporter-modified HCR amplifier polymer bound to the target (see, for example, Figure 73). In some embodiments, additional bridging (using an anti-reporter initiator-labeled bridging probe) and HCR signal amplification (using a reporter-labeled HCR amplifier) ​​can be performed to increase the intensity of the signal bound to the target. For example, a total of M rounds of HCR signal amplification can be performed, where M is 2, 3, 4, 5, 10, 20, 50, or 100 rounds, or any number of rounds in between.In some embodiments, the first reporter, the second reporter, the third reporter, ..., and reporter M are all the same reporter. In some embodiments, the bridging probes used for bridging between rounds of HCR signal amplification are all the same. In some embodiments, the first HCR initiator, the second HCR initiator, the third HCR initiator, ..., and HCR initiator M have all the same sequence. In some embodiments, the first HCR amplifier, the second HCR amplifier, the third HCR amplifier, ..., and HCR amplifier M have all the same sequence. In some embodiments, the reporter supported by the HCR amplifier used in the final round of HCR signal amplification is a fluorophore. In some embodiments, the reporter supported by the HCR amplifier used in the final round of HCR signal amplification is a chromophore. In some embodiments, a reporter supported by an HCR amplifier used in the final round of HCR signal amplification directly or indirectly mediates CARD signal amplification.

[0051] Nonlinear HCR signal amplification of N targets using a bridging probe In some embodiments, N signal probes are used to detect N targets (N is a positive integer) in a sample using nonlinear HCR signal amplification (see reagents in Figures 47B, 52B, and 60D, e.g.). In some embodiments, the j-th target (j=1, ..., N, j is a positive integer) is detected by a j-th initiator-labeled signal probe containing the j-th first HCR initiator (e.g., Figures 52A and 71). In some embodiments, the j-th target is detected by a set of j-th fragmentary initiator signal probes that colocalize with the j-th complete first HCR initiator when the probe in the probe unit specifically binds to the corresponding cognitive binding site on the j-th target (e.g., Figures 38A-38B, 47A, 49A, 60D). 71). In some embodiments, the j-th target is a target complex (or set of adjacent target molecules) that is detected using a set of j-th fragmentary initiator probes and a j-th proximity probe to colocalize the j-th complete first HCR initiator (e.g., Figures 58C, 68AB, 68D-68I, and 69AB). In some embodiments, the j-th target is detected by a j-th anti-target signal probe containing a j-th reporter (e.g., Figure 60C). In some embodiments, the j-th reporter is detected by a j-th anti-reporter signal probe containing a j-th first HCR initiator. In some embodiments, in the linear amplification step, signal amplification for the j-th target is provided by a j-th first HCR amplifier containing a j-th auxiliary reporter, and the growth of a j-th auxiliary reporter-modified HCR amplification polymer linked to the j-th target is initiated using the j-th first HCR initiator. In some embodiments, bridging to a nonlinear amplification step is provided by a j-th anti-auxiliary reporter bridging probe containing a j-th second HCR initiator. In some embodiments, the j-th bridging probe includes a j-th primary bridging probe containing a j-th anti-auxiliary reporter domain and a j-th secondary bridging probe containing a j-th second HCR initiator (e.g., Figures 38A, 45, and 47A). In some embodiments, the j-th bridging probe directly or indirectly bridges the j-th reporter and the j-th second HCR initiator. In some embodiments, the nonlinear amplification step involves HCR signal amplification against the j-th target by a j-th second HCR amplifier containing a j-th tertiary reporter using the j-th second HCR initiator, triggering the growth of a j-th tertiary reporter-modified HCR amplifying polymer linked to the j-th HCR amplifying polymer from the linear amplification step (e.g., Figures 38A and 60C).In some embodiments, the j-th reporter on the j-th signal probe, the j-th auxiliary reporter on the j-th first HCR amplifier, and / or the j-th tertiary reporter on the j-th second HCR amplifier are the same. In some embodiments, the j-th first HCR initiator carried by the j-th signal probe and the j-th second HCR initiator carried by the j-th bridging probe are the same. In some embodiments, the j-th first HCR amplifier in the linear stage and the j-th second HCR amplifier in the nonlinear stage have the same sequence. In some embodiments, additional rounds of bridging and HCR signal amplification are performed (e.g., one additional round of bridging and HCR signal amplification, two additional rounds of bridging and HCR signal amplification, or M additional rounds of bridging and HCR signal amplification; see, for example, Figure 73). In some embodiments, the same j-th bridging probe and j-th HCR amplifier are used for multiple rounds of HCR signal amplification against the j-th target (see, for example, Figure 73). In some embodiments, the j-th tertiary reporter is the j-th fluorophore, the j-th chromosome, or the j-th rare earth element or compound (e.g., Figures 47A-47B, 52A-52B, 60C-60D). In some embodiments, the j-th tertiary reporter mediates CARD signal amplification for the j-th target (e.g., Figure 38A). In some embodiments, a j-th anti-tertiary reporter readout probe containing the j-th enzyme is used to bind to the j-th tertiary reporter on the j-th HCR amplification polymer and to mediate CARD signal amplification directly or indirectly for the j-th target. In some embodiments, the j-th readout probe acts on the j-th CARD substrate to catalyze the deposition of the j-th CARD reporter near the j-th target. In some embodiments, the same enzyme is used to mediate CARD for all targets. In some embodiments, CARD signal amplification generates the j-th fluorescent signal or j-th chromogenic signal for the j-th target.In some embodiments, the j-th signal probe comprises a j-th antibody, a j-th nanobody, a j-th nucleic acid, a j-th molecule containing a j-th target-binding domain, or a j-th molecule containing a j-th reporter-binding domain. In some embodiments, the j-th target is a protein, and HCR imaging is performed. It is used to perform HCR immunohistochemistry (IHC) or immunofluorescence (IF) with or without CARD. In some embodiments, the j-th target is a nucleic acid, and HCR imaging is used to perform HCR in situ hybridization (ISH) with or without CARD.

[0052] In some embodiments, HCR RNA-FISH / IF is performed using the following four-step protocol (e.g., Figure 54) for enzyme-free nonlinear HCR signal amplification: 1) Protein detection step: Incubate all anti-target initiator-labeled signal probes and wash optionally; 2) RNA detection step: Incubate all fragmentary initiator signal probe sets and wash optionally; 3) Linear amplification step for all RNA and protein targets: Incubate all reporter-labeled HCR amplifiers and wash optionally; 4) Nonlinear amplification step for all RNA and protein targets: Incubate all anti-reporter initiator-labeled bridging probes and wash optionally; Incubate all auxiliary reporter-labeled HCR amplifiers and wash optionally.

[0053] In some embodiments, HCR imaging of protein targets, protein:protein target complexes, and RNA targets is performed by enzyme-free nonlinear HCR signal amplification using the following five-step protocol (e.g., Figure 70): 1) Protein detection step: Incubate all anti-target primary signal probes and wash optionally; incubate all anti-target initiator-labeled signal probes and wash optionally (or incubate all anti-target initiator-labeled signal probes and wash optionally); 2) Proximity step: Incubate all proximity probes and wash optionally; 3) RNA detection step: Incubate all fragmentary initiator signal probe sets and wash optionally; 4) Linear amplification step for all targets: Incubate all reporter-labeled HCR amplifiers and wash optionally; 5) Nonlinear amplification step for all RNA and protein targets: Incubate all anti-reporter initiator-labeled bridging probes and wash optionally, and incubate all auxiliary reporter-labeled HCR amplifiers and wash optionally.

[0054] Nonlinear HCR signal amplification using self-bridging hairpins In some embodiments, nonlinear HCR signal amplification is used to detect targets in a sample. In some embodiments, targets are detected by an initiator-labeled signal probe containing a first HCR initiator (e.g., Figures 52A and 71). In some embodiments, targets are detected when they specifically bind to the corresponding cognitive binding site on the target by two or more fragmentary initiator signal probes that colocalize with a complete first HCR initiator (e.g., Figures 38A–38B, 47A, 49A, and 71). In some embodiments, the target is a target complex (or a set of adjacent target molecules) detected using two or more fragmentary initiator probes and one or more proximity probes to colocalize with a complete first HCR initiator (e.g., Figures 58C, 68AB, 68D–68I, and 69AB). In some embodiments, the target is a target complex (or set of adjacent target molecules) detected using two or more fragmentary initiator probes, and the interaction between linking regions on the fragmentary initiator probes colocalizes a complete first HCR initiator. In some embodiments, the target is detected by an anti-target signal probe containing a reporter, and then by an anti-reporter signal probe containing a first HCR initiator (e.g., Figure 60C). In some embodiments, the linear amplification step involves HCR signal amplification using a self-bridging first HCR amplifier containing two or more hairpins, each containing a split initiator tail, and the first HCR initiator (or colocalized complete first HCR initiator) is used within the amplified polymer. This triggers the growth of an HCR-amplifying polymer linked to a target that colocalizes a complete second HCR initiator (e.g., Figures 38B and 71). In some embodiments, the self-bridging first HCR amplifier comprises two HCR hairpins (e.g., Figure 38), each hairpin containing 0, 1, or 2 split initiator tails. In some embodiments, the self-bridging first HCR amplifier comprises four HCR hairpins (e.g., Figures 16C–16D and 71), each hairpin containing 0, 1, or 2 split initiator tails. In some embodiments, the self-bridging first HCR amplifier comprises four HCR hairpins h1, h2, h3, and h4 (see, for example, Figure 71), where h1 and h3 each contain a single split initiator HCR tail, and when the four hairpins polymerize, a complete second HCR initiator i5 colocalizes; and h2 and h4 each contain a single split initiator HCR tail, and when the four hairpins polymerize, a complete second HCR initiator i6 colocalizes, with the polymerization of the four hairpins occurring upon exposure to the first initiator i1. In some embodiments, h1 and h3 each contain a single split initiator tail, or h2 and h4 each contain a single split initiator tail, so that when the four hairpins are exposed to and polymerize with the first initiator i1, h1 and h3 colocalize a complete HCR initiator i5, or h2 and h4 colocalize a complete HCR initiator i6. In some embodiments, the colocalized complete second HCR initiator bridges between the linear and nonlinear amplification stages without the use of a bridging probe. In some embodiments, the nonlinear amplification stage uses a colocalized, complete second HCR initiator to trigger the growth of a tertiary reporter-modified HCR amplification polymer linked to the HCR amplification polymer from the linear amplification stage, and the HCR signal is amplified using the tertiary reporter-labeled second HCR amplification agent (e.g., Figures 38A, 47A, 49A, 52A, and 60C).In some embodiments, the desired reporter on the signal probe and the tertiary reporter on the second HCR amplifier are the same. In some embodiments, additional rounds of nonlinear HCR signal amplification are performed (e.g., one additional round of HCR signal amplification, two additional rounds of HCR signal amplification, or M additional rounds of HCR signal amplification). In some embodiments, the same self-bridging HCR amplifier is used for multiple rounds of HCR signal amplification. In some embodiments, the tertiary reporter is a fluorophore or chromophore or a rare earth element or compound (e.g., Figure 71). In some embodiments, the tertiary reporter mediates CARD signal amplification (e.g., Figure 38B). In some embodiments, an anti-auxiliary reporter enzyme-labeled readout probe is used to bind to the tertiary reporter on the HCR amplification polymer and to directly or indirectly mediate CARD signal amplification (e.g., Figure 38B). In some embodiments, the readout probe acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target. In some embodiments, the signal probe includes antibodies, nanobodies, nucleic acids, molecules containing a target-binding domain, and / or molecules containing a reporter-binding domain. In some embodiments, the target is a protein, and HCR imaging is used to perform HCR immunohistochemistry (IHC) or immunofluorescence (IF) with or without CARD. In some embodiments, the target is a nucleic acid, and HCR imaging is used to perform HCR in situ hybridization (ISH) with or without CARD. In some embodiments, CARD signal amplification generates a fluorescent or chromogenic signal.

[0055] Nonlinear HCR signal amplification of N targets using self-bridging hairpins In some embodiments, N signal probes are used to detect N targets (where N is a positive integer) in a sample using nonlinear HCR signal amplification (see reagents in Figures 47B, 52B, and 60D, e.g.). In some embodiments, the j-th target (j=1, ..., N, where j is a positive integer) is detected by a j-th initiator-labeled signal probe containing the j-th first HCR initiator (e.g., Figures 52A and 71). The j-th target is detected. In some embodiments, the j-th target is detected by a set of j-th fragmentary initiator signal probes that colocalize with the j-th complete first HCR initiator when the probe in the probe unit specifically binds to the corresponding cognitive binding site on the j-th target (e.g., Figures 38A–38B, 47A, 49A, and 71). In some embodiments, the j-th target is a target complex (or set of adjacent target molecules) that is detected using the set of j-th fragmentary initiator probes and the j-th proximity probe, causing the j-th complete first HCR initiator to colocalize (e.g., Figures 58C, 68AB, 68D–68I, and 69AB). In some embodiments, the j-th target is detected by a j-th anti-target signal probe containing a j-th reporter (e.g., Figure 60C). In some embodiments, the j-th reporter is detected by a j-th anti-reporter signal probe containing the j-th first HCR initiator. In some embodiments, during the linear amplification stage, signal amplification to the j-th target is provided by a j-th self-bridging first HCR amplifier including a split initiator tail, which uses the j-th first HCR initiator to trigger the growth of the j-th HCR amplified polymer, colocalizing the j-th complete second HCR initiator within the polymer linked to the j-th target. In some embodiments, the colocalized j-th complete second HCR initiator bridges between the linear and nonlinear amplification stages without the use of a bridging probe. In some embodiments, the nonlinear amplification step involves HCR signal amplification using a colocalized j-th complete second HCR initiator against a j-th target by a j-th second HCR amplifier containing a j-th tertiary reporter, triggering the growth of a j-th tertiary reporter-modified HCR amplifier polymer linked to the j-th HCR amplifier polymer from the linear amplification step (e.g., Figures 38B and 71).In some embodiments, the desired j-th reporter on the j-th signal probe and the j-th tertiary reporter on the j-th second HCR amplifier are the same. In some embodiments, additional rounds of nonlinear HCR signal amplification are performed (e.g., one additional round of HCR signal amplification, two additional rounds of HCR signal amplification, or M additional rounds of HCR signal amplification). In some embodiments, the same j-th self-bridging HCR amplifier is used for multiple rounds of HCR signal amplification against the j-th target. In some embodiments, the j-th tertiary reporter is the j-th fluorophore, the j-th chromophore, or the j-th rare earth element or compound (e.g., Figure 71). In some embodiments, the j-th tertiary reporter mediates CARD signal amplification against the j-th target (e.g., Figure 38B). In some embodiments, a j-th anti-tertiary reporter readout probe containing the j-th enzyme is used to bind to the j-th tertiary reporter on the j-th HCR amplification polymer and to mediate CARD signal amplification directly or indirectly against the j-th target. In some embodiments, the j-th readout probe acts on the j-th CARD substrate to catalyze the deposition of the j-th CARD reporter near the j-th target. In some embodiments, the same enzyme is used to mediate CARD for all targets. In some embodiments, CARD signal amplification generates the j-th fluorescent signal or j-th chromogenic signal for the j-th target. In some embodiments, the j-th anti-target signal probe comprises the j-th antibody, the j-th nanobody, the j-th nucleic acid, the j-th molecule containing the j-th target-binding domain, or the j-th molecule containing the j-th reporter-binding domain. In some embodiments, the j-th target is a protein, and HCR imaging is used to perform HCR immunohistochemistry (IHC) or immunofluorescence (IF) with or without CARD. In some embodiments, the j-th target is a nucleic acid, and HCR imaging is used to perform HCR in situ hybridization (ISH) with or without CARD.

[0056] Complete HCR formed by colocalization of two or more fragmentary initiator probes Nishiator Each set of fragmentary initiator probes that generate a complete HCR initiator (also referred to as a colocalized complete HCR initiator, a complete initiator, or a colocalized complete initiator) is called a probe unit (see, for example, Figures 64 and 65). In some embodiments, a colocalized complete HCR initiator is generated by a pair of fragmentary initiator probes, each carrying a portion of a complete HCR initiator, which together constitute a complete HCR initiator (part f1 for probe p1, part f2 for probe p2, and f1+f2=1). In this case, the probe unit consists of two fragmentary initiator probes (see, for example, Figure 65A). In some embodiments, partial f1 and partial f2 are sufficiently small compared to the complete HCR initiator (e.g., f1=0.5 and f2=0.5, or f1=0.45 and f2=0.55, or f1=0.4 and f2=0.6, or f1=0.3 and f2=0.7), so that if the complete HCR initiator is not colocalized to the target, HCR signal amplification is suppressed.

[0057] In some embodiments, an HCR initiator (i1 or i2) is divided into three fragmentary initiator probes (part f1 of probe P1, part f2 of probe P2, part f3 of probe 3, so f1+f2+f3=1). In this case, the probe unit consists of three fragmentary initiator probes. In some embodiments, an HCR initiator (i1 or i2) is divided into N fragmentary initiator probes (part f1 of probe P1, part f2 of probe P2, ..., part fN of probe PN, so f1+f2+...fN=1; see, for example, Figure 51B), where N is 2, 3, 4, or more. In this case, the probe unit consists of N fragmentary initiator probes. In some embodiments, in any of these N values, if the complete HCR initiator is not colocalized to the target, HCR signal amplification is suppressed.

[0058] In some embodiments, a complete HCR initiator is generated by the colocalization of a pair (or set) of probes, each carrying a portion of the HCR initiator, such that the sum of portion f1 of probe p1 and portion f2 of probe p2 (f1+f2) is sufficiently close to 1 (e.g., f1=0.47, f2=0.47, f1+f2=0.94, or f1=0.44, f2=0.42, f1+f2=0.86). This triggers HCR signal amplification by the colocalized complete initiator resulting from the binding of the pair of probes to the corresponding cognitive binding site on the target. In some embodiments, fragmentary initiator probes within a probe unit generate a complete HCR initiator equivalent to 100% of the HCR initiator. In some embodiments, fragmentary initiator probes within a probe unit generate a sufficient proportion of HCR initiators to provide efficient HCR signal amplification compared to the signal amplification rate when no fragmentary initiator probes are present or when individual fragmentary initiator probes are present but not colocalized with the target. In some embodiments, the proportion of complete HCR initiators generated by colocalized probes within a probe unit is 99%, 95%, 90%, 80%, or 60%, defined as a range greater than any one of the aforementioned values ​​or between any two of the aforementioned values ​​for complete HCR initiators. In some embodiments, a probe unit includes two, three, four, five, or more fragmentary initiator probes. In some embodiments, fragmentary initiators within a probe unit are sufficient to function as HCR initiators when the probes within the probe unit bind to and colocalize with adjacent or near-adjacent cognitive binding sites on the target. In some embodiments, HCR initiators may have a sequence of a specific length (e.g., 15 nucleotides), but fragmentary initiators within a probe unit do not need to be exactly the same length. For example, in some embodiments, if they still function as HCR initiators when colocalized, their total length is 14 Alternatively, it may be 13 nucleotides. In some embodiments, two or more fragmentary initiators can be used, insofar as they together perform the function of an HCR initiator.

[0059] In some embodiments, a complete HCR initiator is generated by the colocalization of a pair of probes, each having a portion of an HCR initiator and further containing one, some, or more sequence modifications, such that the sum of portion f1 of probe p1 and portion f2 of probe p2 (f1+f2) is sufficiently close to 1 (e.g., f1=0.45, f2=0.47, f1+f2=0.92), thereby triggering HCR signal amplification by the colocalized complete initiator resulting from the binding of the pair of probes to an adjacent or near-adjacent cognitive binding site on the target. In some embodiments, fragmentary initiator probes within a probe unit generate a colocalized complete HCR initiator (also referred to as a complete HCR initiator, colocalized complete initiator, or complete initiator) having 100% sequence identity with the HCR initiator. In some embodiments, fragmentary initiator probes within a probe unit generate sufficient sequence identity of the HCR initiator to enable efficient HCR signal amplification compared to the signal amplification rate when fragmentary initiator probes are absent or when individual fragmentary initiator probes are present but not colocalized with the target. In some embodiments, the complete HCR initiator generated by the colocalized probes within a probe unit has 99%, 95%, 90%, 80%, or 60% sequence identity with the HCR initiator, including a range greater than any one of the aforementioned values ​​or a range defined between any two of the aforementioned values.

[0060] Use a helper probe to increase signal intensity. To maintain target selectivity, the probe set size may be limited to one probe or one probe unit or less, two probes or two probe units or less, or N probes or N probe units or less, where N is less than the number of probes or probe units (N+M) preferred to maximize signal intensity. In this scenario, where the probe set size is constrained by selectivity considerations, the amount of signal generated may be less than when detecting the same target using N+M probe units, because the probe set may produce M fewer complete initiators to trigger HCR signal amplification, and the absence of M additional probe units may reduce the binding yield of the remaining N probe units due to synergistic effects.

[0061] In some embodiments, the signal probe carries one or more initiators or fragmentary initiators (see, for example, Figure 64A). In some embodiments, the helper probe does not carry an initiator or fragmentary initiator. In some embodiments, the helper probe does not contain a neighboring domain. In some embodiments, binding one or more helper probes to a target improves accessibility to the target and facilitates binding one or more fragmentary initiator probes to the target. In some embodiments, to increase the signal without reducing selectivity, a signal probe set containing N probe units can be expanded with M helper probes (see, for example, Figure 64D).

[0062] HCR amplifier with two hairpins In some embodiments, the HCR amplifier includes two hairpins (h1 and h2; see, for example, Figures 2A–2B and Figures 17A–17F). In some embodiments, each hairpin includes an input domain with a single-stranded toehold and stem section, and an output domain with a complement of a single-stranded loop and stem section. In some embodiments, In the absence of an HCR initiator (i1 or i2), hairpins h1 and h2 coexist metastable; that is, they are kinetically captured and do not polymerize.

[0063] Initiated by Initiator i1 In some embodiments, initiator i1 includes a domain complementary to the toehold of hairpin h1 and a domain complementary to the stem section of h1. In some embodiments, when hairpin h1 encounters initiator i1, initiator i1 hybridizes to the input domain of hairpin h1 via toehold-mediated substitution, opening hairpin h1 and exposing the output domain of hairpin h1, forming complex i1-h1. In some embodiments, the output domain of hairpin h1 includes a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem section of h2. In some embodiments, when hairpin h2 encounters complex i1-h1, the exposed output domain of h1 hybridizes to the input domain of hairpin h2 via toehold-mediated substitution, opening hairpin h2 and exposing the output domain of hairpin h2, forming complex i1-h1-h2. In some embodiments, the output domain of hairpin h2 includes a domain complementary to the toehold of hairpin h1 and a domain complementary to the stem section of h1. In some embodiments, when the h1 hairpin encounters the complex i1-h1-h2, the exposed output domain of h2 hybridizes with the input domain of hairpin h1 via toehold-mediated chain substitution, opening hairpin h1 and exposing the output domain of hairpin h1, forming the complex i1-h1-h2-h1. In some embodiments, this polymerization process can be repeated alternately through the polymerization steps of h1 and h2 to produce polymers in the form i1-h1-h2-h1-h2-h1-h2-... In the case of a polymer incorporating N alternating copies of hairpins h1 and h2, this can be expressed as i1-(h1-h2) NIt is expressed as follows. For example, in the polymer, several h1 molecules and h2 molecules, dozens of h1 molecules and h2 molecules, hundreds of h1 molecules and h2 molecules, thousands of h1 molecules and h2 molecules, tens of thousands of h1 molecules and h2 molecules, or more may be incorporated. In some embodiments, the polymer may end with either h1 or h2, and thus i1-(h1-h2) N -h1 and i1-(h1-h2) N -h1-h2 are both possible, and the latter is equivalent to i1-(h1-h2) N+1 is equivalent to it.

[0064] Initiation by initiator i2 In some embodiments, initiator i2 includes a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem section of h2. In some embodiments, when the h2 hairpin encounters initiator i2, initiator i2 hybridizes to the input domain of hairpin h2 via toehold-mediated strand displacement, opening hairpin h2 to expose the output domain of hairpin h2 and forming the complex i2-h2. In some embodiments, when the h1 hairpin encounters the complex i2-h2, the exposed output domain of h2 hybridizes to the input domain of hairpin h1 via toehold-mediated strand displacement, opening hairpin h1 to expose the output domain of hairpin h1 and forming the complex i2-h2-h1. In some embodiments, when the h2 hairpin encounters the complex i2-h2-h1, the exposed output domain of h1 hybridizes to the input domain of hairpin h2 via toehold-mediated strand displacement, opening hairpin h2 to expose the output domain of hairpin h2 and forming the complex i2-h2-h1-h2. In some embodiments, this polymerization process can generate a polymer in the form of i2-h2-h1-h2-h1-h2-h1-··· by alternately repeating the polymerization steps of h2 and h1. In the case of a polymer incorporating N alternating copies of h2 and h1, this is i2-(h2-h1) NIt can be written as follows. For example, a polymer may incorporate a few h1 and h2 molecules, tens of h1 and h2 molecules, hundreds of h1 and h2 molecules, thousands of h1 and h2 molecules, tens of thousands of h1 and h2 molecules, or more. In some embodiments, the polymer may end in either h1 or h2, and therefore te i2-(h2-h1) N -h2 and i2-(h2-h1) N -h2-h1 is possible in both cases, and the latter is i2-(h2-h1) N+1 It is equivalent to this.

[0065] Self-bridging HCR amplifier with split initiator tail In some embodiments, the HCR amplifier comprises two or more HCR hairpins, two or more of which contain split initiator tails. The initiation that triggers the growth of the HCR amplifying polymer causes the two split initiator tails at the junction between the hairpins in the amplifying polymer to colocalize, resulting in the production of colocalized complete HCR initiators linked to the resulting HCR amplifying polymer (see, e.g., Figures 16C–16D, 38B, and 71). In some embodiments, the HCR amplifier comprising two or more HCR hairpins with split initiator tails self-bridges between successive rounds of HCR signal amplification because polymerization of the HCR amplifier colocalizes complete HCR initiators that can be used to initiate a new round of HCR signal amplification. In some embodiments, a self-bridging HCR amplifier bridges between one round of HCR signal amplification, which generates one or more colocalized complete HCR initiators linked to the resulting HCR amplification polymer, and another round of HCR signal amplification, which generates new HCR amplification polymers linked to each of the complete HCR initiators colocalized by the previous HCR amplification polymer. In some embodiments, the self-bridging HCR amplifier includes two HCR hairpins, one or both of which include two split initiator tails (e.g., Figure 38B). In some embodiments, the HCR hairpins with two split initiator tails can cause leakage of the HCR amplifier from a kinetically trapped metastable state in the absence of HCR initiators or colocalized complete HCR initiators.In some embodiments, the self-bridging HCR amplifier comprises four HCR hairpins, at least two of which strictly contain one split initiator tail, so that as the HCR amplified polymer grows, the complete HCR initiators linked to the resulting amplified polymer colocalize (see, for example, hairpins h1 and h3 colocalizing the complete HCR initiator i5, and hairpins h2 and h4 colocalizing the complete HCR initiator i6 in Figures 16C and 71), and it is not necessary to use HCR hairpins containing two split initiator tails. In some embodiments, the self-bridging HCR amplifier comprises four HCR hairpins, including two versions of a first hairpin (each version containing a different split initiator tail) and two versions of a second hairpin (each version containing a different split initiator tail), thereby randomly colocalizing complete HCR initiators linked to the resulting amplified polymer as the HCR amplified polymer grows, depending on which version of the hairpin is incorporated adjacent to each junction in the polymer (see, for example, Figure 16D).

[0066] HCR amplifier with four hairpins In some embodiments, the HCR amplifier may include two or more hairpins. For example, the HCR amplifier may include four hairpins h1, h2, h3, and h4 (see, for example, Figures 16A-16C). In some embodiments, as with the 2-hairpin HCR, each hairpin includes an input domain including a single-stranded toehold and stem section, and an output domain including a single-stranded loop and a complement of the stem section. In some embodiments, two or more hairpins include a single split initiator tail (see, for example, Figures 16C and 71), which, when polymerized, colocalizes with a complete second HCR initiator (e.g., i5 or i6 in Figures 16C and 71), and thus self-bridges to a new round of HCR signal amplification. In some embodiments, the new round of HCR signal amplification is configured to be initiated by the colocalized complete second HCR initiator (e.g., hairpins h5 and h6 in Figures 16C and 71). This is carried out using an HCR amplifier containing h6. In some embodiments, in the absence of an HCR initiator (i1, i2, i3, or i4), hairpins h1, h2, h3, and h4 coexist metastable, i.e., they are kinetically captured and do not polymerize. In some embodiments, the output domain of hairpin h1 includes a domain complementary to the toehold of hairpin h2 and a domain complementary to the stem section of hairpin h2; the output domain of hairpin h2 includes a domain complementary to the toehold of hairpin h3 and a domain complementary to the stem section of hairpin h3; the output domain of hairpin h3 includes a domain complementary to the toehold of hairpin h4 and a domain complementary to the stem section of hairpin h4; the output domain of hairpin h4 includes a domain complementary to the toehold of hairpin h1 and a domain complementary to the stem section of hairpin h1. In some embodiments, initiator i1 includes a domain complementary to the toe hold of hairpin h1 and a domain complementary to the stem section of hairpin h1; initiator i2 includes a domain complementary to the toe hold of hairpin h2 and a domain complementary to the stem section of hairpin h2; initiator i3 includes a domain complementary to the toe hold of hairpin h3 and a domain complementary to the stem section of hairpin h3; initiator i4 includes a domain complementary to the toe hold of hairpin h4 and a domain complementary to the stem section of hairpin h4.In some embodiments, as in the case of a 2-hairpin HCR, when hairpin h1 encounters initiator i1, initiator i1 opens hairpin h1 to form complex i1-h1 with exposed h1 output domain, then opens hairpin h2 to form complex i1-h1-h2 with exposed h2 output domain, then opens hairpin h3 with exposed output domain to form complex i1-h1-h2-h3 with exposed h3 output domain, then opens hairpin h4 to expose h4 output A complex i1-h1-h2-h3-h4 with domains is formed, then the hairpin h1 is opened to form a complex i1-h1-h2-h3-h4-h1 with exposed h1 output domains, and so on, until polymerization occurs through alternating polymerization steps of h1, h2, h3, and h4, producing polymers in the form i1-h1-h2-h3-h4-h1-h2-h3-h4-h1-h2-h3-h4... and in the case of a polymer incorporating N alternating copies of h1, h2, h3, and h4, it is i1-(h1-h2-h3-h4). N It can be written as i1-(h1-h2-h3-h4). In some embodiments, the polymer can end in h1, h2, h3, or h4, and thus i1-(h1-h2-h3-h4) N -h1, i1-(h1-h2-h3-h4) N -h1-h2, i1-(h1-h2-h3-h4) N -h1-h2-h3, and i1-(h1-h2-h3-h4) N -h1-h2-h3-h4 are all possible, and the latter is i1-(h1-h2-h3-h4) N+1 This corresponds to: In some embodiments, HCR polymerization can be triggered by any of the cognitive initiators (i1, i2, i3, or i4). For example, initiation by initiator i3 may trigger i3-(h3-h4-h1-h2) NPolymers of this form may be produced. In some embodiments, an HCR amplifier with four hairpins is convenient for generating a signal that is not present in the non-polymerized state but is present in the polymerized state (for example, Figure 16B shows a FRET pair that colocalizes to generate a FRET signal only when the hairpins colocalize within the amplified polymer, and unused hairpins that are not washed away from the sample do not participate in FRET, thus providing a basis for a no-wash method to avoid background generation).

[0067] Self-bridging HCR amplifier with 4 hairpins and up to one split initiator tail per hairpin. In some embodiments, the HCR amplifier includes four HCR hairpins (see, for example, Figures 16C and 71): 1) a first input domain (sequence domain "ab") including a first toehold (sequence domain "a") and a first stem section (sequence domain "b"), and a first loop (sequence domain "c*") and a partner of the first stem section (sequence domain "b*") configured to bond to the first stem section. 1) A first HCR hairpin (h1) including a first output domain (sequence domain "c*-b*") and a first split initiator tail (sequence domain "f"), and 2) a second input domain (sequence domain "bc") including a second toehold (sequence domain "c") and a second stem section (sequence domain "b"), a second output domain (sequence domain "b*-d*") including a second loop (sequence domain "d*") and a partner of the second stem section (sequence domain "b*") configured to join the second stem section, and a second split initiator tail (sequence domain "h"), and a second HCR hairpin (h2); 3) a third split initiator tail (sequence domain "g"), a third toehold (sequence domain "d") and a third stem section (sequence domain 4) A third HCR hairpin (h3) comprising a third input domain (array domain "db") containing "b"), a third output domain (array domain "e*-b*") containing a third loop (array domain "e*") and a partner of the third stem section (array domain "b*") configured to join with the first stem section, and 4) a fourth HCR hairpin (h4) comprising a fourth split initiator tail (array domain "i"), a fourth input domain (array domain "be") containing a fourth toehold (array domain "e") and a fourth stem section (array domain "b"), and a fourth output domain (array domain "b*-a*") containing a partner of the fourth stem section (array domain "b*") configured to join with the fourth loop (array domain "a*") and the fourth stem section. In some embodiments, the first initiator (i1; sequence domain "b*-a*") includes a partner for the first toehold (sequence domain "a*") and a partner for the first stem section (sequence domain "b*"). In some embodiments, the first initiator is configured to bind to the first input domain. In some embodiments, when the first initiator binds to the first input domain, the first HCR hairpin opens, exposing the first output domain.In some embodiments, the exposed first output domain is configured to bind to the second input domain. In some embodiments, binding the exposed first output domain to the second input domain opens the second HCR hairpin, exposing the second output domain. In some embodiments, the exposed second output domain is configured to bind to the third input domain. In some embodiments, binding the exposed second output domain to the third input domain opens the third HCR hairpin, exposing the third output domain. In some embodiments, the exposed third output domain is configured to bind to the fourth input domain. In some embodiments, binding the exposed third output domain to the fourth input domain opens the fourth HCR hairpin, exposing the fourth output domain. In some embodiments, the exposed fourth output domain is configured to bind to the first input domain. In some embodiments, binding the exposed fourth output domain to the first input domain causes HCR polymerization to occur, opening the first, second, third, and fourth HCR hairpins in sequence and periodically adding to the growing polymer. In some embodiments, when initiator i1 initiates the growth of an HCR amplification polymer including the sequential addition and opening of hairpins h1, h2, h3, and h4, the split initiator tails of h1 and h3 colocalize a complete fifth HCR initiator i5 (sequence domain "gf") linked to the HCR amplification polymer, and the split initiator tails of h2 and h4 colocalize a complete sixth HCR initiator i6 (sequence domain "hi") linked to the HCR amplification polymer. In some embodiments, the colocalized complete fifth HCR initiator i5 and the colocalized complete sixth HCR initiator i6 provide a basis for bridging to a new round of HCR signal amplification by triggering the growth of a new HCR amplification polymer linked to the original HCR amplification polymer.

[0068] In some embodiments, the four hairpin amplifiers, including hairpins h1, h2, h3, and h4, have a second toehold (sequence domain "c*") partner and a second stem sector It may also be initiated by a second initiator i2 (sequence domain "c*-b*") including a partner of a sequence domain "b*", a third initiator i3 (sequence domain "b*-d*") including a partner of a third toehold (sequence domain "d*") and a partner of a third stem section (sequence domain "b*"), and / or a fourth initiator i4 (sequence domain "e*-b*") including a partner of a fourth toehold (sequence domain "e*") and a partner of a fourth stem section (sequence domain "b*").

[0069] In some embodiments, a colocalized complete fifth HCR initiator i5 and / or a colocalized complete sixth HCR initiator i6 are configured to trigger polymerization of an HCR amplification polymer comprising two HCR hairpins (see, for example, Figures 16C and 71): 1) a fifth input domain (sequence domain "jk") comprising a fifth toehold (sequence domain "j") and a fifth stem section (sequence domain "k"), and a fifth stem section configured to bond to a fifth loop (sequence domain "l*") and a fifth stem section. 1) A fifth HCR hairpin (h5) including a fifth output domain (sequence domain "l*-k*") including a partner (sequence domain "k*"), and 2) a sixth HCR hairpin (h6) including a sixth input domain (sequence domain "kl") including a sixth toehold (sequence domain "l") and a sixth stem section (sequence domain "k"), and a sixth output domain (sequence domain "k*-j*") including a partner (sequence domain "k*") of a sixth stem section configured to join a sixth loop (sequence domain "j*") and a sixth stem section.

[0070] In some embodiments, a fifth initiator (i5; sequence domain "k*-j*") includes a partner for a fifth toehold (sequence domain "j*") and a partner for a fifth stem section (sequence domain "k*"). In some embodiments, the fifth initiator is configured to bind to a fifth input domain. In some embodiments, the sequence of the fifth HCR initiator i5 can be represented by either "k*-j*" (where the "j*" sequence domain is a partner for the fifth toehold and the "k*" sequence domain is a partner for the fifth stem section) or "gf" (where the "g" sequence domain is a third split initiator tail and the "f" sequence domain is a first split initiator tail). In some embodiments, the chain sequence "k*-j*" is the same as the chain sequence "gf", but the boundary between the sequence domains is at a different point in the chain sequence. In some embodiments, when the fifth initiator binds to the fifth input domain, the fifth HCR hairpin opens, exposing the fifth output domain. In some embodiments, the exposed fifth output domain is configured to bind to the sixth input domain. In some embodiments, when the exposed fifth output domain is bound to the sixth input domain, the sixth HCR hairpin opens, exposing the sixth output domain. In some embodiments, the exposed sixth output domain is configured to bind to the fifth input domain. In some embodiments, when the exposed sixth output domain is bound to the fifth input domain, HCR polymerization occurs, causing the fifth and sixth HCR hairpins to open sequentially and alternately attach to the growing polymer, and the new polymer containing alternately arranged hairpins h5 and h6 is linked to the original polymer containing periodic hairpins h1, h2, h3, and h4. In some embodiments, the first and third split initiator tails are optional (for example, domains "f" and "g" in Figure 16C are optional).

[0071] In some embodiments, a sixth initiator (i6; sequence domain "l*-k*") includes a partner of the sixth toehold (sequence domain "l*") and a partner of the sixth stem section (sequence domain "k*"). In some embodiments, the sixth initiator is configured to bind to the sixth input domain. In this configuration, the sequence of the sixth HCR initiator i6 can be represented as either "l*-k*" (where the "l*" sequence domain is the partner of the sixth toehold and the "k*" sequence domain is the partner of the sixth stem section) or "hi" (where the "h" sequence domain is the second split initiator tail and the "i" sequence domain is the fourth split initiator tail). In some embodiments, the chain sequence "l*-k*" is the same as the chain sequence "hi", but the boundaries between the sequence domains are at different points within the chain sequence. In some embodiments, when the sixth initiator is coupled to the sixth input domain, the sixth HCR hairpin opens and the sixth output domain is exposed. In some embodiments, the exposed sixth output domain is configured to be coupled to the fifth input domain. In some embodiments, when the exposed sixth output domain is coupled to the fifth input domain, the fifth HCR hairpin opens and the fifth output domain is exposed. In some embodiments, the exposed fifth output domain is configured to bond to the sixth input domain. In some embodiments, when the exposed fifth output domain is bonded to the sixth input domain, HCR polymerization occurs, causing the sixth and fifth HCR hairpins to open sequentially and alternately attach to the growing polymer, and the new polymer containing alternately arranged hairpins h6 and h5 is bonded to the original polymer containing periodic hairpins h1, h2, h3, and h4. In some embodiments, the second and fourth split initiator tails are optional (for example, domains "h" and "i" in Figure 16C are optional).

[0072] In some embodiments, the self-bridging HCR amplifier includes four HCR hairpins (see, for example, Figure 16D), including two versions of a first hairpin h1 (a version h1-3' with a 3' split initiator tail and a version h1-5' with a 5' split initiator tail) and two versions of a second hairpin h2 (a version h2-3' with a 3' split initiator tail and a version h2-5' with a 5' split initiator tail). In some embodiments, when initiator i1 triggers polymerization of the HCR-amplifying polymer from hairpins h1-3', h1-5', h2-3', and h2-5', any version of hairpin h1 is randomly incorporated during the h1 polymerization step, and any version of hairpin h2 is randomly incorporated during the h2 polymerization step (see, for example, Figure 16D), thereby generating a colocalized complete third HCR initiator i3 by linking to the amplified polymer at any junction where the 5'-tail of h1-5' and the 3'-tail of h1-3' colocalize within the polymer, and generating a colocalized complete fourth HCR initiator i4 by linking to the amplified polymer at any junction where the 5'-tail of h2-5' and the 3'-tail of h2-3' colocalize. In some embodiments, one or more colocalized complete HCR initiators i3 and / or i4 can then trigger a second HCR amplifier containing hairpins h3 and h4, which in turn can grow an HCR amplifier polymer containing alternating hairpins h3 and h4 linked to the previous HCR amplifier polymer containing alternating hairpins h1 and h2, respectively, linked to colocalized complete HCR initiators i3 or i4 (each hairpin h1 is randomly either hairpin h1-3' or h1-5', and each hairpin h2 is randomly either hairpin h2-3' or h3-5'). In some embodiments, the self-bridging HCR amplifier does not require the use of HCR hairpins containing two split initiator tails, and only requires two hairpin arrays (h1 and h2) (see, for example, Figure 16D).

[0073] HCR amplifier with two or more hairpins More generally, in some embodiments, the HCR amplifier comprises M HCR hairpins (h1, h2, ..., hM), where M is an integer greater than or equal to 2. In the absence of an HCR initiator (i1, i2, ..., iM), the hairpins h1, h2, ..., hM are quasi-safe. They coexist qualitatively, meaning they are kinetically captured and do not polymerize. In the presence of cognitive HCR initiators, polymerization occurs via alternating polymerization steps similar to 2-hairpin or 4-hairpin HCRs. For example, if initiator i1 is a polymer incorporating N alternating copies of h1, h2, ..., hM, then i1-(h1-h2-...-hM) N This leads to the growth of a polymer in this form. The polymer can end in any of h1, h2, ..., hM, and therefore i1-(h1-h2-...-hM) N -h1, i1-(h1-h2-···-hM) N -h1-h2, ..., and i1-(h1-h2-...-hM) N -h1-h2-···-hM are all possible, and the latter is i1-(h1-h2-···-hM) N+1 This is equivalent to HCR polymerization. HCR polymerization can be triggered by any of the cognitive initiators i1, i2, ..., iM). For example, initiation by initiator i3 triggers i3-(h3-...-hM-h1-h2) N In some cases, polymers of this form may be produced.

[0074] Reporter sign HCR hairpin For a given HCR amplifier, each HCR hairpin contains zero, one, or more reporters. Reporters on different hairpins within the amplifier may be the same or different. For example, an amplifier containing hairpins h1 and h2 may have: 1) the same reporter on h1 and h2; 2) different reporters on h1 and h2; 3) a reporter on h1 (no reporter on h2); 4) a reporter on h2 (no reporter on h1); 5) no reporters on either h1 or h2; 6) zero, one, or more reporters on h1, of which zero, one, or more are the same as or different from the zero, one, or more reporters on h2. Similarly, in the case of an HCR amplifier containing hairpins h1, h2, h3, and h4, each hairpin may contain zero, one, or more reporters (e.g., three, five, or ten reporters), of which zero, one, or more may be the same as the zero, one, or more reporters of each other hairpin. In some embodiments, one of the reporters of a particular hairpin may be unique within a mixture of hairpins and / or hairpin reporters. In some embodiments, there may be one, ten, one hundred, one, one, one, one, ten thousand, ten

[0075] In some embodiments, one or more reporters on the reporter-labeled HCR hairpin directly or indirectly contribute to the generation, modification, or removal of the signal. For example, the reporter may be a fluorophore, chromophore, luminescent phore, phosphorescent material, FRET pair, member of a FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, magnetic molecule, enzyme, or other molecule that facilitates the measurement of the signal.

[0076] In some embodiments, a reporter modifying a linked HCR amplification polymer may bind to the reporter-binding domain of a readout probe to directly or indirectly mediate the localization of an auxiliary reporter near the reporter, thereby directly or indirectly mediating the generation of an amplified signal.

[0077] In some embodiments, the reporter may include digoxigenin (DIG) that recruits an anti-DIG antibody as a readout probe, the anti-DIG being directly labeled with one or more auxiliary reporters, or directly labeled with one or more reporters that play a role in directly or indirectly mediating the localization of the auxiliary reporter near the reporter.

[0078] In some embodiments, the reporter may include nucleic acid domains that function as substrates having complete or partial sequence complementarity with the reporter-binding domain in a readout probe carrying one or more auxiliary reporters (see, for example, Figure 17C).

[0079] In some embodiments, the reporter may include a nucleic acid domain that functions as a substrate having complete or fragmentary sequence complementarity with the reporter-binding domain in a readout probe having one or more substrates that play a role in mediating the localization of an auxiliary reporter near the reporter.

[0080] In some embodiments, the reporter may include nucleic acid domains that function as substrates for readout probes that directly or indirectly mediate the localization of an auxiliary reporter near the reporter.

[0081] In some embodiments, the reporter may include a substrate that plays a role in mobilizing a readout probe that indirectly mediates the localization of an auxiliary reporter near the reporter.

[0082] In some embodiments, the reporter may include a substrate that plays a role in recruiting a readout probe containing an enzyme that mediates catalytic reporter deposition (CARD) near the reporter (see, for example, Figure 27A).

[0083] In some embodiments, the reporter may contain biotin that recruits streptavidin (or another biotin-binding molecule) as a readout probe, the streptavidin being directly labeled with one or more auxiliary reporters, or directly labeled with one or more substrates that play a role in directly or indirectly mediating the localization of the auxiliary reporter near the reporter.

[0084] In some embodiments, the reporter may include a hapten that recruits an anti-hapten antibody readout probe, or an anti-hapten nanobody readout probe that directly or indirectly mediates the localization of the reporter near the reporter via CARD signal amplification. For example, the anti-hapten antibody or nanobody readout probe may include an enzyme that mediates CARD (see, for example, Figures 25A to 25E).

[0085] In some embodiments, the reporter may include a hapten that mobilizes an anti-hapten that directly or indirectly mediates the localization of an auxiliary reporter near the hairpin reporter. For example, the anti-hapten readout probe may include an enzyme that mediates CARD (see, for example, Figures 26A-26C).

[0086] In some embodiments, the reporter may include an enzyme that mediates CARD signal amplification and deposits the CARD reporter molecule near the hairpin.

[0087] In some embodiments, the hairpin reporter may include zero, one, or more haptens (see, for example, Figures 17A-17B) that directly or indirectly localize auxiliary reporters near the hapten.

[0088] In some embodiments, a reporter modifying a linked HCR amplification polymer binds to the reporter-binding domain of a bridging probe, directly or indirectly mediating the localization of one or more HCR initiators near the reporter, thereby triggering the growth of a new HCR amplification polymer linked to the original HCR amplification polymer.

[0089] In some embodiments, the reporter may include a hapten that recruits an anti-hapten (e.g., an antibody, nanobody, streptavidin, or another molecule) labeled with an auxiliary reporter.

[0090] In some embodiments provided herein, HCR signal amplification is used to mediate catalytic reporter deposition (CARD), resulting in even higher signal gains. In some embodiments, even higher single gains are values ​​within the range defined by approximately 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000 times, or any two of the aforementioned values.

[0091] Haptens and anti-haptens In some embodiments, hairpin labels that are hapten-containing substrates include, for example, digoxigenin (DIG), dinitrophenyl (DNP), fluorophores, biotin, and any small molecule, biomolecule, or non-biological molecule capable of recruiting anti-haptens. Examples of anti-haptens include antibodies, nanobodies, streptavidin, aptamers, and other molecules or complexes of molecules that selectively bind to haptens.

[0092] Enzymes for HCR-mediated catalytic reporter deposition (CARD) In some embodiments, the reporter-modified HCR amplification polymer mediates signal amplification via catalytic reporter deposition (CARD) by an enzyme that catalyzes the CARD substrate, depositing the CARD reporter near the HCR amplification polymer (see, for example, Figures 25A-25E, 26A-26C, and 27A-27B).

[0093] In some embodiments, the enzyme may be horseradish peroxidase (HRP) (or a polymer HRP containing multiple HRP enzymes) that acts on a CARD substrate to catalyze the deposition of a chromogenic CARD reporter such as AEC, DAB, TMB, or StayYellow, or catalyzes a CARD substrate to catalyze the deposition of a fluorescent CARD reporter such as a fluorophore-labeled tyramide, or catalyzes the deposition of a hapten-labeled CARD substrate, such as a biotin-labeled tyramide, where the hapten plays a role in mediating the localization of the CARD reporter near the reporter-modified HCR amplification polymer.

[0094] In some embodiments, the enzyme may be an alkaline phosphatase (AP) (or a polymer AP containing multiple AP enzymes) that acts on a CARD substrate to catalyze the deposition of a CARD reporter, and may include, but is not limited to, pigment-based CARD reporters such as BCIP / NBT, BCIP / TNBT, Napthol AS-MX phosphate + FastBlue BB, Napthol AS-MX phosphate + FastRed TR, and StayGreen.

[0095] In some embodiments, the enzyme may be a glucose oxidase that acts on a CARD substrate to catalyze the deposition of a CARD reporter, such as NBT.

[0096] In some embodiments, the enzyme may be any molecule or complex that directly or indirectly mediates the localization of the CARD reporter near the reporter-modified HCR amplification polymer.

[0097] In some embodiments, the CARD reporter deposited near the linked HCR amplification polymer is visible to the naked eye. In some embodiments, the CARD reporter deposited near the linked HCR amplification polymer is scanned by an instrument and the signal is read.

[0098] In some embodiments, the CARD-mediated enzyme is inactivated (i.e., deactivated) after CARD reporter deposition (e.g., by using chemical or thermal denaturation). For example, the CARD-mediated enzyme can be deactivated using any combination of the following: 1. Heat (e.g., above 65°C); 2. Fixative (e.g., 4% PFA); 3. Acids (e.g., 0.1M glycine-HCl containing 1% Tween 20 at pH 2.2, 0.2N HCl, 10% acetic acid, 10mM HCl); and 4. Other chemical substances (e.g., hydrogen peroxide (H2O2), hydrogen peroxide + phenol, sodium azide, DEPC, MAB containing 10 mM EDTA).

[0099] In some embodiments, HRP is inactivated using H2O2. In some embodiments, AP is inactivated by a combination of heat and acid. In some embodiments, AP is inactivated with a fixative. In some embodiments, inactivating the CARD-mediated enzyme allows for repeated CARD execution using the same enzyme in combination with different substrates for different targets, enabling multi-target analysis using HCR-mediated CARD. In some embodiments, inactivating the CARD-mediated enzyme allows for repeated CARD execution using different enzymes in combination with different CARD substrates for different targets, enabling multi-target analysis using HCR-mediated CARD.

[0100] In some embodiments, CARD allows stained samples to be stored for more than 10 years and re-analyzed in accordance with regulatory requirements. In some embodiments, CARD-stained samples remain adequately stable for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 years or more, and comply with regulatory requirements. In some embodiments, CARD staining provides long-term preservation of archival samples. In some embodiments, CARD provides preservation of formalin-fixed paraffin-embedded (FFPE) samples for several decades. In some embodiments, CARD provides preservation of pathological samples for several decades for retrospective scientific and medical research. In some embodiments, CARD staining provides long-term preservation of archival samples.

[0101] Target type In some embodiments, the initiator-labeled probe comprises a target-binding domain and further comprises one or more HCR initiators. In some embodiments, the initiator-labeled probe can detect targets including, but not limited to, RNA molecules (e.g., mRNA, rRNA, lncRNA, siRNA, shRNA, microRNA, non-coding RNA, synthetic RNA, or modified RNA), DNA molecules, non-natural nucleic acid molecules, protein molecules, small molecules, biological molecules, chemically modified biological molecules, non-biological molecules, complexes of molecules consisting of any combination of RNA, DNA, proteins, small molecules, and biological molecules, and / or non-biological molecules (e.g., RNA / RNA complexes, RNA / protein complexes, DNA / protein complexes, RNA / DNA / protein complexes, protein / protein complexes), prokaryotic cells, eukaryotic cells, viruses, or any combination of the above.

[0102] In some embodiments, the fragmentary initiator probe includes a target-binding domain and further includes one or more fragmentary initiators. In some embodiments, the probe unit includes two or more fragmentary initiator probes, and the fragmentary initiator probes within the probe unit combine to form a complete HCR initiator. In some embodiments, the probe unit includes RNA molecules (e.g., mRNA, rRNA, lncRNA, siRNA, shRNA, microRNA, non-coding RNA, synthetic RNA, or modified RNA), DNA molecules, non-natural nucleic acid molecules, protein molecules, small molecules, biological molecules, chemically modified biological molecules, non-biological molecules, complexes of molecules consisting of any combination of RNA, DNA, proteins, small molecules, and biological molecules, and / or non-biological molecules (e.g., RNA / RNA complex, RNA / protein complex, DNA / protein complex, RNA / DNA / protein complex, protein / protein complex), and the fragmentary initiator probes constituting the probe unit are adjacent molecules or complexes Targets can be detected that include, but are not limited to, aggregates of adjacent molecules or complexes, prokaryotic cells, eukaryotic cells, viruses, and any combination thereof, such that when each target within the aggregate binds, the fragmentary initiators within the probe unit colocalize to form a complete HCR initiator.

[0103] In any embodiment provided herein, the fragmentary initiator in the probe unit is designed to be (or is) complementary to a non-overlapping region of the HCR hairpin (e.g., a region separated by zero, one, two, or more nucleotides), or to be complementary to a overlapping region of the HCR hairpin (e.g., a region overlapping by one, two, or more nucleotides), or to be substantially complementary to the HCR hairpin (e.g., complementary except for zero, one, two, a few, or some mismatches), or is configured to bind to the HCR hairpin.

[0104] In any embodiment provided herein, the target-binding region within the probe unit is configured to bind to a non-overlapping region of the target (e.g., a region separated by zero, one, two, or more nucleotides, or a region separated by 0 nm, 1 nm, 2 nm, or more nanometers) or to bind to an overlapping region of the target (e.g., a region overlapping by one, two, or more nucleotides, or a region overlapping by 1 nm, 2 nm, or more nanometers).

[0105] Signal probe set for multiplexing In some embodiments, the signal probe set is designed for multiplex experiments using 2, 3, 4, 5, 10, 20, or 100 or more signal probe sets to bind to different targets within the same sample, and 1, 2, 3, 4, 5, 10, 20, or 100 or more signal probe sets include 1 or more probe units, each containing 1 or more initiator-labeled probes or 2 or more fragmentary initiator probes. In some embodiments, the signal probe set is designed for multiplex experiments using 2, 3, 4, 5, 10, 20, or 100 or more signal probe sets in the same sample, and more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, more than 50%, or 100% of the signal probe set includes 1 or more initiator-labeled probes or 1 or more probe units, each containing 2 or more fragmentary initiator probes.

[0106] A signal probe configured to bind to overlapping or non-overlapping regions of a target, and / or a signal probe designed to have a fragment initiator that hybridizes to overlapping or non-overlapping regions of an HCR hairpin. In some embodiments, the probe unit includes two or more fragment initiator probes, each containing a target binding region and a fragment initiator.

[0107] In some embodiments, the fragmented initiator within the probe unit is designed to be complementary to an adjacent region of the HCR hairpin.

[0108] In some embodiments, the fragmented initiator within the probe unit is designed to be complementary to a non-overlapping region of the HCR hairpin (e.g., a region separated by 0, 1, 2, or more nucleotides).

[0109] In some embodiments, the fragmented initiator within the probe unit is designed to be complementary to an overlapping region of the HCR hairpin (e.g., a region where 1, 2, or more nucleotides overlap).

[0110] In some embodiments, the fragmented initiator within the probe unit is designed to be substantially complementary to the HCR hairpin (e.g., complementary except for 0, 1, 2, a few, or some mismatches).

[0111] In some embodiments, the fragmented initiator within the probe unit is designed to hybridize to an adjacent region of the HCR hairpin.

[0112] In some embodiments, the fragmented initiator within the probe unit is designed to hybridize to a non-overlapping region of the HCR hairpin.

[0113] In some embodiments, the fragmented initiator within the probe unit is designed to hybridize to an overlapping region of the HCR hairpin.

[0114] In some embodiments, the fragmented initiator within the probe unit is designed to have a sequence complementary to an adjacent region of the HCR hairpin.

[0115] In some embodiments, the fragmentary initiators within the probe unit are designed to have an arrangement complementary to the non-overlapping regions of the HCR hairpin.

[0116] In some embodiments, the fragmentary initiators within the probe unit are designed to have an arrangement complementary to the overlapping region of the HCR hairpin.

[0117] In some embodiments, the fragmentary initiators within the probe unit are designed to have an arrangement substantially complementary to the adjacent regions of the HCR hairpin.

[0118] In some embodiments, the fragmentary initiators within the probe unit are designed to have an arrangement substantially complementary to the non-overlapping regions of the HCR hairpins.

[0119] In some embodiments, the fragmentary initiators within the probe unit are designed to have an arrangement substantially complementary to the overlapping region of the HCR hairpin.

[0120] In some embodiments, the target-binding region within the probe unit is configured to bind to an adjacent region of the target. In some embodiments, the target-binding region within the probe unit is configured to bind to a non-overlapping region of the target (e.g., a region separated by zero, one, two, or more nucleotides, or a region separated by 0 nm, 1 nm, 2 nm, or more nanometers). In some embodiments, the target-binding region within the probe unit is configured to bind to an overlapping region of the target (e.g., a region with one, two, or more overlapping nucleotides, or a region with 1 nm, 2 nm, or more overlapping nanometers). In some embodiments, the target-binding region within the probe unit is designed to bind to an adjacent region of the target. In some embodiments, the target-binding region within the probe unit is designed to bind to a non-overlapping region of the target. In some embodiments, the target-binding region within the probe unit is designed to bind to an overlapping region of the target, or to have a sequence that hybridizes to an adjacent region of the target. In some embodiments, the target-binding region within the probe unit is designed to have a sequence that hybridizes to a non-overlapping region of the target. In some embodiments, the target binding region within the probe unit is designed to have an array that hybridizes with the overlapping region of the target.

[0121] In some embodiments, the probe unit has two fragmentary initiator probes. In some embodiments, two fragmentary initiator probes bind to a cognitive target, colocalizing a complete HCR initiator. In some embodiments, the colocalized complete HCR initiator then binds to the HCR hairpin of the cognitive to initiate HCR polymerization, with one fragmentary initiator hybridizing to the hairpin to form a first double chain and the other fragmentary initiator hybridizing to the hairpin to form a second double chain. In some embodiments, there is an energetically unfavorable junction between the two double chains, creating a dynamic barrier during the branching transfer process that opens the first HCR hairpin and initiates polymerization. In some embodiments, by configuring a fragmentary initiator to bind to the overlapping region of the hairpin, the junction can be relaxed to an energetically more favorable structure, reducing the height of the dynamic barrier, increasing the efficiency of HCR initiation, and / or increasing the affinity between the colocalized complete HCR initiator and the first HCR hairpin, thereby increasing the amount of amplified HCR signal generated within a given period (e.g., Figures 28 and 31). In some embodiments, the target-binding regions of two probes can be configured to bind to the overlapping region of the target, allowing the intermolecular junction to relax to an energetically favorable structure, thereby increasing the affinity between the two probes and the cognitive target.

[0122] In some embodiments, the probe set is designed for multiplex experiments using 2, 3, 4, 5, 10, 20, or 100 or more probe sets to bind to different targets within the same sample, and 1, 2, 3, 4, 5, 10, 20, or 100 or more probe sets include one or more initiator-labeled probes. In some embodiments, the probe set is designed for multiplex experiments using 2, 3, 4, 5, 10, 20, or 100 or more probe sets in the same sample, and more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, more than 50%, or 100% of the probe sets include one or more initiator-labeled probes.

[0123] In some embodiments, the probe set is designed for multiplex experiments using 2, 3, 4, 5, 10, 20, or 100 or more probe sets to bind to different targets within the same sample, and 1, 2, 3, 4, 5, 10, 20, or 100 or more probe sets include one or more probe units containing fragment initiators designed to hybridize to overlapping regions of HCR hairpins.

[0124] In some embodiments, the probe set is designed for multiplex experiments using 2, 3, 4, 5, 10, 20, or 100 or more probe sets to bind to different targets within the same sample, and 1, 2, 3, 4, 5, 10, 20, or 100 or more probe sets include one or more probe units containing target-binding regions designed to bind to overlapping regions of the target. In some embodiments, the probe set is designed for multiplex experiments using 3, 4, 5, 10, 20, or 100 or more probe sets in the same sample, and more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, more than 50%, or 100% of the probe sets include one or more probe units containing target-binding regions designed to bind to overlapping regions of the target.

[0125] In some embodiments, the probe set is designed for multiplex experiments using 2, 3, 4, 5, 10, 20, or 100 or more probe sets to bind to different targets within the same sample, with one or more probe sets acting as one or more initiators. The probe sets include labeled probes, and each probe set includes one or more probe units, each containing two or more fragmentary initiator probes. In some embodiments, the probe sets are designed for multiplex experiments in which two, three, four, five, ten, twenty, or more than 100 probe sets are used on the same sample, and more than 0.1%, more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, or more than 50% of the probe sets include one or more initiator-labeled probes, and more than 0.1%, more than 1%, more than 2%, more than 5%, more than 10%, more than 30%, or more than 50% of the probe sets include one or more probe units, each containing two or more fragmentary initiator probes.

[0126] In some embodiments, the probe unit includes a fragmentary initiator designed to bind to an overlapping region of the HCR hairpin, where the overlapping region overlaps by one, two, three, four, five, or more bases.

[0127] In some embodiments, the probe unit includes a target-binding region designed to bind to an overlapping region of a target, where the overlapping region overlaps by at least 0.1 nm, or at least 0.2 nm, or at least 0.3 nm, or at least 0.5 nm, or at least 1 nm, or at least 2 nm, or at least 3 nm, or at least 5 nm. In some embodiments, the probe unit includes a target-binding region containing a sequence designed to bind to an overlapping region of a target, where the overlapping region overlaps by at least 1 nucleotide, or 2 nucleotides, or 3 nucleotides, or 4 nucleotides, or 5 nucleotides, or more.

[0128] Material and composition of initiator-labeled probes In some embodiments, the initiator-labeled probe includes one or more target-binding domains and one or more HCR initiators (see, e.g., FIGS. 18A-18N and 19A-19F). In some embodiments, each domain can include one or more materials including DNA, RNA, 2′-OMe-RNA, PNA, XNA, chemically modified nucleic acids, synthetic nucleic acid analogs, amino acids, chemical linkers, synthetic amino acid analogs, and / or other molecules suitable for the purpose of the domain.

[0129] In some embodiments, the initiator-labeled probe can include one or more initiators made of DNA and a target-binding domain made of DNA.

[0130] In some embodiments, the initiator-labeled probe can include one or more initiators made of DNA, a chemical linker, and a target-binding domain made of amino acids (e.g., an antibody, nanobody, or antibody fragment).

[0131] In some embodiments, the initiator-labeled probe can include an initiator made from a synthetic nucleic acid analog and a target-binding domain made from a combination of DNA and 2′-OMe-RNA.

[0132] In some embodiments, the initiator-labeled probe can include an initiator made of 2′-OMe-RNA and a target-binding domain made from a combination of RNA and protein.

[0133] In some embodiments, the initiator-labeled probe can include an initiator made of DNA and a target-binding domain made of PNA.

[0134] In some embodiments, the initiator-labeled probe can include one or more initiators made from any nucleic acid or nucleic acid analog and one or more target-binding domains made from any combination of materials suitable for binding to a target molecule.

[0135] In some embodiments, the initiator-labeled probe may consist of a single covalently bonded molecule, or it may consist of two or more molecules (each covalently bonded) that interact non-covalently to form a complex.

[0136] In some embodiments, the initiator-labeled probe may include a DNA-based initiator covalently bound to a DNA-based target-binding domain.

[0137] In some embodiments, the initiator-labeled probe may comprise one or more initiators made of DNA covalently bound to dCas9 (or another Cas) noncovalently bound to a guide RNA (gRNA), such that the target-binding domain includes a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas).

[0138] In some embodiments, the initiator-labeled probe may comprise one or more initiators made of DNA covalently bound to a gRNA noncovalently bound to dCas9 (or another Cas), such that the target-binding domain includes a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas).

[0139] In some embodiments, the initiator-labeled probe may include an initiator made of nucleic acid or nucleic acid analog covalently or noncovalently bonded to a target-binding domain containing one or more molecules.

[0140] Material and composition of fragmentary initiator probes In some embodiments, a fragmentary initiator probe comprises one or more target-binding domains and one or more fragmentary initiators (see, for example, Figures 20A–20E, 21A–21D, 22A–22E, and 24A–24R). In some embodiments, a fragmentary initiator probe may further comprise one or more adjacent domains. In some embodiments, each domain may comprise one or more materials including DNA, RNA, 2'OMe-RNA, PNA, XNA, chemically modified nucleic acids, synthetic nucleic acid analogs, chemical linkers, amino acids, synthetic amino acid analogs, and / or other molecules suitable for the purpose of the domain.

[0141] In some embodiments, a fragmentary initiator probe may comprise one or more fragmentary initiators made of DNA and a target-binding domain made of DNA.

[0142] In some embodiments, the fragmentary initiator probe may comprise one or more fragmentary initiators made of DNA, a chemical linker, and a target-binding domain made of amino acids (e.g., an antibody, nanobody, or antibody fragment).

[0143] In some embodiments, the fragmentary initiator probe may comprise a fragmentary initiator made from a synthetic nucleic acid analog and a target-binding domain made from a combination of DNA and 2'OMe-RNA.

[0144] In some embodiments, the fragmentary initiator probe may comprise a fragmentary initiator made from 2'OMe-RNA and a target-binding domain made from a combination of RNA and protein.

[0145] In some embodiments, the fragmentary initiator probe may include a fragmentary initiator made of DNA and a target-binding domain made of PNA.

[0146] In some embodiments, a fragmentary initiator probe may comprise one or more fragmentary initiators made from any nucleic acid or nucleic acid analog, and one or more target-binding domains made from any combination of materials suitable for binding to a target molecule.

[0147] In some embodiments, the adjacent domain may include nucleic acids or nucleic acid analogs.

[0148] In some embodiments, the fragmentary initiator probe may consist of a single covalently bonded molecule, or it may consist of two or more molecules (each covalently bonded) that interact non-covalently to form a complex.

[0149] In some embodiments, the fragmentary initiator probe may include a fragmentary initiator made of DNA covalently bound to a target-binding domain made of DNA.

[0150] In some embodiments, the fragmentary initiator probe may comprise one or more fragmentary initiators made of DNA covalently bound to dCas9 (or another Cas) noncovalently bound to a guide RNA (gRNA), such that the target binding domain includes a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas).

[0151] In some embodiments, the fragmentary initiator probe may comprise one or more fragmentary initiators made of DNA covalently bound to a gRNA noncovalently bound to dCas9 (or another Cas), such that the target binding domain includes a gRNA:dCas9 complex (or a gRNA:Cas complex using another Cas).

[0152] In some embodiments, a fragmentary initiator probe may include a fragmentary initiator made of nucleic acid or nucleic acid analog covalently or noncovalently bonded to a target-binding domain containing one or more molecules. Each fragmentary initiator probe in a probe unit may have the same or a different material composition as other fragmentary initiator probes in the probe unit. Each fragmentary initiator probe in a probe unit may have a target-binding region that binds to different detection sites on the same target molecule, different detection sites within a target molecule complex, or different detection sites within a target assembly of a neighboring molecule or complex.

[0153] Material and composition of reporter-labeled probe In some embodiments, the reporter-labeled probe includes one or more reporters. In some embodiments, the reporter includes a hapten, a tag, a small molecule, a fluorophore, a chromophore, a luminescent phore, a phosphorescent body, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a magnetic molecule, an enzyme, a nucleic acid, biotin, DIG, or another molecule that directly or indirectly mediates the generation of a signal. In some embodiments, the reporter-labeled probe is detected using an anti-reporter initiator-labeled probe that can trigger HCR signal amplification. In some embodiments, the anti-target reporter-labeled probe includes the reporter and any molecule or complex containing an antibody, a nanobody, a nucleic acid, or a target-binding domain. In some embodiments, the anti-reporter initiator-labeled probe includes an HCR initiator and any molecule or complex containing an antibody, a nanobody, a nucleic acid, or a reporter-binding domain.

[0154] Materials and composition of bridging probes In some embodiments, the bridging probe bridges between two rounds of HCR signal amplification. In some embodiments, the bridging probe bridges from the reporter-modified HCR amplification polymer generated during the previous round of HCR signal amplification to an initiator that can trigger a new round of HCR signal amplification. In some embodiments, the anti-reporter initiator-labeled bridging probe comprises a reporter-binding domain and one or more HCR initiators. In some embodiments, the anti-reporter initiator-labeled bridging probe comprises a) any molecule or complex containing an antibody, nanobody, nucleic acid, or reporter-binding domain, and b) one or more HCR initiators. In some embodiments, the anti-reporter initiator-labeled bridging probe comprises an anti-reporter primary bridging probe containing a reporter-binding domain and an anti-primary-secondary bridging probe containing one or more HCR initiators. In some embodiments, the primary bridging probe comprises an antibody, nanobody, and / or nucleic acid. In some embodiments, the secondary bridging probe comprises an antibody, nanobody, and / or nucleic acid. In some embodiments, the bridging probe directly or indirectly bridges a reporter that modifies an HCR amplifier from a previous round of HCR signal amplification to an HCR initiator used to trigger a new round of HCR signal amplification.

[0155] Removal of signals from the sample In some embodiments, the HCR signal is removed from the sample after signal detection. In some embodiments, the signal can be removed from the sample by any method of reducing the number of signal-generating reporters and / or auxiliary reporters and / or tertiary reporters and / or first reporters and / or second reporters and / or third reporters.For example, photobleaching of a fluorescent reporter molecule using light and / or chemical reagents; chemically cleaving the reporter from an HCR hairpin and leaching it from the sample (e.g., TCEP); chemically cleaving the reporter from a readout probe and leaching it from the sample; chemically cleaving the hairpin to fragment the HCR amplification polymer and leaching the fragments from the sample; chemically cleaving the probe to release the HCR amplification polymer from the target and leaching the released amplification polymer from the sample; dehybridizing the hairpin from the HCR amplification polymer using an auxiliary chain and leaching the hairpin from the sample; dehybridizing the readout probe from the HCR amplification polymer using an auxiliary chain and leaching the readout probe from the sample; leaching the hairpin from the sample after destabilizing the HCR amplification polymer using a chemical modifier and / or high temperature; leaching the unbound amplification polymer from the sample after destabilizing the interaction between the probe and the target using a chemical modifier and / or high temperature; and destabilizing the interaction between the readout probe and its substrate using a chemical modifier and / or high temperature. This includes destabilizing the signal and then flushing the readout probe from the sample; using an enzyme to degrade the amplification polymer and / or probe and flushing the degraded molecules from the sample; using DNase to degrade the DNA amplification polymer and / or DNA probe and / or DNA target and flushing the resulting molecules from the sample; using RNases to degrade the RNA target and then flushing the amplification polymer from the sample; using a protease to degrade the protein target and then flushing the amplification polymer from the sample; using a combination of RNase for degrading the RNA target and DNase for degrading the DNA amplification polymer and / or DNA probe and then flushing the resulting molecules from the sample; and using two or more of the above methods or other methods for removing the signal from the sample simultaneously or at different times.

[0156] Assay format In some embodiments, the HCR signal can be measured in a variety of assay formats, including but not limited to: blot, Northern blot, Western blot, Southern blot, spot blot, paper assay, flow cytometry assay, lateral flow assay, fluorescence flow cytometry assay, cell sorting assay, fluorescence-activated cell sorting assay, magnetically activated cell sorting assay, microscopy assay, Optical microscopy assays, epifluorescence microscopy assays, confocal microscopy assays, light sheet microscopy assays, microarray assays, bead-based assays, mass spectrometry assays, fluorescence microscopy assays, mass spectrometry microscopy assays, mass spectrometry flow cytometry assays, fluorescence assays, chemiluminescence assays, bioluminescence assays, colorimetric assays, electrochemical impedance assays, electrochemical chemiluminescence assays, energy dissipation assays, assays using the human eye, assays using a mobile phone camera, gel electrophoresis assays, in situ hybridization (ISH) assays, RNA-ISH assays, DNA-ISH assays, immunohistochemistry (IHC) assays, autoradiography assays, and any assay capable of detecting signals generated by HCR amplification polymers.

[0157] Sample type In some embodiments, initiator-labeled probes and / or fragmentary initiator probes can be used with HCR amplification hairpins to detect targets in a sample. Targets include molecules, complexes, or aggregates of adjacent molecules or complexes. Target molecules include, for example, bacteria, zebrafish embryos, chicken embryos, mouse embryos, human biopsy specimens, human tissue sections, FFPE tissue sections, urine samples, blood samples, stool samples, mouse tissue sections, brain sections, sea urchin embryos, nematode larvae, fruit fly embryos, model organisms, non-model organisms, mixtures of multiple organisms, unknown organisms, environmental samples including organism aggregates (e.g., mixtures of protists and bacteria in the intestines of another organism), termites, microbiomes, clinical specimens, diagnostic specimens, and sputum samples. It may be contained in samples, tumor biopsy specimens, research specimens, specimens containing human material, specimens containing pet material (e.g., dogs, cats, rabbits, lizards, snakes, or fish), specimens containing wild animal material (e.g., cheetahs, elephants, rhinos, or chimpanzees), specimens containing extinct animals (e.g., mammoths, dodos, great auks, triceratops, or passenger pigeons), live cells (e.g., bacteria or cultured mammalian cells), or specimens containing living organisms (e.g., live mice or live humans).

[0158] In some embodiments, the target may be freely present in the solution within the sample. For example, the target may be freely present in the solution within a test tube, cells, embryos, organisms, tissue sections, biological specimens, or other samples.

[0159] In some embodiments, the target may be covalently or non-covalently bonded to one or more capture probes covalently or non-covalently bonded to a solid support. For example, it may be directly or indirectly bonded to capture probes covalently bonded to a microarray or beads.

[0160] In some embodiments, the target may be directly or indirectly immobilized, covalently, or non-covalently bonded to a solid support. For example, the target may be bonded, immobilized, or covalently crosslinked to a slide, blot, membrane, paper substrate, or other substrate. The target may be immobilized or covalently crosslinked to cells, embryos, organisms, tissue sections, biological specimens, or other samples. The target may be covalently bonded within an immobilized and permeabilized sample, an immobilized but not permeabilized sample, or an unimmobilized but permeabilized sample.

[0161] In some embodiments, the target may be freely present within living cells, living embryos, organisms, living ecosystems, or consortia of organisms (e.g., the gut microbiota of mammals). The target may be associated with but outside of a cell or organism, or it may be contained within a cell or organism. The target may be covalently crosslinked within living cells, living embryos, organisms, living ecosystems, or consortia of organisms. The target may be present or absent within one or more cell types in a sample. The target may be present or absent within one or more species in a sample. The target may be present in a sample containing one or more off-targets with different degrees of similarity to the target molecule. The target may be present in an enlarged sample. The target may be present in a compressed sample. To increase the spatial separation between molecules, The sample may be magnified before detecting the target. The sample may be compressed before detecting the target to reduce spatial separation between molecules. Target molecules and / or other molecules may be crosslinked into the magnified sample so that the relative positions of molecules within the sample are maintained even when the sample is magnified. To magnify the sample, target molecules and / or other molecules may be crosslinked into a gel, matrix, or other reagent introduced into the sample, while maintaining the relative positions and / or orientation of molecules within the sample even when the sample is magnified. The sample may be selectively magnified and / or compressed with different magnification and / or compression factors in different tissues and / or organs within the sample.

[0162] Sample fixation In some embodiments, the target molecule can be crosslinked to the sample to retain it in subsequent steps of the experiment. For example, the target molecule can be crosslinked to the sample using chemical reagents (e.g., formaldehyde, paraformaldehyde, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)).

[0163] Permeabilization treatment of the sample In some embodiments, sample processing can improve the accessibility of target molecules to HCR probes and amplifiers. For example, samples (e.g., cells, tissue sections, or whole-mount embryos) can be permeabilized using chemical reagents (e.g., methanol, ethanol, or detergent) or enzymes (e.g., proteinase K). Target accessibility can be improved by homogenization of the sample, microdissection, electroporation, sectioning, heat treatment (e.g., 1 It can also be improved by (Gunasekera TS, Barardi CR, Veal D, Vesey G (2004) J Appl Microbiol 96(2):409-417) and / or by microwave treatment (e.g., Lan HY, Mu W, NG YY, Nikolic-Paterson DH, & Atkins RC (1996) J Histochem Cytochem 44(3):281-287). Another option is to deliver the HCR probe and amplification agent across the cell membrane using chemical transfection reagents.

[0164] Washing to remove unbound reagents from the sample In some embodiments, background can be reduced by washing away unused imaging reagents from the sample. For example, washing can be used to remove probes, initiator-labeled probes, fragmentary initiator probes, HCR amplification hairpins, amplification reagents, proximity probes, bridging probes, labeled probes, antibodies, and / or other imaging reagents from the sample. Washing can be performed at a temperature using chemical reagents such that specifically bound imaging reagents are not primarily removed (signal is preserved) and nonspecifically bound imaging reagents are primarily removed (background is reduced). For example, washing buffers may include denaturants (e.g., formamide, urea), salt buffers (e.g., sodium chloride, sodium citrate (SSC), phosphate-buffered saline (PBS)), acids (e.g., citric acid), surfactants (e.g., Tween 20, Triton-X, SDS), or blocking agents (e.g., tRNA, salmon sperm DNA, BSA, Ficol, polyvinylpyrrolidone, heparin). The stringency of the washing process can be optimized by combining the washing buffer with the washing temperature (e.g., 25-80°C).

[0165] Accurate and precise target quantification In some embodiments, HCR probes and HCR amplifiers provide quantitative analysis of target molecules in an anatomical context, generating a signal that changes approximately proportionally to the number of target molecules per imaging voxel. In some embodiments, this quantitative characteristic is obtained from the sum of signals occurring at three levels during imaging: 1) the sum of one or more initiator-labeled probes per target molecule, or 1 or more probes per target molecule. 1) The sum of the probe units (each containing two or more fragmentary initiator probes); 2) The sum of multiple HCR amplification hairpins for each amplification polymer linked to the probe unit of an initiator-labeled probe or a fragmentary initiator probe that colocalizes a complete initiator; 3) The sum of zero, one, or more target molecules in the imaging pixel. In some embodiments, quantitative accuracy can be improved while maintaining intracellular resolution by defining imaging voxels that average the intensity of adjacent pixels (for example, accurate and precise quantitative imaging at intracellular resolution has been demonstrated for mRNA targets in Figures 6A-6B and protein targets in Figures 11A-11C). In some embodiments, the quantitative nature of the HCR signal stems from the binding properties of the HCR probes, the polymerization properties of the HCR amplification hairpins, and the central limit theorem, which together utilize summation and averaging during and after image acquisition to generate a signal that changes approximately proportionally to the abundance of the target. In some embodiments, the same quantitative characteristics are applied to other assay formats, and summation and / or averaging are performed during and / or after data acquisition (e.g., by flow cytometry, blot scanning, or macroscopic lateral flow assay).

[0166] Multiplexing using initiator-labeled probes In some embodiments, an HCR probe set containing initiator-labeled probes and an HCR amplifier including an HCR hairpin can be used for multiplex target analysis (e.g., target analysis by imaging, blotting, flow cytometry, mass cytometry, gel analysis, or other analytical modes) that simultaneously analyzes multiple targets in the same sample. Consider a sample containing some or all of the N target types to be considered, as well as zero, one or more additional off-target species that are not targeted. Each target can be detected using a probe set containing one or more initiator-labeled probes (each containing one or more HCR initiators) that selectively bind to a cognitive target. In some embodiments, the probe set for each of the N target types is labeled with an HCR initiator for a different HCR amplifier. For example, target 1 can be detected with probe set 1 labeled with the HCR initiator of HCR amplifier 1, target 2 can be detected with probe set 2 labeled with the HCR initiator of HCR amplifier 2, and similarly, target 2 can be detected with probe set N labeled with the HCR initiator of HCR amplifier N.

[0167] In some embodiments, the N probe sets operate orthogonally, with each probe set selectively binding to its corresponding target regardless of the presence of other probe sets and / or targets in the sample. In some embodiments, the N amplifiers operate orthogonally, 1) the hairpins of each amplifier coexist metastable in the absence of the cognitive HCR initiator, and 2) each amplifier is selectively triggered and polymerizes in the presence of its cognitive initiator, regardless of the presence of other amplifiers in the sample. In some embodiments, the reporters supported on each HCR amplifier are orthogonal, so that the signal generated by each HCR amplifier can be measured by analytical methods regardless of the presence of other reporters in the sample (e.g., a fluorescence reporter distinguishable using a fluorescence microscope, or a rare-earth reporter distinguishable using mass cytometry). For example, multiplex imaging using initiator-labeled probes and simultaneous HCR signal amplification for all targets are shown in Figures 9, 10, 11, and 50.

[0168] In some embodiments, multiplex target analysis of N target types (type j=1, ..., N) can be performed using N orthogonal HCR initiator-labeled probe sets, allowing all targets to be detected simultaneously (the initiator-labeled probes of probe set j are bound to target j of target type j=1, ..., N).

[0169] In some embodiments, N orthogonal HCR amplifiers are used to target all types of cells. By simultaneously amplifying the gunals, multiplex target analysis of N target types (type j=1, ..., N) can be achieved (the hairpins of the amplifier j polymerize in response to the initiator j, forming amplified polymer j linked to target type j of j=1, ..., N).

[0170] In some embodiments, multiplex target analysis of N target types (type j=1, ..., N) can be achieved by analyzing a sample using a measuring device that detects reporter j directly supported by one or more hairpins of the amplifier j, or reporter j indirectly bound to one or more hairpins of the amplifier j, for target types j=1, ..., N.

[0171] Multiplexing using fragmented initiator probes In some embodiments, an HCR probe set containing a fragmentary initiator probe and an HCR amplifier including an HCR hairpin can be used for multiplex target analysis (e.g., target analysis by imaging, blotting, flow cytometry, mass cytometry, gel analysis, or other analytical modes) that analyzes multiple targets simultaneously in the same sample. Consider a sample containing some or all of the N target types to be considered, as well as zero, one or more additional off-target species that are not to be considered. Each target can be detected using a probe set containing one or more probe units (each containing two or more fragmentary initiator probes) that selectively bind to the cognitive target so that each bound probe unit co-localizes with a complete HCR initiator. In some embodiments, the probe set for each of the N target types co-localizes with a complete HCR initiator using different HCR amplifiers. For example, target 1 can be detected by probe set 1 which colocalizes one or more complete HCR initiators of HCR amplifier 1, and target 2 can be detected by probe set 2 which colocalizes one or more complete HCR initiators of HCR amplifier 2, and so on, with probe set N colocalizing one or more complete HCR initiators of HCR amplifier N.

[0172] In some embodiments, the N probe sets operate orthogonally, with each probe set selectively binding to its corresponding target regardless of the presence of other probe sets and / or targets in the sample. In some embodiments, the N amplifiers operate orthogonally, 1) the hairpins of each amplifier coexist metastable in the absence of a cognitive complete initiator co-localizing with the cognitive target, and 2) each amplifier is selectively triggered to polymerize when a cognitive complete initiator co-localizes with the cognitive target, regardless of the presence of other amplifiers in the sample. In some embodiments, the reporters supported on each HCR amplifier are orthogonal, so that the signal generated by each HCR amplifier can be measured by an analytical method regardless of the presence of other reporters in the sample (e.g., a fluorescence reporter distinguishable using a fluorescence microscope, or a rare-earth reporter distinguishable using mass cytometry). For example, multiplex imaging using fragmentary initiator probes and simultaneous HCR signal amplification for all targets is shown in Figures 4 and 5.

[0173] In some embodiments, multiplex target analysis of N target types (type j=1, ..., N) can be achieved by simultaneously detecting all targets using N orthogonal HCR fragmentary initiator probe sets (fragmentary initiator probes of probe set j bind to target j, and complete HCR initiator j are colocalized for each probe unit in probe set j for target types j=1, ..., N).

[0174] In some embodiments, N orthogonal HCR amplifiers are used to achieve multiplex target analysis of N target types (type j=1, ..., N), and the signals of all target types can be amplified simultaneously (the hairpin of amplifier j is a complete HCR initiator). (It polymerizes in response to j, forming an amplified polymer j linked to target type j of j=1, ..., N).

[0175] In some embodiments, multiplex target analysis of N target types (type j=1, ..., N) can be achieved by analyzing a sample using a measuring device that detects reporter j directly supported by one or more hairpins of the amplifier j, or reporter j indirectly bound to one or more hairpins of the amplifier j, for target types j=1, ..., N.

[0176] Multiplexing using a combination of initiator-labeled probes and fragmented initiator probes. In some embodiments, multiplex analysis is performed by detecting one or more targets (possibly of different types) in a sample using initiator-labeled probes and one or more other targets (possibly of different types) using fragmentary initiator probes. For example, in the same sample, one or more protein targets and one or more small RNA targets can be detected with orthogonal initiator-labeled probes, one or more mRNA targets and / or DNA targets can be detected with orthogonal fragmentary initiator probes, and one or more composite target pairs (including complexes of two or more non-covalent molecules) can be detected with fragmentary initiator probes. In some embodiments, a probe set for each target (including one or more probe units, each containing one or more initiator-labeled probes, or two or more fragmentary initiator probes and optionally one or more neighboring probes) triggers an orthogonal HCR amplifier to generate an orthogonal signal (directly or indirectly). In some embodiments, HCR signal amplification is performed simultaneously for all target types. For example, multiplex imaging, which uses initiator-labeled probes for one or more targets and fragmentary initiator probes for one or more targets to simultaneously amplify the HCR signal for all targets, is shown in Figures 8E-8F, 12A-12B, and 13A-13B.

[0177] Multiplexing using spectral imaging In some embodiments, spectral analysis can increase the number of labels that can be distinguished from one another. For example, if two fluorophores have overlapping emission spectra and the two labels cannot be distinguished by measuring the emission intensity using a bandpass filter, they can be distinguished using spectral imaging, which distinguishes the signals from the two labels using multiple emission measurements at different wavelengths, even if the emission spectra of the labels substantially overlap. Using spectral imaging, in some embodiments, it is possible to spectrally distinguish between 10 fluorescent dyes, or 20 fluorescent dyes, or even 30 or more fluorescent dyes.

[0178] Multiplexing using hybrid spectra with multi-reporter polymers In some embodiments, HCR polymerization proceeds by alternating between h1 polymerization steps and h2 polymerization steps, so that the resulting HCR-amplified polymer includes one of the following: 1) the same number of h1 hairpins and h2 hairpins; 2) one more h1 hairpin; 3) one more h2 hairpin. In some embodiments, as the length of the polymer increases, the proportion of h1 hairpins in the polymer approaches 0.5, and the proportion of h2 hairpins in the polymer also approaches 0.5. In some embodiments, hairpin h1 is labeled with reporter r1, and hairpin h2 is labeled with reporter r2. In some embodiments, the signal produced by the HCR-amplified polymer is a 1:1 blend of the signals produced by reporters r1 and r2 and a new hybrid spectrum. Consider a set of N reporters having different spectra. In some embodiments, N reporters The reporter can be used to create N*(N-1) / 2 hybrid spectra, corresponding to the number of different reporter pairs that can be selected from a set of N reporters. For example, 1) with 6 reporters, 6*5 / 2=15 hybrid reporter spectra can be created; 2) with 8 reporters, 8*7 / 2=28 hybrid reporter spectra can be created; 3) with 15 reporters, 15*14 / 2=105 hybrid reporter spectra can be created; 4) with 50 reporters, 50*49 / 2=1225 hybrid reporter spectra can be created; 5) with 100 reporters, 100*99 / 2=4950 hybrid reporter spectra can be created.

[0179] Computational sequencing design of orthogonal HCR amplifiers using NUPACK In some embodiments, a reaction pathway designer within the NUPACK software suite is used to design a series of orthogonal HCR amplifiers (with or without substrates and / or auxiliary chains). 13、14 In some embodiments, sequence design is formulated as a multistate optimization problem using a set of target test tubes to represent the basic steps of the reaction pathway and to model global crosstalk. 14 In some embodiments, each basic process tube contains a set of desirable on-target complexes (each having a target secondary structure and target concentration) corresponding to the on-pathway hybridization product of a given step, and a set of undesirable off-target complexes (each having a target concentration that disappears) corresponding to the on-pathway reactant of a given step and off-pathway hybridization crosstalk. 56In this scenario, these basic process tubes facilitate the complete conversion of cognitive reactants to cognitive products and counteract local hybridization crosstalk between these same reactants. In some embodiments, a basic process tube is specified for each orthogonal system in order to design N orthogonal systems simultaneously. In some embodiments, a single global crosstalk tube is also specified to counteract off-pathway interactions between systems. 56 In some embodiments, in a global crosstalk tube, the on-target complex corresponds to all reactive species generated in all basic steps of the entire system (e.g., the single-stranded output domain of the HCR hairpin opened by polymerization). In some embodiments, in a global crosstalk tube, the off-target complex corresponds to non-cognitive interactions between these reactive species. In some embodiments, the global crosstalk tube ensemble omits cognitive products that the reactive species are intended to form (they do not appear as either on-target or off-target). In this scenario, all reactive species in the global crosstalk tube can be forced to either not react (remain the desired on-target) or to undergo crosstalk reactions (form undesirable off-targets), providing a basis for minimizing global crosstalk during sequence optimization. In some embodiments, sequence design is carried out according to complementarity constraints inherent to the reaction pathway (e.g., in Figure 1A, domain "a" is complementary to domain "a*", and domain "b" is complementary to domain "b*"). 14 In some embodiments, the sequence is optimized by quantifying the average percentage of nucleotides that mispaired across the entire multitube ensemble and reducing ensemble defects. 14 In some embodiments, defect weights are applied within an ensemble of defects to determine the priority of design efforts. 14Ensemble defect optimization implements both a positive design paradigm, which explicitly designs the fundamental processes on the on-pathway, and a negative design paradigm, which explicitly designs the crosstalk off-pathway. 14

[0180] Multiplexing using repeated reporter detection In some embodiments, the number of targets that can be analyzed within a sample can be increased using the same N labels, allowing for the detection of multiple targets in a series of analysis rounds. For example, N targets can be analyzed using N labels. Using the identifier, imaging can be performed, and then the signal can be removed from the sample to detect another set of N targets using the same N labels. In some embodiments, this repeated imaging technique can be applied when imaging multiple target types within the same sample: 1) regardless of whether the expression levels of different target types are high or low, vary within each target type, and / or vary between different target types; and 2) regardless of whether the expression patterns of different target types spatially overlap or not within the sample. In some embodiments, the selection of probe types may differ for each target and may be mixed within any process as desired. In some embodiments, 1) all targets may be detected with initiator-labeled probes, 2) all targets may be detected with fragmentary initiator probes, or 3) one or more targets may be detected with initiator-labeled probes and other targets may be detected with fragmentary initiator probes. Thus, one option disclosed herein also provides other options and combinations thereof. For example, the statement “provides N probe sets, each comprising either a) one or more HCR initiator-labeled probes, or b) one or more probe units, each comprising two or more HCR fragmentary initiator probes” means that any of the N probe sets may be of either type (a or b), including the possibility that all probe sets are of the same type (all type a or all type b), and that some probe sets are of one type and some are of the other type (some type a, some type b). It is also understood that, in the context of this disclosure, any embodiment where “either…or…” is used may have a mix of types (unless otherwise specified). In some embodiments, the repeated imaging method may be combined with CARD, enzyme inactivation, and / or repeated CARD.

[0181] In some embodiments, any one or more desired steps in any one or more of the methods herein can be combined with any one or more other desired steps in any one or more of the methods herein. In some embodiments, any one or more different steps in any one or more of the methods herein can be combined with any one or more other different steps in any one or more of the methods herein. In some embodiments, any one or more different steps in any one or more of the methods herein can be combined with any one or more other different steps in any one or more of the methods herein.

[0182] Multiplexing using single-molecule barcodes In some embodiments, the number of targets that can be analyzed in a sample can be increased by analyzing each target molecule in multiple analysis rounds. This results in different labels being used for different target types in different analysis rounds, creating different barcodes for each variety of target molecule. The barcode for a particular target molecule is read as a barcode for signal measurement. For example, if single-molecule imaging is used to read the signal for each target molecule as diffraction-limited dots, consider three rounds of imaging. For each target type, consider assigning a probe set containing one or more probe units. This ensures that each probe unit in the probe set co-localizes with a complete HCR initiator corresponding to an HCR amplifier containing an HCR hairpin labeled with a red or green reporter, depending on the target type and the round of imaging. Next, for example, a type 1 target molecule can be read by a barcode (red, red, green; round 1 using an HCR amplifier labeled with a red reporter shows a red dot, round 2 using an HCR amplifier labeled with a red reporter shows a red dot, and round 3 using an HCR amplifier labeled with a green reporter shows a green dot), a type 2 target molecule can have a barcode (red, green, red), a type 3 target molecule can have a barcode (red, red, red), and a type 4 target molecule can have a barcode (green, red, green).

[0183] In some embodiments, the number of targets that can be analyzed in a sample can be increased by detecting each target molecule in only a portion of the barcoding rounds. For example, in a 4-round experiment, type 1 target molecules can be read by the barcode (red, ---, ---, red; round 1 shows a red dot, round 2 shows no dots, round 3 shows no dots, and round 4 shows a red dot), and type 2 target molecules can be read by the barcode (green, red, ---, ---). [Examples]

[0184] The following embodiments are not limiting, and other modifications within the scope of the art of those skilled in the art should be considered.

[0185] Example 1 – Cooperative initiation of HCR using a fragmented initiator probe Figures 3A-3C illustrate the coordinated initiation of HCR using fragmentary initiator probes. Figure 3A shows target-mediated colocalization of the fragmentary initiator probes. Fragmentary initiator probes p1 and p2 each carry a portion of HCR initiator i1. Selective binding of p1 and p2 to the target mRNA leads to colocalization of the complete HCR initiator i1, enabling the coordinated initiation of the HCR amplification cascade. Figure 3B shows in vitro verification of the coordinated initiation of HCR using fragmentary initiator probes p1 and p2. Reaction conditions: Hairpins h1 and h2, 0.5 μM each (lanes 1-7); Oligo-i1, p1, p2, and / or target, 5 nM each (lanes indicated on the gel); 5 × SSCT buffer; react overnight at room temperature. Hairpins h1 and h2 were labeled with Alexa 647 fluorophores. dsDNA 1kb ladders were pre-stained with SYBR Gold. Lane 1: Metastable hairpins h1 and h2 show minimal leakage from kinetically captured state in the absence of HCR initiator i1. Lane 2: In the presence of HCR initiator i1 (i1 as an oligo), HCR hairpin monomers h1 and h2 are completely converted to amplified polymers. Lane 3: In the presence of both target and fragmentary initiator (also known as split initiator) probes p1 and p2, hairpins h1 and h2 are strongly converted to polymers, indicating coordinated initiation of HCR. Lanes 4-6: In the presence of probe p1, probe p2, or both probes p1 and p2, conversion of HCR hairpin monomers h1 and h2 to polymers is minimal, indicating active background suppression. Lane 7: In the presence of target only, conversion of HCR hairpin monomers h1 and h2 to polymers is minimal. Figure 3C shows the quantification of the polymer bands in Figure 3B. Using Multi Gauge software (Fujifilm Corporation), Alexa 647 intensity profiles were calculated for the polymer bands in lanes 1-7. Each intensity profile is displayed with a peak value of 0, within ±3 mm of the gel displacement. Quantified percentages were calculated using Multi Gauge, which features automatic signal and background detection, and the calculated values ​​were normalized to the measurements in lane 2.

[0186] The gel study in Figure 3B demonstrates the coordinated initiation of HCR using a fragmentary initiator probe. Lane 1 shows minimal leakage of hairpins h1 and h2 from a kinetically trapped state in the absence of HCR initiator i1. As a positive control, lane 2 shows the conversion of metastable HCR hairpin monomers h1 and h2 to polymers in the presence of HCR initiator i1 (i1 is a single oligo).

[0187] When using fragmentary initiator (or split initiator) probes, potent conversion of metastable HCR hairpin monomers h1 and h2 to polymers was observed when the target was present with fragmentary initiator probes p1 and p2. However, minimal conversion to polymers was observed when p1 or p2 was present alone (lanes 4 and 5), or when both p1 and p2 were present but the target was absent (lane 6).

[0188] These results demonstrate the active background suppression properties of the fragmentary initiator probe. Probes that are not co-localized by selective hybridization to the target primarily do not trigger HCR amplification. The fact that HCR is not triggered even when both p1 and p2 are present in solution in the absence of the target (lane 6) indicates that the fragmentary initiator probe automatically provides background suppression even without washing.

[0189] Example 2 – Performance comparison of initiator-labeled probes and fragmentary initiator probes in HCR RNA-FISH Figures 4A–4D compare the performance of initiator-labeled probes (referred to as standard probes) and fragmentary initiator probes as the probe set size increases for imaging mRNA targets in whole-mount chicken embryos. In the tests using initiator-labeled probes, an optimized probe set of five probes was used, and these probes were then expanded with additional unoptimized probes to form probe sets of 10 and 20 probes. In the tests using fragmentary initiator probes, probe sets with 5, 10, and 20 probe pairs were used. Each probe pair targeted approximately the same binding site as the corresponding initiator-labeled probe. Increasing the probe set size using initiator-labeled probes resulted in a significant increase in background (Figure 4A), and a corresponding decrease in the signal-to-background ratio as a result of nonspecific binding of some subsets of the additional probes within the sample.

[0190] These data highlight the importance of optimizing probe sets using initiator-labeled probes that do not provide active background suppression. If a probe set contains faulty probes, amplified background is generated, degrading performance. In contrast, using fragmented initiator probes, background remains nearly constant even as the number of probe pairs increases from 5 to 10 and even to 20 (Figure 4A), and the signal-to-background ratio increases monotonically (Figure 4B).

[0191] These data demonstrate the significant advantages of automated background suppression using fragmented initiator probes. Even if a fragmented initiator probe set contains faulty probes, no amplified background is generated; therefore, the signal-to-background ratio can be easily increased by simply increasing the number of probes without optimizing the probe set. Representative images using probe sets with 20 probes (initiator-labeled probes) or 20 probe pairs (fragmented initiator probes) are shown in Figure 4C. Representative pixel intensity distributions for these images are shown in Figure 4D. With fragmented initiator probes, the background and signal-to-background pixel intensity distributions largely do not overlap.

[0192] Figures 4A to 4D are explained in more detail below. They show the background and signal-to-background using initiator-labeled probes and fragmentary initiator probes. Figure 4A shows the fluorescence background using probe sets with 5, 10, or 20 probe pairs (fragmentary initiator probes) for probe sets with 5, 10, or 20 probe pairs (initiator-labeled probes). Unoptimized initiator-labeled probes resulted in nonspecific probe binding, leading to the generation of amplified background. Unoptimized fragmentary initiator probes, which bind nonspecifically within the sample, did not co-localize with complete HCR initiators, thus not triggering HCR signal amplification and avoiding the generation of amplified background. Figure 4B shows the signal-to-background for the probe sets in Figure 4A. The background ratio is shown. Fragmentary initiator probes with automatic background suppression performed better than standard probes that were not optimized for signal-to-background measurements. Figure 4C shows confocal micrographs of the neural crest of fixed whole-mount chicken embryos. Probe set: 20 probes for initiator-labeled probes, 20 probe pairs for fragmentary initiator probes. Figure 4D shows pixel intensity histograms for signal + background (pixels within the solid line boundary in Figure 4C) and background (pixels within the dashed line boundary in Figure 4C). In each image, the total number of pixels within the solid and dashed line boundaries is the same. Fixed embryo: Stage HH10. Target: Sox10.

[0193] Example 3 – Imaging four target mRNAs in whole-mount chicken embryos with high signal-to-background using an unoptimized fragmentary initiator probe. Figures 5A to 5D show imaging results of four target mRNAs in whole-mount chicken embryos with high signal-to-background ratios using unoptimized fragmentary initiator probes. Due to the automatic background suppression characteristics of the fragmentary initiator probes, the signal-to-background ratio for the four target mRNAs is within the range of approximately 25-60 without optimization of the probe set. Figure 5A shows schematic expression diagrams of the four target mRNAs (FoxD3, EphA4, Sox10, Dmbx1). Figure 5B shows four-channel confocal micrographs of the head and neural crest. Figure 5C shows a magnified view of the region shown in Figure 4B. Figure 5D shows the four individual channels from Figure 5C along with signal-to-background measurements. Probe set: 12-20 bare, unoptimized fragmentary initiator probes per target. Amplifiers: Four orthogonal HCR amplifiers carrying spectrally distinct fluorophores (one HCR amplifier per target). Fixed embryo: Stage HH10.

[0194] Example 4 – HCR RNA-FISH using fragmentary initiator probes for quantitative analysis of mRNA expression at intracellular resolution Figures 6A and 6B demonstrate that HCR RNA-FISH using fragmentary initiator probes enables quantitative analysis of mRNA expression at intracellular resolution within whole-mount chicken embryos. Each target mRNA was detected redundantly using two sets of fragmentary initiator probes, each triggering a different, spectrally distinct HCR amplification agent. Plotting a two-channel scatter plot of normalized voxel intensities revealed a tight linear relationship with nearly zero intercepts, indicating that the HCR signal changed proportionally to the number of target mRNAs per imaging voxel. Accuracy improved as the distribution became more linear and the intercepts disappeared, and as the scatter narrowed. 2×2 μm voxels provided cell-level resolution. These results demonstrate that HCR RNA-FISH using fragmentary initiator probes enables accurate and precise relative quantification of mRNA expression at intracellular resolution in an anatomical context without the need for probe set optimization. Figure 6A shows the two-channel redundant detection of target mRNA. Targets: Dmbx1 and EphA4. Confocal microscope: 0.2 × 0.2 μm pixels. Probe set: 20 pairs of fragmentary initiator probes for each target channel. Amplifiers: Two orthogonal HCR amplifiers carrying spectrally distinct fluorophores for each target. Fixed embryos: Stage HH10. Figure 6B shows highly correlated normalized signals (Pearson correlation coefficient, r) for 2 × 2 μm voxels within the selected region of Figure 6A.

[0195] Example 5 – Single-molecule mRNA imaging in whole-mount chicken embryos using HCR RNA-FISH Figure 7 shows single-molecule mRNA imaging using HCR RNA-FISH in whole-mount chicken embryos. Duplicate detection of target mRNA Dmbx1 in whole-mount chicken embryos. Probe set: 25 fragmentary initiator probes per channel. Lobe pair. No probe set optimization. Pixel size: 0.1 × 0.1 μm. Embryos fixed at stage HH8. Each target mRNA was detected using two probe sets, each probe set initiating spectrally distinct orthogonal HCR amplifiers (Ch1, Alexa 647, Ch2, Alexa 594). Representative field of view from confocal micrographs. Gray circles in the left panel: Dots detected in Ch1. Gray circles in the center panel: Dots detected in Ch2. White circles in the right panel: Dots detected in both channels. Co-localization represents the proportion of dots within a single channel that were detected in both channels (mean ± SEM, N=3 embryos).

[0196] Example 6 – Multiplex imaging of protein targets in FFPE mouse brain sections Figures 9A-9B and 10A-10B show multiplex HCR immunofluorescence (IF) for imaging protein targets in formalin-fixed paraffin-embedded (FFPE) mouse brain sections.

[0197] Figures 9A-9B show the use of initiator-labeled primary antibody probes, as shown in Figure 8A. Figure 8A shows a two-step HCR IF protocol consisting of a protein detection step (initiator-labeled primary antibody probe binds to the protein target, followed by washing) and an amplification step (initiator triggers self-assembly of fluorophore-labeled HCR hairpins into a linked fluorescence amplification polymer, followed by washing). Figure 8B shows a multiplexing timeline in which the same two-step protocol is used regardless of the number of target proteins. Figure 9A shows a 4-channel epifluorescence micrograph of FFPE mouse brain sections (four target proteins: TH, GFAP, MBP, MAP2), and Figure 9B shows a magnified view of the region shown in Figure 9A. The approach in Figures 9A-9B has the advantage of achieving a high level of multiplexing using initiator-labeled primary antibodies, which carry orthogonal HCR initiators in which antibody probes for different protein targets operate simultaneously and independently within the sample. Because it is not necessary to generate primary antibodies in different organisms, it becomes more flexible to construct large libraries of primary antibodies against various targets.

[0198] Figures 10A and 10B show unmodified primary antibody probes and initiator-labeled secondary antibody probes, as shown in Figure 8C. Figure 8C shows a two-step HCR IF protocol consisting of a protein detection step (primary antibody probe binds to the protein target, washes, initiator-labeled secondary antibody probe binds to the primary antibody probe, washes) and an amplification step (initiator triggers self-assembly of fluorophore-labeled HCR hairpins into a fluorescence amplification polymer, washes). Figure 8D shows a multiplexed timeline where the same two-step protocol is used regardless of the number of target proteins. Figure 10 shows quadruple HCR IF in FFPE mouse brain sections. Figure 10A shows a 4-channel epifluorescence microscope image of FFPE mouse brain sections (four target proteins: TH, GFAP, PVALB, MBP), and Figure 10B shows a magnified view of the region shown in Figure 10A.

[0199] The advantage of the approach shown in Figures 10A and 10B is that it allows the use of validated libraries of initiator-labeled secondary antibodies, where different secondary antibodies support orthogonal HCR initiators that operate simultaneously and independently within the sample, without modifying the primary antibody. In this approach, the primary antibodies must be of different isotypes or produced in different organisms so that only one type of initiator-labeled secondary antibody can detect each primary antibody.

[0200] Example 7 – Quantitative analysis of protein expression at intracellular resolution using HCR IF Figures 11A–11C demonstrate that HCR IF enables quantitative analysis of protein expression at intracellular resolution in an anatomical context. Each target protein was detected using two initiator-labeled primary antibody probes, each triggering a spectrally distinct HCR amplification agent. Two-channel scatter plots of normalized voxel intensities are shown. Lotting revealed a tight linear relationship with nearly zero intercepts, indicating that the HCR signal changed proportionally to the number of target proteins per imaging voxel. Figure 11A shows the two-channel overlap detection of the target protein. The target protein was detected using two initiator-labeled primary antibody probes that bind to different epitopes, each spectrally distinct from an orthogonal HCR amplifier (Ch1: Alexa 546, Ch2: Alexa 546). Initiate step 647). Figure 11B shows an epifluorescence microscope image of an FFPE mouse brain section. Figure 11C shows the highly correlated normalized signals (Pearson correlation coefficient, r) of intracellular 2×2 μm voxels within the area enclosed by the rectangle in Figure 11B. In this scatter plot, precision corresponds to linearity where the intercept is zero, and precision corresponds to scatter around the line. The 2×2 μm voxels provided cell-level resolution. These results demonstrate that HCR IF enables accurate and precise relative quantification of protein expression at intracellular resolution in an anatomical context.

[0201] Example 8 – Multiplex HCR RNA-FISH / IF with simultaneous HCR signal amplification of all mRNA and protein targets Figures 12A-12B and 13A-13B show multiplex HCR RNA-FISH / IF in which HCR signal amplification was performed simultaneously on all mRNA and protein targets.

[0202] Figures 12A and 12B show multiplex HCR RNA-FISH / IF using initiator-labeled primary antibody probes and fragmentary initiator DNA probes, as shown in Figure 8E. Figure 8E shows a three-step HCR RNA-FISH / IF protocol consisting of a protein detection step (initiator-labeled primary antibody probe binds to the protein target, wash), an RNA detection step (fragmentary initiator DNA probe binds to the RNA target, wash), and an amplification step (initiator triggers self-assembly of fluorophore-labeled HCR hairpins into a fluorescent amplification polymer, wash). The same three-step HCR RNA-FISH / IF protocol is used regardless of the number of target proteins and target RNAs. Figure 12A shows a 4-channel epifluorescence microscope image of an FFPE mouse brain section (two target proteins (TH, MBP) and two target mRNAs (Prkcd, Slc17a7)), and Figure 12B shows a magnified view of the region shown in Figure 12A.

[0203] Figures 13A and 13B show multiplex HCR RNA-FISH / IF using a primary antibody probe, an initiator-labeled secondary antibody probe, and a fragmentary initiator DNA probe. Figure 8F shows a three-step HCR RNA-FISH / IF protocol consisting of a protein detection step (primary antibody probe binds to the protein target, washes, initiator-labeled secondary antibody probe binds to the primary antibody probe, washes), an RNA detection step (fragmentary initiator DNA probe binds to the RNA target, washes), and an amplification step (initiator triggers self-assembly of fluorophore-labeled HCR hairpins into a fluorescent amplification polymer, washes). The same three-step HCR RNA-FISH / IF protocol is used regardless of the number of target proteins and target RNAs. Figure 13A shows a 4-channel epifluorescence microscope image of an FFPE mouse brain section (containing two target proteins (TH, MBP) and two target mRNAs (Prkcd, Slc17a7)), and Figure 13B shows a magnified view of the region shown in Figure 13A.

[0204] Example 9 – Optimization of a coordinated probe junction to enhance the suppression and conversion of fragmented initiator HCRs Figures 31A to 31C illustrate the use of coordinated probe junctions to enhance fragmentary initiator HCR suppression (off state) and conversion (on state). In Figure 31A, fragmentary initiator probes p1 and p2 are complementary to the overlapping region of the HCR hairpin h1 (containing domains u* and v*, which are complementary to domains u and v of h1, respectively), and the junction The cushion is relaxed to achieve an energetically favorable conformation, reducing the dynamic barrier when opening the first HCR hairpin and initiating HCR polymerization. In Figure 31B, leakage (off state) is assayed on an agarose gel using probes with varying degrees of overlap (u* = 0, 1, or 2 nucleotides, v* = 0, 1, or 2 nucleotides). The off state is characterized by testing for HCR initiation with individual probes p1 or p2 (Figure 31B, left) and quantification of the resulting polymerization band in the gel (Figure 31B, right). In Figure 31C, conversion (on state) is assayed on an agarose gel using probes with varying degrees of overlap (u* = 0, 1, or 2 nucleotides, v* = 0, 1, or 2 nucleotides). The on state is characterized by testing for HCR initiation with probes p1 and p2 and the target (Figure 31C, left) and quantification of the resulting polymerization band in the gel (Figure 31C, right). The reaction conditions in Figure 28 were as follows: when hairpins h1 and h2 were off, the concentrations were 500 nM each; when they were on, the concentrations were 60 nM each (all lanes); and the initiator i1, probes p1 and p2, and / or DNA target were at 0.01 times the hairpin concentration.

[0205] By testing different junction designs, probes p1 and p2 were generated in a clean off state (fragmentary initiator HCR suppression was approximately 0.5% of standard initiator HCR suppression, within the frame in Figure 31B) and a strong on state (fragmentary initiator HCR conversion was approximately 99% of standard initiator HCR conversion, within the frame in Figure 31C). These results demonstrate that replacing the standard probe (v2.0) with a pair of fragmentary initiator probes (v3.0) dramatically reduces the amplified background (lane 2 vs. lanes 4 and 9 in Figure 31B) without significantly reducing the amplified signal (lane 2 vs. lane 6 in Figure 31C). In this probe junction, the domain dimensions are u*=0 nucleotides and v*=2 nucleotides, which corresponds to a fragmentary initiator within the probe unit hybridizing to the binding site of the HCR hairpin h1, where two nucleotides overlap.

[0206] Example 10 - Dehybridization of HCR hairpins from HCR amplification polymer using auxiliary chains Figure 32A shows in vitro verification of dehybridization of HCR hairpins from HCR-amplified polymers using an auxiliary chain. Reaction conditions: Hairpins h1 and h2 at 0.6 μM each (lanes 1-5); HCR initiator i1 at 0.6 μM (lane 2), 60 nM (lane 3), and 6 nM (lanes 4 and 5); auxiliary chain at 6 μM (lane 5); 5 × SSCT buffer; HCR amplification overnight at room temperature; auxiliary chain incubated at room temperature for 1 hour. Hairpins h1 and h2 labeled with Alexa 647 fluorophores. Lane 1: Metastable hairpins h1 and h2 show minimal leakage in the absence of HCR initiator i1. Lane 2: Formation of short HCR-amplified polymers at equimolar HCR initiator i1 concentrations. Lanes 3 and 4: Formation of longer HCR-amplified polymers at lower HCR initiator concentrations. Lane 5: Dehybridization of hairpins from the HCR-amplified polymer using auxiliary chains generates polymer fragments consisting of h1, h2, and auxiliary chains.

[0207] Table 1 below shows some embodiments of sequences used for dehybridization of HCR hairpins from HCR amplification polymers (e.g., Figure 32A). These sequences can be used in any embodiment of the methods described herein. [Table 1]

[0208] Figure 32B shows imaging of three target mRNAs in a pellet of FFPE-cultured human cells (HEK293) on a slide using a repeat reporter detection method. Target mRNAs: GAPDH, ACTB, U6. All three target probe sets were introduced simultaneously during the detection phase. In the first round of amplification, only GAPDH and ACTB were detected using amplification agents labeled with spectrally distinct fluorophores (amplifier 1 was labeled with reporter 1 (Alexa 647) for GAPDH, and amplification agent 2 was labeled with reporter 2 (Alexa 546) for ACTB). Only amplification agent 1, used for GAPDH, contained the toehold for auxiliary strand nucleation. After image acquisition, the sample was incubated with the auxiliary strand of amplification agent 1 at room temperature for 1 hour, and the Alexa 647-labeled hairpin was dehybridized from the amplification polymer linked to the GAPDH mRNA, and the resulting polymer fragment was washed away from the sample. In the second round of amplification, the U6 signal was amplified using another HCR amplifier labeled with the same reporter as GAPDH (Amplifier 3 labeled with reporter 1 (Alexa 647)).

[0209] The image shows that the cytoplasmic reporter 1 (Alexa 647) signal of GAPDH was completely removed by the addition of the auxiliary chain of amplifier 1, followed by washing, and replaced by the nuclear reporter 1 (Alexa 647) signal of U6 in the second round of imaging. The ACTB signal intensity (amplifier 2 labeled with reporter 2 (Alexa 546)) remained comparable between rounds of imaging.

[0210] Example 11 - Ultra-high sensitivity HCR signal amplification using CARD Figures 33 and 35A show ultra-sensitive HCR RNA-CISH using catalytic reporter deposition (CARD). RNA targets are detected using fragmentary initiator probes that specifically bind to the corresponding cognitive binding site on the target, colocalizing the complete HCR initiator i1. HCR signal amplification is performed using reporter-labeled HCR amplifiers. CARD is mediated by an anti-reporter enzyme-labeled readout probe that acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target.

[0211] Figures 34A and 34B show ultra-high sensitivity HCR using branched DNA (bDNA) CARD in 5 μm FFPE tissue sections compared with weak staining using CARD and damaged tissue morphology. This shows RNA-CISH. HCR RNA-CISH does not require protease pretreatment and preserves the morphology of the sample. On the other hand, bDNA CARD requires protease pretreatment in order for the large bDNA preamplifier and amplification reagent to penetrate the sample.

[0212] Figure 35B shows the reagents for quadruple HCR RNA-CISH using CARD with the approaches shown in Figures 33 and 35A. The j-th target (j=1, ..., 4, where j is a positive integer) is the j-th fragmentary initiator probe that colocalizes the j-th complete initiator when the probe specifically binds to the corresponding cognitive binding site of the j-th target. Detection is performed using a 3D printer. Signal amplification of the j-th target is provided by the j-th HCR amplifier labeled with the j-th reporter. Readout of the j-th target is provided by the j-th anti-reporter readout probe labeled with an enzyme that acts on the j-th CARD substrate to mediate the deposition of the j-th CARD reporter near the j-th target.

[0213] Figures 36A and 36B show dual HCR RNA-CISH by CARD in 5 μm FFPE mouse duodenal tissue sections using the approaches shown in Figures 33 and 35A and the reagents shown in Figure 35B.

[0214] Figure 38A shows nonlinear HCR RNA-CISH using CARD (Approach 1). RNA targets are detected using fragmentary initiator probes that specifically bind to the corresponding cognitive binding site on the target, colocalizing the complete HCR initiator. In the linear amplification stage, HCR signal amplification is performed using a reporter-labeled first HCR amplifier. Bridging to the nonlinear amplification stage is performed using an anti-reporter primary antibody bridging probe, followed by an anti-primary initiator-labeled secondary antibody bridging probe. In the nonlinear amplification stage, HCR signal amplification is performed using a reporter-labeled second HCR amplifier. CARD is mediated by an anti-reporter enzyme-labeled readout probe that acts on the CARD substrate to catalyze the deposition of the CARD reporter near the target.

[0215] Figure 38B shows nonlinear HCR RNA-CISH (Approach 2) using CARD, employing a fragmentary initiator probe for detecting RNA targets, a self-bridging HCR amplifier in a linear amplification step that colocalizes complete HCR initiator i3 and complete HCR initiator i4 during polymerization (each hairpin carries two split initiator tails), and an anti-reporter enzyme-labeled readout probe for mediating CARD.

[0216] Figures 39A and 39B show ultra-high-sensitivity HCR IHC using direct detection (Figure 39A) or indirect detection (Figure 39B) of protein targets with initiator-labeled antibody signal probes, HCR signal amplification with reporter-labeled HCR amplifiers, and then CARD signal amplification with anti-reporter enzyme-labeled readout probes. Figures 40A and 40B show that sensitivity is enhanced when using HCR IHC with CARD compared to two conventional IHC products.

[0217] Figures 41A and 41B show the reagents for quadruple HCR IHC using CARD. Figure 42 shows double HCR IHC using CARD in 5 μm FFPE human tonsil tissue.

[0218] Figure 43 shows an overview of the multiplex HCR RNA-CISH / IHC protocol using CARD for simultaneous imaging of RNA and protein targets within the same sample.

[0219] Protease pretreatment is not required for the small HCR amplification hairpin to penetrate 5 μm FFPE tissue sections. This prevents the degradation of protein targets as part of the RNA imaging workflow, allowing protein targets to be imaged using conventional IHC protocols following HCR RNA-CISH. Figure 44A (left) shows the results of performing conventional IHC without protease pretreatment after HCR RNA-CISH using CARD. High signal-to-background ratios are observed for both RNA and protein targets. Figure 44A (right) shows the decrease in protein signal that occurs when the same experiment is performed with a protease pretreatment step from the bDNA RNA-CISH product. (This results in damage to protein targets). Figure 44B shows HCR RNA-CISH / conventional IHC in 5 μm FFPE human breast cancer tissue without protease pretreatment.

[0220] Example 12 - HCR imaging using a reporter-labeled signal probe Figures 56A to 56N and 57A to 57B show target detection using reporter-labeled signal probes that bind directly or indirectly to the target. Figures 58A and 59A to 59F show the binding of reporter-labeled signal probes to anti-reporter initiator-labeled signal probes. Figure 58B shows the binding of reporter-labeled signal probes to two anti-reporter fragment initiator signal probes.

[0221] Figures 58C and 68D show the detection of two targets within or in proximity of a complex using a pair of anti-target reporter-labeled signal probes, a pair of anti-reporter fragment initiator signal probes, and a proximity probe. Figures 68F to 68I show the detection of two targets within or in proximity of a complex using a proximity probe and different signal probe compositions for each of the two targets within or in proximity of the complex, where at least one of the two targets within or in proximity of the complex is detected using a reporter-labeled signal probe, and that reporter-labeled signal probe is detected by an anti-reporter signal probe.

[0222] Figure 60A shows the use of reporter-labeled probes for HCR IHC using CARD. The target protein is detected with an anti-target reporter-labeled primary antibody signal probe, and then with an anti-reporter initiator-labeled primary antibody signal probe. Signal amplification is performed using reporter-labeled HCR amplifiers. These reporters are then bound to an anti-reporter enzyme-labeled readout probe that mediates CARD signal amplification.

[0223] Figure 60B shows the reagents for quadruple HCR IHC using CARD with the approach in Figure 60A. The j-th target (j=1, ..., 4, where j is a positive integer) is detected using the j-th anti-target primary antibody labeled with the j-th reporter, which is then conjugated by the j-th anti-reporter primary antibody labeled with the j-th initiator. Signal amplification of the j-th target is provided by the j-th HCR amplifier labeled with the j-th reporter. Readout of the j-th target is provided by the j-th anti-reporter readout probe, which is labeled with an enzyme that acts on the j-th CARD substrate to mediate the deposition of the j-th CARD reporter near the j-th target.

[0224] Figure 60C shows the use of reporter-labeled probes for enzyme-free nonlinear HCR IF. The target protein is detected with an anti-target reporter-labeled primary antibody signal probe, and then with an anti-reporter initiator-labeled primary antibody signal probe. In the linear amplification step, signal amplification is performed using a reporter-labeled first HCR amplifier. Bridging to the nonlinear amplification step is provided by an anti-reporter initiator-labeled primary antibody bridging probe. In the nonlinear amplification step, signal amplification is performed using a fluorophore-labeled second HCR amplifier.

[0225] Figure 60D shows the reagents for quadruple enzyme-free nonlinear HCR IF using the approach in Figure 60C. The j-th target (j=1, ..., 4, where j is a positive integer) is detected using the j-th anti-target primary antibody labeled with the j-th reporter, and the j-th reporter is detected with the j-th anti-reporter primary antibody labeled with the j-th initiator. In the linear amplification stage, signal amplification of the j-th target is provided by the j-th primary HCR amplifier labeled with the j-th reporter. Bridging to the nonlinear amplification stage is performed by a bridging probe of the j-th anti-reporter primary antibody labeled with the j-th initiator. This is then provided for the j-th target. In the nonlinear amplification stage, signal amplification of the j-th target is provided by the j-th second HCR amplifier labeled with the j-th fluorophore.

[0226] Figure 66 shows a multiplex HCR IF using reporter-labeled probes according to the approaches in Figures 58A and 59C. The j-th target (j=1, 2) is detected using a signal probe of the j-th anti-target primary antibody labeled with the j-th reporter, and the j-th reporter is detected with a signal probe of the j-th anti-reporter primary antibody labeled with the j-th initiator. Signal amplification of the j-th target is provided by the j-th HCR amplifier labeled with the j-th fluorophore.

[0227] Example 13 - Ultra-high sensitivity enzyme-free nonlinear HCR signal amplification Figures 45 and 47A show enzyme-free HCR RNA-FISH (Approach 1) using a fragmentary initiator probe for detecting RNA targets, a reporter-labeled HCR amplifier in the linear stage, an anti-reporter primary antibody bridging probe, followed by an anti-primary initiator-labeled secondary antibody bridging probe, followed by a fluorophore-labeled amplifier in the nonlinear amplification stage.

[0228] Figure 46 shows the enhanced sensitivity achieved using nonlinear HCR signal amplification (following Approach 1 in Figures 45 and 47A) compared with linear HCR signal amplification (left) in FFPE human colorectal cancer sections (right).

[0229] Figure 47B shows the reagents for quadruple enzyme-free nonlinear HCR RNA-FISH using Approach 1 in Figures 45 and 47A. The j-th target (j=1, ..., 4, where j is a positive integer) is detected using a set of j-th fragmentary initiator probes that colocalize the j-th complete HCR initiator when specifically bound to the cognitive binding site on the target. In the linear amplification stage, signal amplification of the j-th target is provided by the j-th first HCR amplifier labeled with the j-th reporter. Bridging to the nonlinear amplification stage is provided for the j-th target by the j-th anti-reporter primary antibody bridging probe, followed by the j-th anti-primary-secondary antibody bridging probe labeled with the j-th initiator. In the nonlinear amplification stage, signal amplification of the j-th target is provided by the j-th second HCR amplifier labeled with the j-th fluorophore.

[0230] Figure 48 shows triple imaging of target mRNA in FFPE mouse duodenal sections using enzyme-free nonlinear HCR signal amplification, following Approach 1 in Figures 45 and 47A, with the multiplexing reagent in Figure 47B.

[0231] Figure 49A shows enzyme-free HCR RNA-FISH (Approach 2) using a fragmentary initiator probe for detecting the RNA target, a reporter-labeled HCR amplifier in the linear amplification stage, an anti-reporter initiator-labeled primary antibody bridging probe, followed by a fluorophore-labeled HCR amplifier in the nonlinear amplification stage.

[0232] Figure 67 shows the enhanced sensitivity achieved using nonlinear HCR signal amplification (following Approach 2 in Figure 49A) compared with linear HCR signal amplification (left) in FFPE human tonsil tissue sections (right).

[0233] Figure 49B shows enzyme-free HCR RNA-FISH (Approach 3) using a fragmented initiator probe to detect the RNA target, and a self-bridging HCR amplifier during the linear amplification step that colocalizes complete HCR initiator i3 and complete HCR initiator i4 during polymerization (each hairpin contains two split initiator tails). ), and a fluorophore-labeled HCR amplifier in the nonlinear amplification stage are shown.

[0234] Figures 51A and 52A show an enzyme-free HCR IF using an anti-target initiator-labeled primary antibody signal probe for detecting the target protein, a reporter-labeled HCR amplifier in the linear amplification stage, an anti-reporter initiator-labeled primary antibody bridging probe, followed by a fluorophore-labeled HCR amplifier in the nonlinear amplification stage.

[0235] Figure 52B shows the reagents for quadruple enzyme-free nonlinear HCR IF using the approaches of Figures 51A and 52A. The j-th target (j=1, ..., 4, where j is a positive integer) is detected using a j-th anti-target primary antibody probe labeled with the j-th initiator. In the linear amplification stage, signal amplification of the j-th target is provided by a j-th first HCR amplifier labeled with the j-th reporter. Bridging to the nonlinear amplification stage is provided for the j-th target by a j-th anti-reporter primary antibody bridging probe containing the j-th initiator. In the nonlinear amplification stage, signal amplification of the j-th target is provided by a j-th second HCR amplifier labeled with the j-th fluorophore.

[0236] Figure 53 shows the dual enzyme-free HCR IF in FFPE human tonsil tissue sections.

[0237] Figure 54 shows an overview of the enzyme-free nonlinear HCR RNA-FISH / IF protocol. In the protein detection stage, the j-th protein target is detected by a signal probe of the j-th anti-target primary antibody labeled with the j-th initiator. In the RNA detection stage, the j-th RNA target is detected using a set of j-th fragmentary initiator probes that colocalize the j-th initiator when specifically hybridized to the cognitive binding site on the j-th RNA target. In the linear amplification stage, signal amplification of the j-th target (j is a positive integer) is provided by the j-th first HCR amplifier labeled with the j-th reporter. Bridging between the linear and nonlinear amplification stages of the j-th target is provided by a bridging probe of the j-th anti-reporter primary antibody labeled with the j-th initiator. In the nonlinear amplification stage, signal amplification of the j-th target is provided by the j-th second HCR amplifier labeled with the j-th fluorophore.

[0238] Figure 55 shows enzyme-free HCR RNA-FISH / IF in FFPE human breast cancer tissue sections.

[0239] Figure 70 outlines a protocol for simultaneous HCR imaging of protein targets, protein:protein target complexes, and RNA targets using enzyme-free nonlinear HCR signal amplification for all target classes. In the protein detection stage, when imaging protein targets individually, the j-th protein target is detected by the j-th anti-target primary antibody signal probe, and then by the j-th anti-primary / secondary antibody signal probe labeled with the j-th initiator (where j is a positive integer). Alternatively, when imaging the protein target as part of the j-th protein:protein target complex, the protein is detected by the anti-target primary antibody signal probe, and then by the j-th anti-primary / secondary antibody signal probe labeled with the j-th fragmentary initiator. In the proximity stage, the j-th complete HCR initiator is co-localized by hybridizing the j-th proximity probe to the j-th fragmentary initiator signal probe indirectly bound to the target protein within the j-th target complex. In the RNA detection stage, the j-th RNA target is detected using a set of j-th fragmentary initiator probes that co-localize the j-th initiator when specifically hybridized to the cognitive binding site on the j-th RNA target. In the linear amplification stage, signal amplification of the j-th target is provided by the j-th first HCR amplifier labeled with the j-th reporter. Bridging between the amplification phase and the nonlinear amplification phase is provided by a bridging probe of the j-th anti-reporter primary antibody labeled with the j-th initiator. In the nonlinear amplification phase, signal amplification of the j-th target is provided by a j-th second HCR amplifier labeled with the j-th fluorophore.

[0240] Figures 16C and 71 illustrate enzyme-free nonlinear HCR signal amplification for the detection of RNA targets, protein targets, and / or target complexes. In the detection stage, targets are detected by either a fragmentary initiator probe (and optionally a proximity probe) or an initiator-labeled probe. In the linear amplification stage, signal amplification is provided by a self-bridging type 4-hairpin HCR amplifier (containing hairpins h1, h2, h3, and h4), each hairpin containing a single split initiator tail, and initiation by HCR initiator i1 triggers the growth of an amplification polymer containing periodic hairpins h1, h2, h3, and h4, where h1 and h3 colocalize with a complete HCR initiator i5 linked to the HCR amplification polymer, and h2 and h4 colocalize with a complete HCR initiator i6 linked to the HCR amplification polymer. In the nonlinear amplification stage, signal amplification is carried out by a fluorophore-labeled HCR amplifier (containing hairpins h5 and h6), which is initiated by colocalizing complete HCR initiators i5 and i6 periodically arranged along an HCR amplification polymer containing periodically repeating hairpins h1, h2, h3, and h4, leading to the growth of new HCR amplification polymers containing periodically repeating hairpins h5 and h6, each linked to a colocalizing complete HCR initiator i5 or i6, along the HCR amplification polymer containing the periodically repeating hairpins h1, h2, h3, and h4.

[0241] Figures 72A–72C show the enhanced sensitivity of HCR RNA-FISH achieved by using nonlinear HCR signal amplification (right, following the approach in Figure 71) compared with linear HCR signal amplification (left) for three different RNA targets in HeLa cells. ACTB target (highly expressed mRNA): 10 fragmentary initiator probe pairs. HTT target (lowly expressed mRNA): 30 fragmentary initiator probe pairs. U6 (highly expressed small nuclear RNA): 2 fragmentary initiator probe pairs.

[0242] Figure 73 shows the detection of the target by enzyme-free nonlinear HCR signal amplification with three rounds of HCR signal amplification. The target is detected by an anti-target initiator-labeled probe. The first round of HCR signal amplification (also referred to as the linear amplification stage) is performed using a reporter-labeled HCR amplifier. Bridging to the second round of HCR signal amplification is performed using an anti-reporter initiator-labeled bridging probe. The second round of HCR signal amplification (also referred to as the first nonlinear amplification stage or second-order amplification stage) is performed using the same reporter-labeled HCR amplifier used for the first round of HCR signal amplification. Bridging to the third round of HCR signal amplification is performed using the same anti-reporter initiator-labeled bridging probe used for bridging between the first and second rounds of HCR signal amplification. The third round of HCR signal amplification (also referred to as the second round of nonlinear signal amplification or third-order amplification stage) is performed using a fluorophore-labeled HCR amplifier having the same sequence as the reporter-labeled HCR amplifier used in the first and second rounds of HCR signal amplification.

[0243] Additional arrangements Arrangement 1. Hybridization chain reaction (HCR) method, a) providing a sample containing a target; b) contacting the sample with a probe set comprising one or more probe units, each containing two or more fragmentary initiator probes; c) contacting the sample with an HCR amplifier labeled with a reporter; and d) the reporter A method comprising detecting a signal directly or indirectly from a reporter, wherein each of the two or more fragmentary initiator probes comprises a target binding region, a linker region, and a fragmentary initiator, the target binding regions on the two or more fragmentary initiator probes are configured to bind to different binding sites on the target, the linker regions on the two or more fragmentary initiator probes are configured to bind to each other, the fragmentary initiators on the two or more fragmentary initiator probes are configured to bind to different binding sites on an HCR hairpin, the probes within each probe unit are configured to form a cooperative probe junction when binding to the target and the HCR hairpin, the HCR amplifier comprises two or more HCR hairpins; one HCR hairpin comprises an input domain comprising a single-stranded toehold and a stem section, and an output domain comprising a single-stranded loop and a complement of the stem section, and at least one HCR hairpin further comprises a reporter.

[0244] Arrangement 2: The method according to Arrangement 1, wherein the washing process is performed between any of steps (b) to (d).

[0245] Arrangement 3: The method according to either arrangement 1 or 2, wherein each of the two or more fragmentary initiator probes further comprises a neighboring domain.

[0246] Arrangement 4: The method according to any one of arrangements 1 to 3, wherein the proximity domain is configured to bind to the proximity probe.

[0247] Arrangement 5: The method according to any one of Arrangements 1 to 4, further comprising bringing the sample into contact with one or more proximity probes after step (b) and before step (c).

[0248] A method according to any of arrangements 1 to 5, wherein after providing arrangement 6:1 or multiple proximity probes, a washing step is performed to remove unbound proximity probes.

[0249] Arrangement 7: The method according to any one of arrangements 1 to 6, wherein the coordinated probe junction further includes the one or more proximity probes.

[0250] Arrangement 8: The method according to any one of Arrangements 1 to 7, wherein one or more auxiliary reporter-labeled readout probes are provided after step (c).

[0251] The method according to any of arrangements 1 to 8, wherein after providing arrangement 9:1 or a plurality of auxiliary reporter-labeled readout probes, a cleaning step is performed to remove unbound auxiliary reporter-labeled readout probes.

[0252] Arrangement 10: The method according to any one of Arrangements 1 to 9, further comprising detecting a signal from the auxiliary reporter.

[0253] Arrangement 11: The method according to any one of arrangements 1 to 10, further comprising step (e) in which the signal is removed after step (d).

[0254] Arrangement 12: The method according to any one of Arrangements 1 to 11, further comprising repeating any of the steps of the method to detect another target in the sample, wherein the target-binding domain is for a different target.

[0255] Arrangement 13: Target binding on the two or more fragmentary initiator probes The method according to any one of arrangements 1 to 12, wherein the region is configured to bind to the overlapping binding site on the target.

[0256] Arrangement 14: The method according to any one of arrangements 1 to 13, wherein the target binding regions on the two or more fragmentary initiator probes are configured to bind to non-overlapping binding sites on the target.

[0257] Arrangement 15: The method according to any one of arrangements 1 to 14, wherein the fragment initiators on the two or more fragment initiator probes are configured to bind to the overlapping binding sites on the HCR hairpin.

[0258] Arrangement 16: The method according to any one of arrangements 1 to 15, wherein the fragment initiators on the two or more fragment initiator probes are configured to bind to non-overlapping binding sites on the HCR hairpin.

[0259] Arrangement 17: The method according to any one of Arrangements 1 to 16, wherein the target includes an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

[0260] Arrangement 18: The reporter and the auxiliary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carburetors A method according to any one of arrangements 1 to 17, comprising: bombrak (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

[0261] Arrangement 19: A composition for detecting a target in a sample, comprising: an anti-target signal probe comprising a reporter and configured to bind to the target; an anti-reporter signal probe labeled with an initiator and configured to bind to the reporter; and an HCR amplifier labeled with an auxiliary reporter and configured to be triggered by the initiator to grow an auxiliary reporter-modified HCR amplification polymer linked to the target, wherein the auxiliary reporter directly or indirectly mediates the generation of a signal.

[0262] Arrangement 20: The composition according to Arrangement 19, further comprising an anti-auxiliary reporter tertiary reporter labeled readout probe configured to bind to the auxiliary reporter modified amplification polymer.

[0263] Arrangement 21: A composition according to any one of the embodiments of Arrangement 19 or 20, wherein the tertiary reporter directly or indirectly ...

Claims

1. Hybridization chain reaction (HCR) method, a) To provide a sample containing the target; b) Contacting the sample with a probe set comprising one or more probe units, each containing two or more fragmentary initiator probes; c) Contacting the sample with an HCR amplifier labeled with a reporter; and d) Including detecting a signal directly or indirectly from the reporter, Each of the two or more fragmented initiator probes is It includes a target binding region, a linker region, and a fragmentary initiator. The target binding regions on the two or more fragmentary initiator probes are configured to bind to different binding sites on the target. The linker regions on the two or more fragmentary initiator probes are configured to connect with each other. The fragment initiators on the two or more fragment initiator probes are configured to bind to different binding sites on the HCR hairpin. The probes within each probe unit are configured to form a coordinated probe junction when coupled to the target and the HCR hairpin. The HCR amplifier comprises two or more HCR hairpins, One HCR hairpin includes an input domain comprising a single-strand toehold and a stem section, and an output domain comprising a single-strand loop and a complement of the stem section, and A method in which at least one HCR hairpin includes further reporters.

2. The method according to claim 1, wherein the cleaning step is performed during any of steps (b) to (d).

3. The method according to claim 1, wherein each of the two or more fragmentary initiator probes further comprises a neighboring domain.

4. The method according to claim 3, wherein the proximity domain is configured to couple with the proximity probe.

5. The method according to claim 4, further comprising bringing the sample into contact with one or more proximity probes after step (b) and before step (c).

6. The method according to claim 5, wherein after providing one or more proximity probes, a cleaning step is performed to remove any uncoupled proximity probes.

7. The method according to claim 5, wherein the coordinated probe junction further comprises the one or more proximity probes.

8. The method according to claim 1, wherein one or more auxiliary reporter-labeled readout probes are provided after step (c).

9. The method according to claim 8, wherein a cleaning step is performed after providing one or more auxiliary reporter-labeled readout probes to remove uncoupled auxiliary reporter-labeled readout probes.

10. The method according to claim 8, further comprising detecting a signal from the auxiliary reporter.

11. The method according to claim 1, further comprising step (e) in which the signal is removed after step (d).

12. The method according to claim 11, further comprising repeating any of the steps of the method to detect another target in the sample, wherein the target-binding domain is for a different target.

13. The method according to claim 1, wherein the target binding regions on the two or more fragmentary initiator probes are configured to bind to overlapping binding sites on the target.

14. The method according to claim 1, wherein the target binding regions on the two or more fragmentary initiator probes are configured to bind to non-overlapping binding sites on the target.

15. The method according to claim 1, wherein the fragment initiators on the two or more fragment initiator probes are configured to bind to overlapping binding sites on the HCR hairpin.

16. The method according to claim 1, wherein the fragment initiators on the two or more fragment initiator probes are configured to bind to non-overlapping binding sites on the HCR hairpin.

17. The method according to claim 1, wherein the target includes an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

18. The method according to claim 8, wherein the reporter and the auxiliary reporter may be the same or different, and each comprises a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, magnetic carbon nanotube, gold nanoparticle (AuNP), gold nanoshell, gold nanorod, silver shell gold nanoparticle, latex, magnetic nanoparticle, silica nanoparticle, fluorophore-supported nanoparticle, dye-supported nanoparticle, hapten, ligand, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzyme, combination thereof, or a molecule that directly or indirectly mediates the generation of a signal.

19. A composition for detecting a target in a sample, a) an anti-target signal probe comprising a reporter and configured to bind to the target; b) an anti-reporter signal probe labeled with an initiator and configured to bind to the reporter; and c) comprising an HCR amplifier, which is labeled with an auxiliary reporter and is triggered by the initiator to grow an auxiliary reporter-modified HCR amplifying polymer linked to the target, The auxiliary reporter is a composition that directly or indirectly mediates the generation of a signal.

20. The composition according to claim 19, further comprising an anti-auxiliary reporter tertiary reporter labeled readout probe configured to bind to the auxiliary reporter modified amplification polymer.

21. The composition according to claim 20, wherein the tertiary reporter directly or indirectly mediates the generation of a signal.

22. The composition according to claim 19, wherein the target comprises an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

23. The reporter, the auxiliary reporter, and the tertiary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetism The composition according to claim 20, comprising molecules, carbon black (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

24. A composition for detecting N targets in a sample, where N is a positive integer. a) The j-th anti-target signal probe (j = 1, ..., N, where j is a positive integer) containing the j-th reporter and configured to bind to the j-th target; b) a j-th anti-reporter signal probe comprising the j-th HCR initiator and configured to bind to the j-th reporter; and c) comprising a j-th auxiliary reporter and configured to grow a j-th auxiliary reporter-modified HCR amplification polymer linked to the j-th target, triggered by the j-th HCR initiator, A composition wherein the j-th auxiliary reporter directly or indirectly mediates the generation of a signal for the j-th target.

25. The composition according to claim 24, further comprising a j-th tertiary reporter and a j-th anti-auxiliary reporter readout probe configured to bind to the j-th auxiliary reporter modified amplification polymer.

26. The composition according to claim 25, wherein the j-th tertiary reporter directly or indirectly mediates the generation of a signal for the j-th target.

27. The composition according to claim 24, wherein the j-th target includes an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

28. The j-th reporter, the j-th auxiliary reporter, and the j-th tertiary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, The composition according to claim 25, comprising magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

29. Hybridization chain reaction (HCR) method, To provide a sample containing the target; The sample is brought into contact with at least one reporter-labeled signal probe, which includes a target binding region and at least one reporter; The sample is brought into contact with at least one anti-reporter initiator-labeled signal probe, which includes a reporter binding region and at least one initiator; The sample is brought into contact with a first HCR hairpin, which includes a first input domain comprising a first toehold and a first stem section, and a first output domain comprising a first hairpin loop and a complement of the first stem section; The sample comprises a second input domain including a second toehold and a second stem section, Contacting a second HCR hairpin which includes a second hairpin loop and a second output domain which includes a complement of the second stem section, wherein at least one of the first HCR hairpin and the second HCR hairpin further includes an auxiliary reporter, and A method comprising detecting a signal directly or indirectly from the reporter and / or the auxiliary reporter.

30. The method according to claim 29, wherein the cleaning step is performed during any of the above steps.

31. The method according to claim 29, wherein the signal is removed after detection.

32. The method according to claim 29, wherein the first HCR hairpin includes a first auxiliary reporter.

33. The method according to claim 32, wherein the first auxiliary reporter directly or indirectly mediates the generation of a signal.

34. The method according to claim 29, wherein the second HCR hairpin includes a second auxiliary reporter.

35. The method according to claim 34, wherein the second auxiliary reporter directly or indirectly mediates the generation of a signal.

36. a) The reporter-labeled signal probe comprises an anti-target primary antibody or a nanobody, b) The reporter includes a hapten, and c) The method according to claim 29, wherein the anti-reporter initiator-labeled signal probe comprises an anti-hapten primary antibody or a nanobody.

37. The method according to claim 29, further comprising: attaching the first HCR hairpin to the at least one initiator; attaching the second HCR hairpin to the first HCR hairpin; contacting the sample with an anti-auxiliary reporter readout probe containing a CARD-mediating enzyme; contacting the sample with one or more CARD substrates; and measuring the signal from one or more deposited CARD reporters generated from the CARD substrates by the CARD-mediating enzyme.

38. The method according to claim 29, wherein the anti-auxiliary reporter readout probe comprises a primary antibody or nanobody that binds to the auxiliary reporter, and further comprises a secondary antibody or nanobody (labeled with one or more CARD-mediated enzymes) that binds to the primary antibody or nanobody.

39. The method according to claim 31, further comprising repeating any of the steps of the method to detect another target in the sample.

40. The method according to claim 29, wherein the target includes an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

41. The reporter and the auxiliary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, ker The method according to claim 29, comprising: bombrak (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

42. A composition for amplification of nonlinear hybridization chain reaction (HCR) signals, a) First HCR initiator; b) A first HCR amplifier comprising two or more HCR hairpins, at least one of which contains a reporter; c) an anti-reporter bridging probe comprising a reporter-binding domain and a second HCR initiator; and d) A second HCR amplifier comprising two or more HCR hairpins, at least one of which includes an auxiliary reporter, A composition wherein the first HCR initiator is configured to trigger the HCR hairpin of the first HCR amplifier to grow a reporter-modified first HCR amplifying polymer linked to the first HCR initiator; the anti-reporter bridging probe is configured to bind to the reporter that modifies the first HCR amplifying polymer and to be modified by the second HCR initiator; the second HCR initiator is configured to trigger the HCR hairpin of the second HCR amplifier to grow an auxiliary reporter-modified second HCR amplifying polymer linked to the first HCR amplifying polymer; and the reporter and / or auxiliary reporter are configured to directly or indirectly mediate the generation of an amplified signal.

43. The composition according to claim 42, wherein the first HCR initiator is coupled to a signal probe configured to bind directly or indirectly to a target.

44. The composition according to claim 42, wherein the first HCR initiator is a colocalized complete first HCR initiator formed when two or more fragmentary initiator probes specifically bind to their cognitive binding sites on a target.

45. The composition according to claim 42, wherein the first HCR initiator is a colocalized complete first HCR initiator formed when two or more fragmentary initiator probes are compounded or specifically bind to their cognitive binding sites on two adjacent targets.

46. The composition according to claim 45, further comprising one or more proximity probes configured to be coupled to the two or more fragmentary initiator probes.

47. The composition according to claim 42, wherein the auxiliary reporter is the same as the reporter.

48. The composition according to claim 42, wherein the first HCR initiator has the same sequence as the second HCR initiator, and the first HCR amplifier has the same sequence as the second HCR amplifier.

49. The composition according to claim 42, wherein the reporter and the auxiliary reporter may be the same or different, and each comprises a hapten, a fluorophore, a chromophore, or a rare earth element or compound.

50. The composition according to claim 42, wherein the auxiliary reporter is configured to mediate catalytic reporter deposition (CARD).

51. The composition according to claim 42, further comprising an anti-auxiliary reporter readout probe containing a tertiary reporter.

52. The composition according to claim 51, wherein the tertiary reporter comprises an enzyme.

53. The composition according to claim 52, wherein the enzyme is configured to act on a CARD substrate to catalytically deposit a CARD reporter that directly or indirectly generates a fluorescent signal or a chromogenic signal.

54. The composition according to claim 42, wherein the target includes an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

55. The reporter, the auxiliary reporter, and the tertiary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetism The composition according to claim 51, comprising molecules, carbon black (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

56. A composition for detecting N targets in a sample using nonlinear HCR signal amplification, wherein N is a positive integer, and the composition is a) The j-th signal probe set (j = 1, ..., N, j is a positive integer) configured to bind to the j-th target, i. A signal probe labeled with the j-th initiator, including the j-th first HCR initiator, or ii. A set of j-th fragment initiator signal probes configured to colocalize the j-th complete first HCR initiator when the probe in the probe unit specifically binds to the cognitive binding site on the j-th target, or iii. A set of j-th fragmentary initiator signal probes configured such that the probe within the probe unit specifically binds to the j-th target complex or the j-th set of nearby targets, and colocalizes the j-th complete first HCR initiator when bound by one or more j-th nearby probes, or iv. A j-th signal probe set comprising a j-th anti-target reporter-labeled primary signal probe containing the j-th reporter, and a j-th anti-reporter initiator-labeled secondary signal probe containing the j-th first HCR initiator; b) A j-th first HCR amplifier comprising two or more HCR hairpins, at least one of which contains the j-th auxiliary reporter; c) a j-th anti-auxiliary reporter bridging probe comprising the j-th auxiliary reporter binding domain and the j-th second HCR initiator; and d) A second HCR amplifier comprising two or more HCR hairpins, at least one of which contains the j-th tertiary reporter, The j-th first HCR initiator is configured to trigger the HCR hairpin constituting the j-th first HCR amplifier to grow a first HCR amplifying polymer modified with a j-th auxiliary reporter linked to the j-th target, and the j-th anti-auxiliary reporter bridging probe is configured to bind to the j-th auxiliary reporter that modifies the j-th first HCR amplifying polymer, thereby modifying the j-th first HCR amplifying polymer with the j-th second HCR initiator, the j-th first A composition wherein the j-th second HCR initiator modifying the HCR amplification polymer is configured to trigger the HCR hairpin constituting the j-th second HCR amplifier to grow a second HCR amplification polymer modified by a j-th tertiary reporter linked to the j-th first HCR amplification polymer, and the j-th reporter, the j-th auxiliary reporter, and / or the j-th tertiary reporter are configured to directly or indirectly mediate the generation of the j-th amplified signal at the site of the j-th target.

57. The composition according to claim 56, wherein the j-th target includes an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

58. The j-th reporter, the j-th auxiliary reporter, and the j-th tertiary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon This includes elementary atoms, chemical linkers, magnetic molecules, carbon black (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation. The composition according to claim 56.

59. A composition for nonlinear HCR signal amplification, a) First HCR initiator; b) A self-bridging type first HCR amplifier comprising two or more HCR hairpins, each containing one split initiator tail; and c) comprising a second HCR amplifier comprising at least one HCR hairpin containing a reporter, A composition wherein the first HCR initiator is configured to trigger the HCR hairpins constituting the self-bridging first HCR amplifier to form a first HCR amplifying polymer linked to the first HCR initiator; the split initiator tails on the two or more HCR hairpins constituting the self-bridging first HCR amplifying polymer are configured to colocalize complete second HCR initiators within the polymer; each of the colocalized complete second HCR initiators within the first HCR amplifying polymer is configured to trigger the HCR hairpins constituting the second HCR amplifier to grow a reporter-modified second HCR amplifying polymer linked to the first HCR amplifying polymer; and the reporter is configured to directly or indirectly mediate the generation of an amplified signal.

60. The composition according to claim 59, wherein the target comprises an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

61. The composition according to claim 59, wherein the reporter comprises a fluorophore, a chromophore, a light-emitting phore, a phosphorescent body, a FRET pair, a member of a FRET pair, a quencher, a fluorophore / quencher pair, a rare earth element or compound, a radioactive molecule, a nucleotide, an amino acid, an oligonucleotide, DNA, RNA, 2'OMe-RNA, a chemically modified nucleic acid, a synthetic nucleic acid analog, a chemically modified protein, a synthetic protein analog, a peptide, a binding substrate, a carbon atom, a chemical linker, a magnetic molecule, carbon black (CB), a carbon nanotube, a magnetic carbon nanotube, a gold nanoparticle (AuNP), a gold nanoshell, a gold nanorod, a silver-shelled gold nanoparticle, a latex, a magnetic nanoparticle, a silica nanoparticle, a fluorophore-supported nanoparticle, a dye-supported nanoparticle, a hapten, a ligand, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, an enzyme, a combination thereof, or a molecule that directly or indirectly mediates the generation of a signal.

62. A method for detecting a target by amplification of a nonlinear hybridization chain reaction (HCR) signal, a) To provide a sample containing one or more targets; b) Bringing the sample into contact with a probe set containing one or more initiator-labeled probes, or with one or more probe units, each containing two or more fragmentary initiator probes, so that the target is bound to the target binding region of the probe set; c) Contacting the sample with a first HCR amplifier labeled with a reporter to initiate a linear HCR amplification step; d) Contacting the sample with an anti-reporter initiator-labeled bridging probe containing one or more HCR initiators; e) Contacting the sample with a second HCR amplifier labeled with an auxiliary reporter to initiate a nonlinear HCR amplification step, and f) Detect one or more signals from the reporter and / or the auxiliary reporter. A method that includes the act of releasing.

63. The method according to claim 62, wherein the probe set includes one or more initiator-labeled probes, and one initiator-labeled probe includes a target binding region and one or more HCR initiators.

64. The method according to claim 62, wherein the probe set comprises one or more probe units, each containing two or more fragmentary initiator probes, and one fragmentary initiator probe contains a target binding region and a fragmentary initiator.

65. The method according to claim 62, comprising an additional HCR signal amplification round, each additional round comprising repeating steps (c) to (d).

66. The method according to claim 62, wherein the reporter comprises a hapten and the bridging probe comprises an anti-hapten primary antibody or a nanobody.

67. The method according to claim 62, wherein the auxiliary reporter is a fluorophore, a chromophore, or a rare earth element or compound.

68. The method according to claim 62, wherein the anti-reporter bridging probe comprises a primary antibody or nanobody that binds to the reporter, and further comprises an initiator-labeled secondary antibody or nanobody that binds to the primary antibody or nanobody.

69. The method according to claim 62, wherein the first HCR amplifier and the second HCR amplifier have the same sequence.

70. The method according to claim 62, wherein the reporter and the auxiliary reporter are the same.

71. Contacting the sample with an anti-auxiliary reporter readout probe containing a CARD-mediated enzyme; Contacting the sample with one or more CARD substrates; and The method according to claim 62, further comprising measuring the signals from the catalytically deposited one or more CARD reporters.

72. The method according to claim 62, wherein the cleaning step is performed during any of steps (b) to (f).

73. The method according to claim 62, further comprising step (g) of removing the signal after step (f).

74. The method according to claim 62, wherein the auxiliary reporter molecule directly or indirectly mediates the generation of a signal.

75. The method according to claim 62, wherein one HCR amplifier comprises two or more HCR hairpins.

76. The method according to claim 75, wherein one HCR hairpin includes an input domain comprising a single-chain toehold and a stem section.

77. The method according to claim 76, wherein one HCR hairpin includes an output domain comprising a single-chain loop and the complement of the stem section.

78. The method according to claim 75, wherein at least one HCR hairpin of the first HCR amplifier further comprises one or more reporters, and at least one HCR hairpin of the second HCR amplifier further comprises one or more auxiliary reporters.

79. The method according to claim 73, further comprising repeating any of the steps of the method to detect another target in the sample.

80. The method according to claim 62, wherein the target includes an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

81. The reporter and the auxiliary reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, ker The method according to claim 62, comprising: bombrak (CB), carbon nanotubes, magnetic carbon nanotubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

82. A method for amplifying a nonlinear hybridization chain reaction (HCR) signal, a) Contacting the sample with a first HCR initiator or two or more fragmentary initiators that together constitute a complete first HCR initiator; b) Contacting the sample with a first HCR amplifier labeled with a first reporter and incubating it to initiate the growth of a first reporter-modified HCR amplification polymer linked to the first HCR initiator; c) Contacting the sample with an anti-first reporter bridging probe containing a second HCR initiator and incubating it so that the first HCR amplification polymer is modified with the second HCR initiator; d) Contacting the sample with a second reporter-labeled HCR amplifier and incubating it to initiate the growth of a second reporter-modified HCR amplifier polymer linked to each second HCR initiator that modifies the first HCR amplifier polymer linked to the first HCR initiator; and e) including detecting signals directly or indirectly generated by the first reporter and / or the second reporter, A method wherein at least one HCR hairpin of the first reporter-labeled HCR amplifier contains one or more first reporters, and at least one HCR hairpin of the second reporter-labeled HCR amplifier contains one or more second reporters.

83. The method according to claim 82, further comprising preparing an anti-second reporter bridging probe containing a third HCR initiator and incubating the second HCR amplification polymer so that it is modified with the third HCR initiator.

84. A third reporter-labeled HCR amplifier is prepared, and the third reporter-modified HCR amplifier, linked to each of the third HCR initiators, is incubated to initiate the growth of the third reporter-modified HCR amplifier, which modifies the first HCR amplifier polymer linked to the first HCR initiator, which modifies the second HCR amplifier polymer linked to the second HCR initiator, which modifies the second HCR initiator linked to the second HCR initiator. The method according to claim 83, further comprising, wherein at least one HCR hairpin of the third reporter-labeled HCR amplifier comprises one or more third reporters.

85. The method according to claim 84, wherein the third reporter directly or indirectly mediates the generation of a signal.

86. The method according to claim 84, wherein some or all of the first reporter, the second reporter, and the third reporter are the same.

87. The method according to claim 84, wherein part or all of the first reporter-modified HCR amplifier, the second reporter-modified HCR amplifier, and the third reporter-modified HCR amplifier have the same sequence.

88. The method according to claim 84, wherein the first, second, and third reporters may be the same or different, and each comprises a hapten, a fluorophore, a chromophore, or a rare earth element or compound.

89. The method according to claim 84, wherein the third reporter is used to mediate CARD signal amplification.

90. The method according to claim 84, wherein the anti-reporter initiator-labeled bridging probe used to bridge between different rounds of HCR signal amplification is the same.

91. The method according to claim 82, further comprising performing a wash during any of steps (a) to (e).

92. The method according to claim 82, further comprising step (f) of removing a signal after step (e).

93. The method according to claim 92, wherein any of steps (a) to (f) is repeated.

94. The method according to claim 82, wherein one HCR amplifier comprises two or more HCR hairpins.

95. The method according to claim 94, wherein one HCR hairpin includes an input domain comprising a single-chain toehold and a stem section.

96. The method according to claim 94, wherein one HCR hairpin further comprises an output domain including a single-chain loop and the complement of the stem section.

97. The method according to claim 82, wherein the target includes an RNA molecule, a DNA molecule, a protein, a small molecule, a chemical substance, a biological molecule, a pathogen, a molecular complex, or a set of adjacent molecules.

98. The first reporter, the second reporter, and the third reporter may be the same or different, and each may be a fluorophore, chromophore, luminescent phore, phosphorescent body, FRET pair, member of FRET pair, quencher, fluorophore / quencher pair, rare earth element or compound, radioactive molecule, nucleotide, amino acid, oligonucleotide, DNA, RNA, 2'OMe-RNA, chemically modified nucleic acid, synthetic nucleic acid analog, chemically modified protein, synthetic protein analog, peptide, binding substrate, carbon atom, chemical linker, magnetic molecule, carbon black (CB), carbon nanotube, magnetic carbon nanotube The method according to claim 84, comprising notubes, gold nanoparticles (AuNP), gold nanoshells, gold nanorods, silver-shelled gold nanoparticles, latex, magnetic nanoparticles, silica nanoparticles, fluorophore-supported nanoparticles, dye-supported nanoparticles, haptens, ligands, digoxigenin (DIG), fluorescein isothiocyanate (FITC), biotin, dinitrophenol, aniline, enzymes, combinations thereof, or molecules that directly or indirectly mediate signal generation.

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