Application of click chemistry for signal amplification in IHC and ISH assays

A method of detecting a first target in a biological sample, comprising:

• (i) introducing to the biological sample a first detection probe specific to the first target in the biological sample to form a first detection probe-target complex;
• (ii) introducing to the biological sample a first labeling conjugate specific for the first detection probe of the formed first detection probe-target complex, wherein the first labeling conjugate comprises a first enzyme, and wherein the introduction of the first labeling conjugate permits the formation of a first-detection probe-target complex labeled with the first enzyme;
• (iii) introducing to the biological sample a first member of a first pair of click conjugates, wherein the first member of the first pair of click conjugates has the structure of Formula (Id):
• wherein A is a first reactive functional group, “Linker” is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S;
• and “Tissue Reactive Precursor Moiety” is a first tissue reactive precursor moiety selected from (i) a tyramide or a derivative or analog thereof, and (ii) a quinone methide precursor moiety;
• wherein the first enzyme of the formed first-detection probe-target complex labeled with the first enzyme converts the first tissue reactive moiety to a first reactive intermediate which covalently bonds to the biological sample proximal to or directly on the first target in the biological sample to form a first immobilized tissue-click conjugate complex comprising the first reactive functional group;
• (iv) introducing to the biological sample a second member of a first pair of click conjugates, wherein the second member of the first pair of click conjugates has the structure of Formula (IV):
• ALinkerZ (IV),
• wherein A is a second reactive functional group, “Linker” is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S;
• and Z comprises at least one first detectable moiety;
• wherein the second reactive functional group is capable of reacting with the first reactive functional group of the first immobilized tissue-click conjugate complex comprising the first reactive functional group through a click chemistry such that a covalent bond is formed between the first reactive functional group of the first immobilized tissue-click conjugate complex comprising the first reactive functional group and the second reactive functional group of the second member of the first pair of click conjugates to form a first tissue-click conjugate adduct including the at least one first detectable moiety;
• and
• (v) detecting signals from the at least one first detectable moiety of first tissue-click conjugate adduct.