Multiplexed immunosignal amplification using hybridization chain reaction-based method

Abstract

The invention provides a method for optimizing isHCR for multiplexed labeling, which combines binder-biomolecule interactions with hybridization Chain Reaction (HCR).

1 - 33 . (canceled) 
     
     
         34 . A method for detecting multiple target biomolecules, which combines binder-biomolecule interactions with Hybridization Chain Reaction (HCR), wherein orthogonal binders for conjugating orthogonal initiators and targeting multiple target biomolecules, and orthogonal initiators directed to orthogonal binders respectively are used in HCR to allow HCR amplification of multiple target biomolecules. 
     
     
         35 . The method of  claim 34 , wherein the orthogonal binders are orthogonal antibodies, orthogonal nanobodies, or orthogonal antibody fragments of antibodies, wherein the antibodies are biotinylated antibodies, and the orthogonal HCR initiators are biotinylated initiators for conjugating the vacant binding sites of streptavidin, which is capable of conjugating to the biotinylated antibodies in order to sequentially amplify multiple target biomolecules. 
     
     
         36 . The method of  claim 35 , wherein the orthogonal HCR initiators are directly conjugated to the orthogonal antibodies using chemical linkers, said chemical linker is selected from an amine-reactive linker containing a succinimidyl ester group, a thiol-reactive linker or a click chemistry linker. 
     
     
         37 . The method of  claim 36 , wherein the click chemistry linker is selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker. 
     
     
         38 . The method of  claim 35 , wherein the antibody is a secondary antibody that reacts with a primary antibody specific to an analyte, the secondary antibody is a IgG or a Nanobody, and the primary antibody is a IgG, a Nanobody or a scFv. 
     
     
         39 . The method of  claim 35 , wherein the orthogonal binders are genetically-engineered protein tags for labeling different target biomolecules, and the orthogonal HCR initiators are conjugated to tag binding partners respectively, which are capable of binding tags, preferably, the tag has a chemical group nonreactive toward a biomolecule, and said chemical group is selected from an amine moiety, a carboxyl moiety, a thiol moiety and a glycosylated modification moiety, preferably, the tag is CLIP-tag or Halo-tag. 
     
     
         40 . The method of  claim 39 , wherein the HCR initiators are conjugated to tag binding partners selected from SpyCatcher, SnoopCatcher, benzylguanine (BG), and scFv, and subsequently are used to detect the subcellular localization of the genetically-encoded tags selected from SpyTag, SnoopTag, SNAP-tag, and GCN4-tag respectively. 
     
     
         41 . The method of  claim 34 , wherein an amplifier or a pair of amplifiers are terminally modified or internally modified with a chemical group and/or a fluorescent dye, which allows initiating further rounds of amplification, said chemical group is selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazine, Alkyne, and/or DBCO, and said fluorescent dye is selected from FITC, Cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluro 546, Alexa Fluor 488, and Alexa Fluor 647, preferably, a pair of fluorophore-tagged amplifiers are added to the final round of the multiple-round HCR for visualization. 
     
     
         42 . The method of  claim 41 , wherein the amplifiers are modified at internal positions, which are accessible to streptavidin and which serve as anchors for each successive round of branching in multi-round isHCR. 
     
     
         43 . The method of  claim 34 , further comprising using grapheme oxide (GO) for absorbing unassembled HCR amplifiers; or further comprising using grapheme oxide (GO) for absorbing unassembled HCR amplifiers and quenching the fluorescence, wherein the amplifiers are terminally modified and/or internally modified with fluorescent dye. 
     
     
         44 . The method of  claim 43 , wherein GO has a particle size of <500 nm. 
     
     
         45 . A kit for detecting multiple target biomolecules, which comprises (1) orthogonal binders; (2) orthogonal HCR initiators; and (3) orthogonal pairs of HCR amplifiers, wherein each of HCR initiators has a region for hybridizing with a HCR amplifier, and a region for conjugating a binder, and the orthogonal binders target multiple target biomolecules respectively to allow HCR amplification directed to multiple target biomolecules. 
     
     
         46 . The kit of  claim 45 , wherein the orthogonal binders are orthogonal antibodies, orthogonal nanobodies, or orthogonal antibody fragments of antibodies, preferably, the orthogonal antibodies are orthogonal biotinylated antibodies, and the orthogonal HCR initiators are biotinylated initiators for conjugating the vacant binding sites of streptavidin, which is capable of conjugating to the biotinylated antibodies in order to sequentially amplify multiple target biomolecules. 
     
     
         47 . The kit of  claim 46 , wherein the orthogonal HCR initiators are directly conjugated to the orthogonal antibodies using chemical linkers, said chemical linker is selected from an amine-reactive linker containing a succinimidyl ester group, a thiol-reactive linker or a click chemistry linker, preferably, the click chemistry linker is selected from NHS-Azide linker, NHS-DBCO linker, maleimide-azide linker, and maleimide-DBCO linker. 
     
     
         48 . The kit of  claim 46 , wherein the antibody is a secondary antibody that reacts with a primary antibody specific to an analyte, the secondary antibody is a IgG or a Nanobody, and the primary antibody is a IgG, a Nanobody or a scFv. 
     
     
         49 . The kit of  claim 45 , wherein the orthogonal binders are genetically-engineered protein tags for labeling different target biomolecules, and the orthogonal HCR initiators are conjugated to tag binding partners, which are capable of binding tags, preferably, the tag has a chemical group nonreactive toward a biomolecule, said chemical group is selected from an amine moiety, a carboxyl moiety, a thiol moiety and a glycosylated modification moiety, preferably, the tag is CLIP-tag or Halo-tag. 
     
     
         50 . The kit of  claim 45 , wherein the HCR initiators are conjugated to tag binding partners selected from SpyCatcher, SnoopCatcher, benzylguanine (BG), and scFv, and subsequently are used to detect the subcellular localization of the genetically-encoded tags selected from SpyTag, SnoopTag, SNAP-tag, and GCN4-tag respectively. 
     
     
         51 . The kit of  claim 45 , wherein an amplifier or a pair of amplifiers are terminally modified or internally modified with a chemical group and/or a fluorescent dye, which allows initiating further rounds of amplification, said chemical group is selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazinc, Alkyne, and/or DBCO, and said fluorescent dye is selected from FITC, Cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluro 546, Alexa Fluor 488, and Alexa Fluor 647, preferably, a pair of fluorophore-tagged amplifiers is added to the final round of the multiple-round HCR for visualization. 
     
     
         52 . The kit of  claim 51 , wherein the amplifiers are modified at internal positions, which are accessible to streptavidins and which serve as anchors for each successive round of branching in multi-round isHCR. 
     
     
         53 . The kit of  claim 45 , further comprising grapheme oxide (GO) for absorbing unassembled HCR amplifiers; or further comprising grapheme oxide (GO) for absorbing unassembled HCR amplifiers and quenching the fluorescence, wherein the amplifiers are terminally modified and/or internally modified with fluorescent dye, preferably, GO has a particle size of <500 nm.