Chromogenic peroxidase substrates

Abstract

The present disclosure relates to chromogenic conjugates including a chromogenic moiety and a peroxidase substrate moiety, methods of chromogenic analysis, such as ImmunoHistoChemistry (IHC) and Chromogen In Situ Hybridization (CISH), and methods of making chromogenic conjugates.

Description

CHROMOGENIC PEROXIDASE SUBSTRATES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Application No.63 / 640,052, filed April 29, 2024, the content of which is hereby incorporated by reference in its entirety. COPYRIGHT STATEMENT

[0002] A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. FIELD

[0003] The present disclosure relates to chromogenic conjugates, methods of chromogenic analysis, such as ImmunoHistoChemistry (IHC) and Chromogen In Situ Hybridization (CISH), and methods of making chromogenic conjugates. BACKGROUND

[0004] When analyzing tissue samples on a microscope slide, staining the tissue or certain parts of the tissue with a colored dye can aid the analysis. The ability to visualize or differentially identify microscopic structures is frequently enhanced through the use of histological stains. Hematoxylin and eosin (H&E) stain is the most commonly used stain in light microscopy for histological samples. Hematoxylin is used to stain nuclei blue, and eosin stains cytoplasm and the extracellular connective tissue matrix pink. In addition to H&E stains, other stains or dyes have been applied to provide more specific staining and provide a more detailed view of tissue morphology. Immunohistochemistry (IHC) stains have great specificity, as they use an antibody targeted against specific proteins and either a peroxidase substrate or alkaline phosphotase (AP) substrate for IHC stainings, providing a uniform staining pattern that appears to the viewer as a homogeneous color with intracellular resolution of cellular structures, e.g. membrane, cytoplasm, and nucleus.

[0005] Formalin Fixed Paraffin Embedded (FFPE) tissue samples, metaphase spreads or histological smears are typically analyzed by staining on a glass slide, where a particularbiomarker, such as a protein or nucleic acid of interest, can be stained with a colored dye, hereafter “chromogen” or “chromogenic moiety”. IHC staining is a common tool in evaluation of tissue samples for the presence of specific biomarkers. IHC stains are precise in the recognition of specific targets throughout the sample and allow quantification of these targets.

[0006] IHC staining employs chromogenic and / or fluorescent reporters that mark targets in histological samples. This is carried out with antibodies that recognize the biomarker directly. The antibodies are typically visualized either directly through the conjugated enzyme or indirectly with an secondary antibody conjugated to an enzyme. The most commonly used enzymes for this visualization are either Horse Radish Peroxidase (HRP) or Alkaline Phosphatase (AP), that subsequently catalyzes the formation of an insoluble colored precipitate, at the location of the biomarker from a soluble suitable enzyme substrate, which exhibits a color.

[0007] In blotting / capture assays, the biomarker is extracted into solution from its original location and then re-immobilized on a membrane, gel, or chip array, but the biomarker is also stained with a visible color, a chromogen, typically by action of the same HRP or AP enzymes.

[0008] Compared to other detection techniques, such as radioactivity, chemo-luminescence or fluorescence, chromogens, have the advantage of a permanent, plainly visible color which can be visually observed, such as with bright field microscopy.

[0009] One limitation of enzyme-based chromogenic detection of targets in solid biological samples or targets that are immobilized onto or into a solid support include that there is a very limited number of chromogenic HRP and AP substrates that can be used for target staining, which limits use of these target visualization systems for detection of multiple targets in samples. Also, some chromogens, like the HRP substrate 3,3′-diaminobenzidine (DAB), are not characterized by well-defined spectral features, but rather appear as insoluble light adsorbing brown precipitates. Moreover, where visualization of multiple targets is concerned, it often requires one to use a combination of multiple enzyme-based visualization systems, such as HRP and AP. These limitations make sample staining procedures complex, less robust and expensive and also complicates automated detection of targets and image analyses of stained samples.

[0010] Triarylmethane dyes, Rhodamines, rhodols and fluoresceins are intensely colored and fluorescent. They come in virtually any color, dependent on halogenation and / or substitution pattern. They have been known for more than a century, and several are used as special stains which stain tissue samples without any enzyme activity. For example, Malachite Green is used to stain bacterial spores, Rhodamine 110 is used as a mitochondrial stain,TetraBromoFluorescein, also referred to as Eosin, is used extensively in Haematoxilin / Eosine (H and E) double stains, where Haematoxilin stains nuclei blue and Eosin stains essentially any protein pink or red. So while derivatives of these compounds would seem attractive as potential chromogens due to their distinct and bright color, unspecific tissue staining, even in the absence of any enzyme activity, is an impediment to their use as chromogens. Another impediment is to provide derivatives with suitable solubility in aqueous environments.

[0011] Rhodamine and fluorescein compounds are also stable and require forcing conditions to undergo further reaction. A solution has been to introduce extra reactive groups, such as an IsoThioCyanate as in FluoresceinIsoThioCyanate (“FITC”) and Tetramethyl Rhodamine IsoThioCyanate (“TRITC”) or CarboxyFluorescein and SulphoRhodamine. However, the addition of these reactive groups is not done easily and results in a mixture of two almost inseparable isomers. FITC has become widely associated with reactive fluorescein, the proven way to prepare fluorescein derivatives of antibodies and nucleic acid probes. However such derivatives are expensive, some prohibitively expensive.

[0012] In the last decade, there has been progress made within the field of rhodamine and fluorescein 2′-ester derivatives. See, for example Beija, Mariana et al., “Synthesis and applications of Rhodamine derivatives at fluorescent probes.” Chem. Soc. Rev., 2009, 38, 2410-2433; Afonso, A. M. Carlos, et al., “An Expedient Synthesis of Cationic Rhodamine fluorescent Probes Suitable for Conjugation to Amino Acids and Peptides.” Synthesis, 2003, 17, 2647-2654; Xi Chen et al., “An efficient and versatile approach for the preparation of a rhodamine B ester bioprobe library.” Dyes and Pigments 2012, 94, 296-303, the contents of each of which are incorporated herein by reference in its entirety.

[0013] Both rhodamines and fluoresceins possess a 2′ carboxylic acid that can be derivatized as esters or amides under certain conditions. However, 2′ primary amides of rhodamines of fluoresceins collapse into colorless spirolactam or spirolactone tautomers, making such derivatives unsuitable as chromogens. Esters and amides of secondary amines do not undergo this tautomerization as they lack the labile N—H proton.

[0014] A method of preparing amides of the secondary amine piperazine of Rhodamines and Fluorescein have been reported. See Nguyen T. et al., “Practical synthetic route to rhodamine dyes”, Org. Lett.2003, 18, 3245-48; Huang, Chusem, et al.; “Versatile Probes for the Selective Detection of Vicinal-Dithiol-Containing proteins: Design, Syntheses, and Applications in Living Cells”. Chem. Eur. J. 2013, 19, 7739-7747, the contents of each of which are incorporated herein by reference in its entirety.

[0015] Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in many applications employing fluorescence, such as flow cytometry. FITC comprises a fluorescein molecule functionalized at its 4′ position with a isothiocyanate reactive group (—N═C═S) on the monocyclic phenyl of the structure. This derivative is reactive towards nucleophiles including amine and sulfhydryl groups on proteins.

[0016] Use of fluorescein as the detectable part of HRP substrates in histochemical detection of targets has recently been described. WO2007 / 015168 to Lohse, the contents of which are incorporated herein by reference in its entirety, relates to monomeric or polymeric linker molecules useful in biological and chemical applications, their synthesis, and the synthesis and use of derivatives of the linkers conjugated to a variety of detectable labels and other substrates. The linkers may be used, for example, in conjunction with fluorescent labels, nucleic acid or nucleic acid analog probes, and solid phase systems, and to enhance the solubility of the conjugated molecules. WO2009 / 03670, WO2010 / 094283, WO2010 / 084284, WO2011 / 047680 and WO2012 / 143010, the contents of each of which are incorporated herein by reference in its entirety, relate to HRP substrates that are conjugated via linker of WO2007 / 015168 to fluorescein at its 4′ position. The latter conjugates are colorless but fluorescent and can be used either for direct fluorescent or indirect histochemical detection of targets: the conjugates are deposited in target sites labeled with HRP activity via the enzymatic reaction, and then the deposited conjugates may be detected optically as fluorescent stain or immunochemically as haptens. Deposition of the conjugates by HRP is enhanced in the presence of certain amounts of DAB, ferulic acid or alpha-cyano-4-hydroxycinnamic acid (ACHCA) in the deposition medium. SUMMARY

[0017] An embodiment of the present disclosure provides chromogenic conjugate molecules capable of serving as substrates of an enzyme with peroxidase activity, and describes their utility for detecting molecular targets in samples. 1. One embodiment of the present disclosure provides a chromogenic conjugate of the present disclosure comprising: (a) a chromogenic moiety, and (b) a peroxidase substrate moiety,wherein the chromogenic moiety and the peroxidase substrate moiety are linked together via a linker, wherein the conjugate is a compound of Formula Ia, Ib, Ic, or a salt thereof:c wherein, X is independently selected from –OH, -ORXor –NRXRXX; Y is independently selected from O or =N+RYRYY; Z, Z’, Z1, or Z1’are independently selected from O, S, or NRZ; wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R1’, R2’, R3’, R4’, R5’, R6’, R7’, R31, R32, R31’, R32’, RX, RXX, RY, RYY, and RZare independently selected from hydrogen, OH, and a substituent having less than 40 atoms, wherein three or more atoms can be connected to form a cyclic structure;m or p’ is an integer selected from 0 or 1; n or n’ is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is an integer selected from 0, 1, 2 or 3; L is a linker comprising 5 to 29 connected atoms; and PS is H, NH2, OH, or a peroxidase substrate moiety represented by the following Formula II, provide that at least one of PS is a peroxidase substrate moiety:Formula II wherein, L and PS are connected through R26; R21is -H -OR34, or -NR34R35; R22is -H, -OR34, or -NR34R35; R23is -OH; R24is -H, -OR34, or -NR34R35; R25is - H, -OR34, or -NR34R35; and R26is –C(=Z3)-(Z4)p-; wherein, each Z3and Z4is independently O, S, or NR36; each R34, R35, or R36is independently H, alkyl or aryl; and p is 0 or 1.2. In one embodiment, R1is selected from hydrogen, OH, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5- C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, R1may be taken together with R2to form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; R2is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, R2may be taken together with R1, to form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; RX, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; RXX, when present, is selected from (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R3is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R4is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, when Y is –NRYRYY, R4may be taken togetherwith RYYto form a 5- or 6-membered ring which is optionally substituted with one or more of the same or different R13or suitable R14groups; RYY, when present, is selected from (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively RYYmay be taken together with R4to form a 5- or 6-membered ring which is optionally substituted with one or more of the same or different R13or suitable R14groups; RY, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, RYmay be taken together with R5to form a 5- or 6-membered ring optionally substituted with one or more of the same or different R13or suitable R14groups; RZ, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R5is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, R5may be taken together with R6to form part of a benzo, naptho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, when Y is –NRYRYY, R5may be taken together with RYto form a 5- or 6-membered ring optionally substituted with one or more of the same or different R13or suitable R14groups; R6is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or differentR13or suitable R14groups, or, alternatively, R6together with R5may form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; R7, R8and R9are each, independently of one another, selected from hydrogen, R11, (C1- C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R10is selected from selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, halo, haloalkyl, –OR12, –SR12, –SOR12, – SO2R12, and nitrile; R11is selected from –NR15R15, –OR16, –SR16, halo, haloalkyl, –CN, –NC, –OCN, – SCN, –NO, –NO2, –N3, –S(O)R16, –S(O)2R16, –S(O)2OR16, –S(O)NR15R15, –S(O)2NR15R15– OS(O)R16, –OS(O)2R16, –OS(O)2NR15R15, –OP(O)2R16, –OP(O)3R16R16, –P(O)3R16R16, – C(O)R16, –C(O)OR16, –C(O)NR15R15, –C(NH)NR15R15, –OC(O)R16, –OC(O)OR16, – OC(O)NR15R15and –OC(NH)NR15R15; R12is selected from (C1-C20) alkyls or heteroalkyls optionally substituted with lipophilic substituents, (C5-C20) aryls or heteroaryls optionally substituted with lipophilic substituents and (C2-C26) arylalkyl or heteroarylalkyls optionally substituted with lipophilic substituents; R13is selected from hydrogen, (C1-C8) alkyl or heteroalkyl, (C5-C20) aryl or heteroaryl and (C6-C28) arylalkyl or heteroarylalkyl; R14is selected from –NR15R15, =O, –OR16, =S, –SR16, =NR16, =NOR16, halo, haloalkyl,OC(O)OR16, –OC(O)NR15R15and –OC(NH)NR15R15; each R15is independently hydrogen or R16, or alternatively, each R15is taken together with the nitrogen atom to which it is bonded to form a 5- to 8-membered saturated or unsaturated ring which may optionally include one or more of the same or different additionalheteroatoms and which may optionally be substituted with one or more of the same or different R13or R16groups; each R16is independently R13or R13substituted with one or more of the same or different R13or R17groups; and each R17is selected from –NR13R13, –OR13, =S, –SR13, =NR13, =NOR13, halo, haloalkyl,OC(O)OR13, –OC(O)NR13R13and –OC(NH)NR13R13. 3. In another embodiment, the chromogenic moiety is a derivative of Cyanine, triarylmethane, fluorescein, or a salt thereof. 4. In a specific embodiment, the chromogenic moiety is a derivative of Cyanine or a salt thereof. 5. In another specific embodiment, the chromogenic moiety is a derivative of triarylmethane or a salt thereof. 6. In yet another specific embodiment, the chromogenic moiety is a derivative of fluorescein or a salt thereof. 7. In some embodiment, the chromogenic moiety is selected from the group consisting of Cy-3, Cy-5 , triarylmethanes, fluorescein, and O-carboxymethyl fluorescein. 8. In some specific embodiment, the chromogenic moiety is a 2'-piperazine amide derivative. 9. In another specific embodiment, R23is –OH, and R24is –H. 10. Yet in another specific embodiment, either R21or R25is –OH, R22and R24are –H, and R23is –OH. 11. In some embodiment, the peroxidase substrate is a residue of ferulic acid, cinnamic acid, caffeic acid, sinapinic acid, 2,4-dihydroxycinnamic acid or 4-hydroxycinnamic acid (coumaric acid). 12. In some embodiment, L comprises 1 or 2 repeats of Formula IVa, IVb, IVc, IVd, or IVe:,wherein each R37is independently selected from methyl, ethyl, propyl, OCH2, CH2OCH2, (CH2OCH2)2, S, NH, NHCH2, NH(CH2)2, CH2NHCH2, cycloalkyl, alkyl-cycloalkyl, alkyl- cycloalkyl-alkyl, heterocycloalkyl, alkyl-heterocyclyl, alkyl-heterocyclyl-alkyl; each R38is independently NH or O; w is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each R39is independently CH2or CH2NH. 13. In some embodiment, the linker is selected from: ,,,,,. 15. The present disclosure also provide a composition comprising at least one chromogenic conjugate according to claim 1 and a solvent, and optionally one or more of (i) an organic modifier; (ii) an enzyme enhancer; (iii) an iron chelator; (iv) a detergent; (v) an anti-microbial agent; (vi) organic or inorganic salt; or (vii) an enzyme substrate.16. A composition of the present disclosure comprising the chromogenic conjugate of Formula Ia provides yellow color. 17. Another composition of the present disclosure comprising Formula Ib of the chromogenic conjugate provides cyan or blue color. 18. Yet another composition of the present disclosure comprising Formula Ic of the chromogenic conjugate provides yellow or turquoise color. 19. A composition of the present disclosure comprises more than one chromogenic conjugates for detection of a target having peroxidase activity or linked to a peroxidase enzyme, in a sample; or wherein, in an embodiment, the composition comprises a compound of Formula Ia, Ib, or a salt thereof; or wherein the composition comprises a compound of Formula Ia, Ic, or a salt thereof. 20. A composition comprising 5-50 wt % of magenta chromogenic conjugate and 50-95 wt% of the chromogenic conjugate of claim 1. 21. Some embodiment provides a method for preparing the chromogenic conjugate of the present disclosure by: reacting a peroxidase substrate moiety represented by Formula IIIa having a protected phenol and a carboxylic acid moiety with a linker moiety having two amino group with one of the amine with a protecting group P2represented by Formula IIIb to prepare P2-HN-L-PS- P1represented by Formula IIIc; removing the protecting group P2to prepare Formula IIId; reacting Formula Iaa, Formula Ibb, or Formula Icc with Formula IIId to prepare Formula IIIe, Formula IIIf, or Formula IIIg, respectively; and removing a protecting group P1to prepare the chromogenic conjugate of claim 1 represented by Formula Ia, Ib or Ic:Formula IIIaFormula IIIbwherein, provided that at least one of RPSor RPS’isg. 22. Another embodiment provides another method for preparing the chromogenic conjugate of the present disclosure by: reacting Formula Iaa, Formula Ibb, or Formula Icc with Formula IIIb; removing a protecting group P2to prepare Formula Idd, Iee, or Iff; reacting a peroxidase substrate moiety represented by Formula IIIa having a protected phenol and a carboxylic acid moiety with Idd, Iee, or Iff to prepare chemical compound represented by Formula IIIe, IIIf, or IIIg; and removing a protecting group P1to prepare the chromogenic conjugate of claim 1 represented by Formula Ia, Ib or Ic,Formula IIIbff,, provided that at least one of RPSor RPSisFormula IIIg. 23. In a method for preparing the chromogenic conjugate of the present disclosure, a large excess of H2N-L-NH2 is used instead of P2-HN-L-NH2. 24. The present disclosure also provides a method for detection of a target in a sample by chromogenic detection comprising: incubating a sample comprising a target in an aqueous solution, wherein the target comprises peroxidase activity or the target is directly or indirectly linked to a peroxidase enzyme, wherein the aqueous solution comprises: a) at least one chromogenic conjugate of the present disclosure; b) a peroxide compound, at a time and temperature sufficient to form a colored precipitate of the chromogenic conjugate; detecting the colored precipitate of the chromogenic conjugate in the sample, thereby detecting the target in the sample, and optionally incubating the sample two or more cycles with a chromogenic conjugate having Formula Va, Vb, Vc or Vd:Formula Vawherein all variables are as defined in claim 1, wherein each cycle comprises same or different chromogenic conjugates in same or different ratios. 25. An embodiment of the present disclosure provides a method of performing chromogenic in situ hybridization comprising: contacting a nucleic acid target with a probe that hybridizes with the nucleic acid target under hybridization conditions, wherein the probe comprises (1) a nucleic acid sequence at least partially complementary to the nucleic acid target and (2) a peroxidase enzyme or a first member of a specific binding pair; wherein the target and probe form a complex;when the probe comprises (2), contacting the complex with a second member of the specific binding pair, wherein the second member of the specific binding pair, wherein the second member is directly or indirectly linked to a peroxidase enzyme, and specifically binds to the first member; incubating the complex with at least one of the chromogenic conjugates of the present disclosure; for a time and temperature sufficient to form a color precipitate at the target; detecting the color precipitate. 26. Another embodiment of the present disclosure provides a method of detecting two or more targets comprising: providing peroxidase activity at a first target in a sample; contacting the sample with a first chromogenic conjugate; forming a first colored precipitate at the first target; removing the peroxidase activity from the first target; removing non-precipitated first chromogenic conjugate from the sample; providing peroxidase activity at a second target in the sample; contacting the sample with a second chromogenic conjugate; forming a second colored precipitate at the second target; and detecting the first colored precipitate and the second colored precipitate, thereby detecting the first and second targets in the sample, wherein at least one of the first and second chromogenic conjugates is a conjugate of the present disclosure, and wherein the first chromogenic conjugate molecule and the second chromogenic conjugate have one or more spectral characteristics that are different from each other, or wherein the method further comprises: removing the peroxidase activity from the second target; removing non-precipitated second chromogenic conjugate from the sample; providing peroxidase activity at a third target in the sample; contacting the sample with a third chromogenic conjugate having one or more spectral characteristics that are different from those of the first and second chromogenic conjugates. 27. Yet another embodiment of the present disclosure provides a method for preparing a chromogenic conjugate of the present disclosure comprising: reacting a chromogenic moiety with a secondary amine compound containing an ester to prepare a secondary amide compound of the chromogenic moiety; converting the ester in the secondary amide compound of the chromogenic moiety to a carboxylic acid; andreacting the secondary amide compound of the chromogenic moiety having the carboxylic acid with a linker compound having an amine group to form the chromogenic conjugate. 28. Some embodiment of the disclosure provides an immunohistochemical staining composition comprising two or more of chromogenic conjugates selected from the group consisting of a chromogenic conjugate of Formula Ia, a chromogenic conjugate of Formula Ib, a chromogenic conjugate of Formula Ic of claim 1, and a chromogenic conjugate of Formula Id:wherein all variables are as defined above; wherein, alternatively, the composition provides red, orange, grass green, green, blue, or purple color; or wherein, alternatively, the color is determined by the ratio of the first, the second, the third and the fourth chromogenic conjugates. 29. Some embodiment provides a kit for detection of a target having peroxidase activity or linked to a peroxidase enzyme in a sample comprising at least one chromogenic conjugate of the present disclosure, wherein the kit comprises more than one chromogenic conjugate of the present disclosure to stain multiple targets in different colors, or wherein the kit comprises a composition comprising 5-50 wt % of magenta chromogenic conjugate and 50-95 wt% of the chromogenic conjugate of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present teachings are best understood from the following detailed description when read with the accompanying drawing figures. The features are not necessarily drawn to scale. Wherever practical, like reference numerals refer to like features.

[0019] FIG.1 presents staining result of a tonsil sample with the yellow chromogen.

[0020] FIG.2 presents results of mixed staining color of yellow chromogen with magenta.

[0021] FIG.3 presents staining result of tonsil sample with turquoise chromogen.

[0022] FIG.4 presents staining result of colon carcinoma with turquoise chromogen.

[0023] FIG.5 presents staining result of kidney tissue with turquoise chromogen.

[0024] FIG. 6 presents staining result of tonsil tissue with S100 (yellow) and lambda light chain (cyan).

[0025] FIG. 7 presents staining result of colon tissue with S100 (yellow) and lambda light chain (cyan). DEFINITION OF TERMINOLOGY

[0026] It is to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The defined terms are in addition to the technical and scientific meanings of the defined terms as commonly understood and accepted in the technical field of the present teachings. For example, definitions of common terms in molecular biology may be found in Benjamin Lewin, GenesVII, published by Oxford University Press, 2000 (ISBN019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994(ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN0471186341); and other similar references, the disclosures of each of which are incorporated herein by reference in its entirety.

[0027] As used in the specification and appended claims, the terms “a”, “an” and “the” include both singular and plural referents, unless the context clearly dictates otherwise. Thus, for example, “a moiety” includes one moiety and plural moieties.

[0028] As used in the specification and appended claims, and in addition to their ordinary meanings, the terms “substantial” or “substantially” mean to within acceptable limits or degree to one having ordinary skill in the art. For example, “substantially cancelled” means that one skilled in the art considers the cancellation to be acceptable.

[0029] As used in the specification and the appended claims and in addition to its ordinary meaning, the terms “approximately” and “about” mean to within an acceptable limit or amount to one having ordinary skill in the art. The term “about” generally refers to plus or minus 15% of the indicated number. For example, “about 10” may indicate a range of 8.7 to 11.5. For example, “approximately the same” means that one of ordinary skill in the art considers the items being compared to be the same.

[0030] A “moiety” is a portion of a molecule that retains chemical and / or physical and / or functional features of the entire molecule, that are relevant for performance of the chromogenic conjugates; fr example, a “peroxidase substrate moiety” is a portion of a molecule capable of serving as substrate of an enzyme with peroxidase activity; “peroxidase moiety” is a portion of a molecule that has inherent peroxidase activity, for example an enzyme.

[0031] A “conjugate” refers to two or more molecules (or two moieties of two or more molecules) that are covalently linked into a larger construct.

[0032] The term “linked” in the present context means connected via a chemical bond.

[0033] A “target” is an object in a test sample to be detected by use of the present chromogenic conjugates and methods; present targets include chemical and biological molecules and structures. Embodiments of present targets are discussed herein.

[0034] A “biological marker” refers to one or more biological objects such as molecules, molecular complexes, structures, particles or organisms which are associated with features that are characteristic for a particular cell type, tissue, cellular structure, physiological condition, etc. Such biological objects are often considered markers of that particular cell type, tissue, cellular structure, or physiological condition. Non-limited examples of such biological markers include but are not-limited to particular nucleotide sequences, proteins or other biological molecules, e.g. carbohydrates or lipids, chromosomal or membrane structures, viruses, bacteria, microorganisms etc. In some embodiments, the term target is used interchangeably with the term biological marker and relates to a molecule, molecular complex, structure or particle that is characteristic for a particular cell type, tissue, physiologic condition, etc., wherein the total population of any of the latter biological markers in the test sample is considered to be the target.

[0035] “Spectral characteristics” are characteristics of electromagnetic radiation emitted or absorbed due to a molecule or moiety making a transition from one energy state to another energy state, for example from a higher energy state to a lower energy state. Only certain colors appear in a molecule's or moiety's emission spectrum, since certain frequencies of light are emitted and certain frequencies are absorbed. Spectral characteristics may be summarized or referred to as the color of the molecule or moiety.

[0036] The term “rhodamine” can refer to the family of related dyes based on xanthene, which includes, but is not limited to, Rhodamine 6G and Rhodamine B; or the term “rhodamine” can refer to the specific compound:

[0037] The term “fluorescein” can refer to the family of related dyes based on xanthene, which includes, but is not limited to, fluorescein isothiocyanate, NHS-fluorescein and O- carboxyfluorescein; or the term “fluorescein” can refer to the specific compound:

[0038] Certain abbreviations are used for the sake of brevity: “Rho” refers to rhodamine; “TMRho” refers to tetra methyl rhodamine: “Flu” refers to fluorescein; “Pip” refers to piperazine; “Cou” refers to coumarin: “Caf” refers to caffeic acid; “Fer” refers to ferulic acid; “Cin” refers to cinnapinic acid; “Tyr” refers to tyrosine: “Et” refers to ethyl. Other abbreviations may also appear in this disclosure.

[0039] “Spectrally narrow” refers to a chromogen having less than 50 nm broadness at its maximum absorbance at half peak height by UV-VIS spectroscopy, measured in +99% water at pH, 6-8 in 10 μM concentration. A “magenta chromogen” is a spectrally narrow chromogen with peak absorbance between 525 and 530 nm, measured in +99% water at pH, 6.0-8.0 in 10 μM concentration. A “greenish-yellow chromogen” is a chromogen with peak absorbance below 475 nm and less than 10% absorbance at 530 nm or above relative to the peak absorbance, measured in +99% water at pH, 6.0-8.0 in 10 μM concentration. A “yellow chromogen” is a chromogen with peak absorbance below 505 nm and less than 10% absorbance at 530 nm or above relative to the peak absorbance, measured in +99% water at pH, 6.0-8.0 in 10 μM concentration. A “cyan chromogen” is a chromogen having maximal absorbance above 615 nm and less than 10% relative absorbance at any wavelength between 530 and 400 nm relative to the peak absorbance, measured in +99% water at pH, 6.0-8.0 m 10 μM concentration. A “blue chromogen” is a chromogen having maximal absorbance above 500 nm. An example of blue chromogen such as Brilliant Blue FCF (Blue No. 1) by Shimadzu has a maximum absorption at about 625 nm. An “orange chromogen” is a “spectrally narrow” chromogen that has maximum absorbance between 495 and 520 nm, measured in +99% water at pH, 6-8 in 10 μM concentration. A “dichroic chromogen” is a chromogen that has at least two absorbance maxima, separated by a local minimum at least 50 nm wide and one further global minimum between 390 and 700 nm measured in water between pH 6 and 8. DETAILED DESCRIPTION

[0040] In the following detailed description, for purposes of explanation and not limitation, representative embodiments disclosing specific details are set forth in order to provide a thorough understanding of the present teachings. Descriptions or details of known systems, compounds, materials, methods of use and methods of manufacture may be omitted so as to avoid obscuring the description of the exemplary embodiments. Nonetheless, systems, moieties and methods that are within the purview of one of ordinary skill in the art may be used in accordance with the representative embodiments.

[0041] The present disclosure provides chromogenic peroxidase substrate conjugates (interchangeably termed herein as “chromogenic conjugates”, “conjugate molecules” or “reporter molecules”). In some embodiments, the present conjugate molecules combine one or more of the virtues of DAB, without one or more of the disadvantages.

[0042] Embodiments of the present chromogenic conjugates include molecules comprising a peroxidase substrate moiety linked to a chromogenic moiety, that are (a) non-toxic, (b) precipitated from solutions via HRP-mediated reaction as bright and spectrally narrow and intense stains; (c) are stable in aqueous solutions for more than 24 hours once mixed; and / or (d) are insoluble in organic solvents when precipitated.

[0043] In some embodiments, the chromogenic conjugates of the present disclosure absorb and / or emit light in the range from about 400 nm to about 700 nm. In some embodiments, the chromogenic conjugate is yellow and absorbs light at 460 nm. In some embodiments, the chromogenic conjugate is cyan and absorbs light at 637 nm. The embodiments of the present chromogenic conjugates can serve as substrates of a peroxidase enzyme, e.g. HRP, and they are spectrally narrow, non-dichromatic, and do not substantially change their spectral characteristics upon precipitation; the stains produced via enzymatic precipitation of the chromogenic conjugates are poorly soluble, if at all, in water or organic solutions and do not bleach when exposed to light sources used for imaging of stained samples. These features make the present chromogenic conjugates particularly suitable for automated image analyses and multiplexing. Further, the molecules of the chromogenic conjugates have well-defined chemical structures and can easily be produced by the processes described herein. Chromogenic Conjugate Molecules

[0044] Some embodiments of the present chromogenic conjugate may be represented by the general formula: (Z)-L-(S)wherein S is a peroxidase substrate moiety, Z is a chromogenic moiety, L is a linker, wherein the chromogenic conjugate molecules have one, two, three, four, five or all, and preferably all, of the following features: (1) comprise one substrate moiety of a peroxidase enzyme, such as HRP substrates; (2) comprise one chromogenic moiety, which is a Rho, Flu, Blue, triarylmethane, Cyanine 2′-ester or 2′-secondary amide derivative thereof; (3) the enzyme substrate and chromogenic moieties are linked together via a water soluble linker compound and are distanced away from each other by at least 5 consecutively interconnected atoms, (4) the linker compound (also termed herein as “linker molecule”, “linker” or “L”) comprises a chain of 5-29 interconnected atoms (correspondingly abbreviated as “L5-L29”); wherein, in some preferred embodiments, the linker compound comprises two consecutive carbons followed by an oxygen or nitrogen atom; (5) the conjugate is substantially soluble in aqueous solutions; (6) the conjugate is substantially stable as a chromogen both in solution and as precipitate.

[0045] The chromogenic conjugates may include a chromogenic moiety capable of providing visible color. The chromogenic moiety may be a xanthene, a cyanine derivative, or a triarylmethane such as, fluorescein, rhodol, patent blue or rhodamine residue.

[0046] The chromogenic conjugates of the present disclosure may express yellow, blue, cyan, or turquoise color.

[0047] In some embodiments, the chromogenic conjugate comprises (a) a chromogenic moiety, and (b) a peroxidase substrate moiety, wherein the chromogenic moieties and the peroxidase substrate moieties are linked together via a linker. For example, the chromogenic conjugate can be a compound of wherein the conjugate is a compound of Formula Ia, Ib, Ic, or a salt thereof:Formula Iawherein, X is independently selected from –OH, -ORXor –NRXRXX; Y is independently selected from O or =N+RYRYY; Z, Z’, Z1, or Z1’are independently selected from O, S, or NRZ; wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R1’, R2’, R3’, R4’, R5’, R6’, R7’, R31, R32, R31’, R32’, RX, RXX, RY, RYY, and RZare independently selected from hydrogen, OH, and a substituent having less than 40 atoms, wherein three or more atoms can be connected to form a cyclic structure;m or p’ is an integer selected from 0 or 1;n or n’ is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is an integer selected from 0, 1, 2 or 3; L is a linker comprising 5 to 29 connected atoms; and PS is H, NH2, OH, or a peroxidase substrate moiety represented by the following Formula II, provide that at least one of PS is a peroxidase substrate moiety:Formula II wherein,

[0048] In some embodiments of the chromogenic conjugate of the present disclosure, R1is selected from hydrogen, OH, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, R1may be taken together with R2to form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups;R2is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, R2may be taken together with R1, to form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; RX, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; RXX, when present, is selected from (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R3is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R4is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, when Y is –NRYRYY, R4may be taken together with RYYto form a 5- or 6-membered ring which is optionally substituted with one or more of the same or different R13or suitable R14groups; RYY, when present, is selected from (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively RYYmay be taken together with R4to forma 5- or 6-membered ring which is optionally substituted with one or more of the same or different R13or suitable R14groups; RY, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, RYmay be taken together with R5to form a 5- or 6-membered ring optionally substituted with one or more of the same or different R13or suitable R14groups; RZ, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R5is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, R5may be taken together with R6to form part of a benzo, naptho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, when Y is –NRYRYY, R5may be taken together with RYto form a 5- or 6-membered ring optionally substituted with one or more of the same or different R13or suitable R14groups; R6is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, R6together with R5may form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; R7, R8and R9are each, independently of one another, selected from hydrogen, R11, (C1- C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same ordifferent R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R10is selected from selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, halo, haloalkyl, –OR12, –SR12, –SOR12, – SO2R12, and nitrile; R11is selected from –NR15R15, –OR16, –SR16, halo, haloalkyl, –CN, –NC, –OCN, – SCN, –NO, –NO2, –N3, –S(O)R16, –S(O)2R16, –S(O)2OR16, –S(O)NR15R15, –S(O)2NR15R15– OS(O)R16, –OS(O)2R16, –OS(O)2NR15R15, –OP(O)2R16, –OP(O)3R16R16, –P(O)3R16R16, – C(O)R16, –C(O)OR16, –C(O)NR15R15, –C(NH)NR15R15, –OC(O)R16, –OC(O)OR16, – OC(O)NR15R15and –OC(NH)NR15R15; R12is selected from (C1-C20) alkyls or heteroalkyls optionally substituted with lipophilic substituents, (C5-C20) aryls or heteroaryls optionally substituted with lipophilic substituents and (C2-C26) arylalkyl or heteroarylalkyls optionally substituted with lipophilic substituents; R13is selected from hydrogen, (C1-C8) alkyl or heteroalkyl, (C5-C20) aryl or heteroaryl and (C6-C28) arylalkyl or heteroarylalkyl; R14is selected from –NR15R15, =O, –OR16, =S, –SR16, =NR16, =NOR16, halo, haloalkyl, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)R16, –S(O)2R16, –S(O)2OR16, – S(O)NR15R15, –S(O)2NR15R15, –OS(O)R16, –OS(O)2R16, –OS(O)2NR15R15, –OS(O)2OR16, – OS(O)2NR15R15, –C(O)R16, –C(O)OR16, –C(O)NR15R15, –C(NH)NR15R15, –OC(O)R16, – OC(O)OR16, –OC(O)NR15R15and –OC(NH)NR15R15; each R15is independently hydrogen or R16, or alternatively, each R15is taken together with the nitrogen atom to which it is bonded to form a 5- to 8-membered saturated or unsaturated ring which may optionally include one or more of the same or different additional heteroatoms and which may optionally be substituted with one or more of the same or different R13or R16groups; each R16is independently R13or R13substituted with one or more of the same or different R13or R17groups; and each R17is selected from –NR13R13, –OR13, =S, –SR13, =NR13, =NOR13, halo, haloalkyl, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)R13, –S(O)2R13, –S(O)2OR13, – S(O)NR13R13, –S(O)2NR13R13, –OS(O)R13, –OS(O)2R13, –OS(O)2NR13R13, –OS(O)2OR16, –OS(O)2NR13R13, –C(O)R13, –C(O)OR13, –C(O)NR13R13, –C(NH)NR15R13, –OC(O)R13, – OC(O)OR13, –OC(O)NR13R13and –OC(NH)NR13R13.

[0049] In some other embodiments, the chromogenic conjugate moiety is a salt comprising an anion such as, but not limited to, I-. Cl−, Br−, CH3CO2−, CF3CO2−, NO3−, F-, SO42-, HSO4-, (CO3)2-, HCO3-, HCO2-, (PO4)3-, H(PO4)2-, H2(PO4)-, CN-, OH-, CCl3CO2- or ClO4−.

[0050] In another embodiments, the chromogenic conjugate moiety is a salt comprising an cation such as, but not limited to, Na+, K+, Li+, Ca2+, EtNH3+, Et2NH2+, Et3NH+, diisopropylethylammonium, pyridinium, imidazolium, Mg2+, Fe2+, Fe3+, Cu+, Cu2+, Zn2+, Al3+or NH4+.

[0051] In another embodiment, the chromogenic moiety is a derivative of Cyanine, triarylmethane, fluorescein, Cy-3, Cy-5, fluorescein, and O-carboxymethyl fluorescein, 2'- piperazine amide derivative or a salt thereof.

[0052] In some embodiments, R1, R3, R4, R5, R6, R7, R8, R9, and R10, are independently selected from —H, -halogen, -methyl, -ethyl, -propyl, -isopropyl, -vinyl, —SO3H, —PO3H, —NO2, — COOH, —NH2, —CN, —OH, —OMe and —OEt.

[0053] In a specific embodiment, R23,R21or R25is independently –OH, R22or R24is independently –H.

[0054] In a certain embodiment, the chromogenic conjugate is selected from:,,Linkers

[0055] In the present disclosure, a linker (“L”) is a water soluble molecular moiety comprising a chain from 5 to less than 30 contiguous atoms, such as 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 contiguous atoms. In some embodiments the chain of atoms may be linear, in other embodiments it may comprise one or more ring structures. In preferred embodiments, a linker molecule comprises a contiguous chain of 5 to 29 atoms, wherein every two connected carbon atoms are followed by a heteroatom, such as an atom of oxygen or nitrogen. In some embodiments, the linker has no more than two consecutively repeating oxyethylene or -CH2CH2O- groups.

[0056] In some embodiments, the tinker is a compound that comprises 1 or 2 repeats of the following formula:wherein R37is selected from CH2, CH2CH2, CH2CH2CH2, OCH2, CH2OCH2, (CH2OCH2)2, NHCH2, NH(CH2)2, CH2NHCH2, cycloalkyl, alkyl-cycloalkyl, alkyl- cycloalkyl-alkyl, heterocyclyl (such as nitrogen-containing rings of 4 to 8 atoms), alkyl- heterocyclyl, alkyl-heterocycyl-alkyl, and wherein no more than three consecutively repeating -CH2CH2O- groups, or R37and R38are independently elected from NH and O; and each R39is independently CH2or CH2NH.

[0057] More particularly, in some embodiments, the linker is selected from: ,.

[0058] Properties of the linker can be modified to obtain desired performance, such as by altering the length or branching of the linker. Furthermore, the linker may be chemically modified to carry various substituents. The substituents may be further chemically protected and / or activated, allowing the linker to be derivatized further. Peroxidase Substrate Moieties

[0059] The present chromogenic conjugates comprise a peroxidase substrate moiety. The term “peroxidase” relates to an enzyme having enzymatic activity catalyzing a reaction of the form: ROOR′+electron donor (2e−)+2H+→ROH+R′OH

[0060] For many peroxidases the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as organic peroxides. The nature of the electron donor is very dependent on the structure of the enzyme, e.g. Horseradish peroxidase (HRP) / EC 1.11.1.7) can use a variety of organic compounds both as electron donors and acceptors. HRP has an accessible active site, and many compounds can reach the site of the reaction.

[0061] The enzyme with peroxidase activity may be represented by a molecule of a peroxidase enzyme which is directly or indirectly linked to the molecule of a binding agent, or a fragment of the enzyme containing the enzymatic activity, e.g. 51% to 99.9% of the full size of the peroxidase molecule, or less than 51%.

[0062] The peroxidase may be directly or indirectly conjugated with other molecules, e.g. agents that are capable of binding targets of interest in a sample, such as a biological sample, e.g. a histological sample. The term “directly conjugated” means that the enzyme moiety is linked (e.g. chemically conjugated) to another molecule via a chemical bond; the term “indirectly conjugated” means that the peroxidase is linked to the molecule via a linker molecule, which has one chemical bond with a binding agent and another chemical bond with the peroxidase. Methods of conjugating enzyme moieties are well known in the art.

[0063] In one embodiment the moiety of peroxidase is a moiety of HRP, e.g. the whole HRP molecule or a fragment thereof that is capable of the HRP enzymatic activity. It may also be a recombinant protein comprising the part of HRP that possesses the enzymatic activity, etc. In another embodiment the peroxidase may be soybean peroxidase (SP).

[0064] Non-limiting examples of agents which comprise an enzyme with peroxidase activity may be an antibody molecule, such as a primary or secondary antibody molecule or a derivative thereof, e.g. a Fab, directly or indirectly conjugated with one or more moieties of HRP, and nucleic acid binding agents conjugated with HRP. Such binding agents may bind directly or indirectly to targets and form thereby complexes, each comprising a target and one or more molecules of binding agents that comprise an enzyme with peroxidase activity.

[0065] The number of HRP or other peroxidase moiety per molecule of binding agent may vary from 1 to 10 or more, such as 20-50 or more.

[0066] A location of a solid sample (such as a histological sample) or a solid support (such as a membrane or microscopic slide) comprising peroxidase activity is sometimes termed herein “target site”. In one embodiment the target site may comprise a peroxidase activity, such as a moiety of a peroxidase enzyme, which is directly immobilized onto or within a solid support. In another embodiment the target site may comprise a peroxidase activity which is immobilized onto or within a solid support indirectly, such as when a moiety of a peroxidase enzyme islinked to an agent capable of directly or indirectly binding to a target that is immobilized onto or within the support.

[0067] In some embodiments, the peroxidase substrate moiety is a moiety of a substrate of horse radish peroxidase (HRP) or soybean peroxidase (SP). In some embodiments, the peroxidase substrate moiety (also identified as S or PS in some formulas) is a moiety of a non- chromogenic or colorless HRP or SP substrate. In some embodiments, the moiety of peroxidase enzyme substrate has the following formula (Formula II):Formula II wherein chromogenic moiety and L are connected through R26; R21is -H -OR34, or -NR34R35; R22is -H, -OR34, or -NR34R35; R23is -OH; R24is -H, -OR34, or -NR34R35; R25is - H, -OR34, or -NR34R35; and R26is –C(=Z3)-(Z4)p-; wherein, each Z3and Z4is independently O, S, or NR36; each R34, R35, or R36is independently H, alkyl or aryl; and p is 0 or 1.

[0068] In a certain embodiment, R34or R35is H or alkyl; Z3is O or S, and Z4is NR34R35.

[0069] In some embodiment, S or PS is a residue of ferulic acid.

[0070] In other embodiments S or PS is a residue of caffeic acid.

[0071] In other embodiments S or PS is a residue of sinapinic acid. In one preferred embodiment S is a residue of coumaric acid. Composition Comprising Chromogenic Conjugate

[0072] As another aspect, a composition comprising any of the chromogenic conjugates described herein (termed herein as “chromogenic composition” or “chromogenic medium”) isprovided. Some compositions include one of the chromogenic conjugates described herein. Other compositions include two or more of the chromogenic conjugates described herein, for example exactly two chromogenic conjugates (that is, two types of chromogenic molecules, rather than exactly two molecules) or exactly three chromogenic conjugates, or exactly four chromogenic conjugates.

[0073] The compositions may comprise one or more solvents, salts, detergents, and other components. In different embodiments the chromogenic composition may further comprise one or more of the following: (i) an organic modifier; (ii) an enzyme enhancer; (iii) an iron chelator; (iv) a detergent; (v) an anti-microbial agent; (vi) organic or inorganic salt; (vii) an enzyme substrate, e.g. peroxidase substrate. The list of additives to the chromogenic composition is not limiting, and any compound that is capable of enhancing or attenuating the performance of the present chromogenic conjugates as peroxidase substrates or chromogenic molecules is contemplated as a part of the composition depending on the context of its use.

[0074] In some embodiments, the chromogenic composition or medium may comprise any liquid solvent, preferably aqueous solvent (water), where the chromogenic conjugate is initially soluble but capable of reacting to form an insoluble precipitate at the site of peroxidase activity. The liquid solvent may comprise a primary solvent such as water and an organic cosolvent, such as NMP, 1,2- propanediol, ethanol or 2-pyrrolidone, or a mixture thereof. The liquid solvent can comprise a buffer with a suitable buffering capacity, for example, phosphate buffered saline (PBS), Tris buffer, HEPES, EPPS and / or imidazole buffer. The composition can be a buffered aqueous solution that has a pH in the range from 3 to 9, alternatively from about 3 to about 6, alternatively about 4 to about 7, alternatively about 5 to about 8, or about 7.5.

[0075] In some embodiments, the chromogenic composition or medium may comprise an organic or inorganic salt. The inorganic salt may be selected from, but not limited to, sodium chloride, magnesium chloride, potassium chloride, calcium chloride, sodium phosphate, or ammonium sulfate, and combinations thereof. The organic salt may be selected from sodium acetate, ammonium acetate or imidazole salts, for example, imidazole hydrochloride, or others. The concentration of salt may range from approximately 10−3M to saturation, for example, from approximately 20 mM to approximately 200 mM, or from approximately 25 mM to approximately 100 mM. In some embodiments, the media may comprise salt in a concentration of approximately 10 mM, 20 mM, 50 mM, 75 mM or 100 mM.

[0076] In some embodiments the chromogenic composition may comprise a detergent, for example, polyethylenglycol-p-isooctyphenyl ether (NP-40) or a surfactant, for example,selected from the surfactants based on polyoxyethylene sorbitan monolaurate (Tween®), or a surfactant based on block copolymers (Ecosurf®, Pluronic®etc.) or others. The amount of the detergent may vary from about 0.001% to about 5%, either v / v or w / v.

[0077] In some embodiments, an organic modifier may be present in the composition in an amount from about 0% to about 80%, alternatively about 0% to about 75%, about 0% to about 70%, about 0% to about 60%, about 0% to about 50%, or about 1% to about 50% (v / v or w / v), however, in some embodiments higher concentrations of the organic modifier may be required. For example, yellow chromogenic conjugate may use about 75 % of organic modifier, while some chromogenic conjugate such as cyan does not need an organic modifier. An example of yellow chromogenic solution for IHC staining is prepared by dissolving 150 mg HRP yellow chromogenic conjugate in 20 mL propanediol / HEPES at pH 7.7 at 3:1 v / v ratio. For IHC staining, the HRP yellow solution is further mixed with Envision Flex Substrate Buffer (Agilent # GV825; DM843) in 1:1 v / v before use. The organic modifier may for example be propanediol. Other examples include but are not limited to organic modifiers selected from the group consisting of C1-C4 alcohols, N-Methyl pyrrolidone (NMP), dimethylsulphoxide (DMSO), polyethylene glycol (PEG) , mono- and diethylene glycol, sulpholane, N,N- dimethylformamide (DMF) and combinations thereof. In some embodiments it may be advantageous to use polyethylene glycol (PEG), for example, PEG2000, or propylene glycol. The amount of polyethylene glycol or other organic modifier in the composition may vary from about 0.1% (v / v) to about 20% (v / v), for example from about 1% (v / v) to about 15% (v / v), such as 5-10% (v / v).

[0078] By the term “enzyme enhancer” is meant any compound which enhances the catalytic activity of a peroxidase. Such enzyme enhancers may be selected from the group consisting of phenylboronic acid derivatives and divalent metal ions such as nickel or calcium. The concentration of the enzyme enhancer may vary from about 10-7to about 10-3M.

[0079] Examples of an iron chelator include ethylene diamine tetra acetic acid (EDTA) or ethylene diamine hydroxyl phenylacetic acid type chelator (EDHPA). The concentration of the iron chelator may vary from about 10-9to about 10-6M.

[0080] The chromogenic composition can be provided as a stable solution. The term “stable” in the present context means that the capability of the chromogenic composition to serve as reaction media for the peroxidase-mediated conjugate deposition and of the chromogenic conjugate(s) to maintain substantially the same spectral characteristics remain substantially unchanged during substantial periods of time; for example, in some embodiments, the compositions may be prepared and kept for at least 4 hours at room temperature before the use.The compositions may also be prepared and preserved for longer periods of time, such as from 12 hours to 12 months, or for longer periods of time. To prolong the shelf-life of the chromogenic composition, it may be useful to store the composition at temperatures below 20 °C., for example, at 4-10 °C., and / or to add to the composition an anti-microbial compound. The anti-microbial compound may be any anti-microbial compound commonly used with chromogenic compounds.

[0081] The concentration of the chromogenic conjugate in the composition may vary from about 10-9M to about 10-2M, depending on the nature of the method of use. For example, in some embodiments, the concentration of the chromogenic conjugate in the composition may be from about 10-5M to about 10-2M, such as from about 10-3M to about 10-2M.

[0082] In some embodiments, the chromogenic composition may also comprise a peroxide compound. For example the medium can include hydrogen peroxide (H2O2) for example, at a concentration of 0.0002% to 0.04%, or from around 0.5 mM to around 1.5 mM.

[0083] In some embodiments, the present composition are substantially free of peroxidase substrates other than the peroxidase substrate moieties of the chromogenic conjugates of the present disclosure and peroxide compound.

[0084] As mentioned above, in some embodiments, the present compositions may comprise a detergent. In some preferred embodiments, the detergent is a nonionic, non-denaturing detergent. In some embodiments, the composition comprises from 0.001% to 10% detergent, alternatively 0.01% to 1%, alternatively 0.05% to 0.5%.

[0085] In some embodiments, a two-component formulation is provided as a kit or used in a method. A first component comprises a chromogenic conjugate as described herein in a buffer with a pH close to neutral (such as pH of 7 to 8, for example, pH 7.5) to avoid hydrolysis over time. The second component is a buffered solution of hydrogen peroxide with a higher pH (such as a pH of 7-8, for example, pH 7.4). The two components when mixed form a working solution having an intermediate pH (such as pH of 6-8). Such a two component formulation can be mixed by instrument or manually and can have a shelf life of more than 2 years at cold room temperature. The formulation has 1 week stability when mixed together at room temperature.

[0086] In some embodiments, the chromogenic composition comprises an organic cosolvent, for example 2-50% v / v organic co-solvent, which has been found to yield a staining intensity which is significantly enhanced. Suitable organic co-solvents include Propanediol, 2- pyrrolidone and NMP.Method of Preparation of Chromogenic Conjugates

[0087] The chromogenic conjugates of the present disclosure may be synthesized by reacting derivatives of a chromogenic moiety containing a chromogen and a linker with a peroxidase substrate moiety. Alternatively, a peroxidase substrate may be modified to contain a linker moiety followed by reacting with a chromogenic moiety to provide the chromogenic conjugates of the present disclosure.

[0088] Examples of preparation methods for the chromogenic conjugates are provided below:

[0089] In one embodiment, the present disclosure provides a method for preparing the chromogenic conjugate of the present disclosure comprising: reacting a peroxidase substrate moiety represented by Formula IIIa having a protected phenol and a carboxylic acid moiety with a linker moiety having two amino groups with one of the amine with a protecting group P2represented by Formula IIIb to prepare P2-HN-L-PS- P1represented by Formula IIIc; removing the protecting group P2to prepare Formula IIId; reacting Formula Iaa, Formula Ibb, or Formula Icc with Formula IIId to prepare Formula IIIe, Formula IIIf, or Formula IIIg, respectively; and removing a protecting group P1to prepare the chromogenic conjugate of claim 1 represented by Formula Ia, Ib or Ic:Formula IIIaFormula IIIbFormula IIIc, provided that at least one of RPSor RPS’is, provided that at least one of RPSor RPSisFormula IIIg.

[0090] In another embodiment, the present disclosure provides a method for preparing the chromogenic conjugate of the present disclosure comprising: reacting Formula Iaa, Formula Ibb, or Formula Icc with Formula IIIb; removing a protecting group P2to prepare Formula Idd, Iee, or Iff; reacting a peroxidase substrate moiety represented by Formula IIIa having a protected phenol and a carboxylic acid moiety with Idd, Iee, or Iff to prepare chemical compound represented by Formula IIIe, IIIf, or IIIg; removing a protecting group P1to prepare the chromogenic conjugate of claim 1 represented by Formula Ia, Ib or Ic:, provided that at least one of RPSor RPS’iso ua dd, provided that at least one of RPSor RPSisg, wherein all variables are as defined above.

[0091] Alternatively, the above method may employ a large excess of H2N-L-NH2 instead of P2-HN-L-NH2.

[0092] In another alternate embodiment, one or more chemically reactive functional groups in the reactants may be protected with an appropriate protecting groups.

[0093] An example of synthetic method of the chromogenic conjugates of the present disclosure is provided in Scheme 1: Scheme 1.

[0094] Another example of synthesis of chromogenic conjugate of the present disclosure, such as yellow chromogen (compound 11 or Formula IIId), is described in the following Scheme 2: Scheme 2.

[0095] Yet another example of synthesis of chromogenic conjugate of the present disclosure, such as cyan chromogenic conjugates, is provides in Scheme 3: Scheme 3.Methods of Use

[0096] The present disclosure provides a method for detection of a target in a sample by chromogenic detection comprising: incubating a sample comprising a target in an aqueous solution, wherein the target comprises peroxidase activity or the target is directly or indirectly linked to a peroxidase enzyme, wherein the aqueous solution comprises: a) at least one chromogenic conjugate described herein; b) a peroxide compound, at a time and temperature sufficient to form a colored precipitate of the chromogenic conjugate;detecting the colored precipitate of the chromogenic conjugate in the sample, thereby detecting the target in the sample.

[0097] The above method may further comprise incubating the sample two or more cycles with a chromogenic conjugate having Formula Va, Vb, Vc or Vd:VcFormula Vd, wherein all variables are as defined previously, and wherein each cycle comprises a same or different chromogenic conjugates in a same or different ratios.

[0098] Another aspect of the present disclosure provides a method of performing chromogenic in situ hybridization comprising: contacting a nucleic acid target with a probe that hybridizes with the nucleic acid target under hybridization conditions, wherein the probe comprises (1) a nucleic acid sequence at least partially complementary to the nucleic acid target and (2) a peroxidase enzyme or a first member of a specific binding pair; wherein the target and probe form a complex; when the probe comprises (2), contacting the complex with a second member of the specific binding pair, wherein the second member is directly or indirectly linked to a peroxidase enzyme, and specifically binds to the first member; incubating the complex with at least one of the chromogenic conjugates of the present disclosure for a time and temperature sufficient to form a color precipitate at the target; and detecting the color precipitate.

[0099] Yet another embodiment of the present disclosure provides a method of detecting two or more targets comprising: providing peroxidase activity at a first target in a sample; contacting the sample with a first chromogenic conjugate; forming a first colored precipitate at the first target; removing the peroxidase activity from the first target; removing non-precipitated first chromogenic conjugate from the sample; providing peroxidase activity at a second target in the sample; contacting the sample with a second chromogenic conjugate; forming a second colored precipitate at the second target; anddetecting the first colored precipitate and the second colored precipitate, thereby detecting the first and second targets in the sample, wherein at least one of the first and second chromogenic conjugates is any of the conjugates described herein, and wherein the first chromogenic conjugate molecule and the second chromogenic conjugate have one or more spectral characteristics that are different from each other, wherein, the method may further comprise: removing the peroxidase activity from the second target; removing non-precipitated second chromogenic conjugate from the sample; providing peroxidase activity at a third target in the sample; contacting the sample with a third chromogenic conjugate having one or more spectral characteristics that are different from those of the first and second chromogenic conjugates.

[0100] An embodiment of the present disclosure also provides a method for preparing a chromogenic conjugate of the present disclosure comprising: reacting a chromogenic moiety with a secondary amine compound containing an ester to prepare a secondary amide compound of the chromogenic moiety; converting the ester in the secondary amide compound of the chromogenic moiety to a carboxylic acid; and reacting the secondary amide compound of the chromogenic moiety having the carboxylic acid with a linker compound having an amine group to form the chromogenic conjugate.

[0101] Another embodiment of the present disclosure provides an immunohistochemical staining composition comprising two or more of chromogenic conjugates selected from the group consisting of a chromogenic conjugate of Formula Ia, a chromogenic conjugate of Formula Ib, a chromogenic conjugate of Formula Ic, and a chromogenic conjugate of Formula Id:Formula Iawherein, all variables are as defined above; the immunohistochemical staining composition may provide red, orange, dark green, lime, pea green, aqua, blue, cyan or purple color; color is determined by the ratio of the first, the second, the third or the fourth chromogenic conjugates, wherein the chromogenic conjugates of the present disclosure may be mixed in certain ratios to provide a desired color.

[0102] By using the combination of a mixture of the chromogenic conjugates of the present disclosure in various ratios, a various colors can be generated to provide stains of any desired color. Examples of mixed color and the results are provided as following and also in FIG.3:

[0103] As another aspect, methods are provided for using the chromogenic conjugates described herein and compositions comprising those conjugates in various analyses, techniques and steps where a sample, tissue, or portions thereof are stained or dyed. For example, the present chromogenic conjugates are useful for detection of molecular targets, such biological or chemical molecules, molecular structures, etc, in samples using a host of experimental schemes for detecting and visualizing such targets, for example, immunohistochemistry (IHC), in situ hybridization (ISH), ELISA, Southern, Northern, and Western blotting. Generally, the present chromogenic conjugates may be used in any analysis in which DAB has been used as stain for visualization of targets. Compared to conjugates described in WO2009 / 036700, WO2010 / 094283, WO 2010 / 094284, WO2011 / 047680 and WO2012 / 143010, the disclosures of each of which are incorporated herein by reference in its entirety, the present chromogenic conjugates do not demand the presence of a cross-linker such as DAB, ACHC or ferulic acid to mediate precipitation of the conjugates from chromogenic described above in the presence of peroxidase. The chromogenic conjugates can be used for detection of molecular targets in solid or semi-solid samples or targets that are immobilized onto or into solid supports, such as a microscopic slide, nitrocellulose membrane, microarray chip, gel and other supports.

[0104] For example, the present chromogenic conjugates can be used to stain or dye formalin Fixed Paraffin Embedded (FFPE) tissue samples, metaphase spreads or histological smears. The present chromogenic conjugates can be used in immunohistochemical analytical methods, where a protein of interest is detected or identified by color. The present chromogenic conjugates can be used in chromogenic in situ hybridization (CISH), where a nucleic acid of interest is detected or identified by color.

[0105] The present methods are carried out by linking a target with a molecule or moiety with peroxidase activity, typically Horse Radish Peroxidase (HRP) or fragments having the enzymatic activity, then subsequently catalyzing the formation of an insoluble colored precipitate at the location of the target using any of the soluble chromogenic conjugates described herein.

[0106] The present conjugates are superior to chromogens such as DAB for use with cells, tissues and other types of samples which have brown coloring. For example, melanomas are already brown by nature, as are many lung cancer specimens due to smoking or urban air pollution. For these samples, DAB is especially ill suited for staining specimens from patients afflicted with these two major types of cancers. Accordingly, as another aspect, methods are provided for staining specimens having a natural brown color by applying the present conjugates, particularly a red, yellow or blue chromogenic conjugate. In some embodiments, one, two, or more of the present chromogenic conjugates are contacted with a sample comprising a brown tissue, for example, a sample comprising lung cancer cells, melanoma cells, melanocytes, mole tissue, tonsil tissue, or liver tissue. The present conjugates can also advantageously be used for detection of multiple targets in a sample.

[0107] In the present methods, one or more binding agents may be applied to a sample before the present conjugates are applied to the sample. The term “binding agent” designates a molecule that is capable of direct or indirect binding to a target, wherein the term “directly” means that the binding agent has affinity to the target and is capable of recognizing the target and specifically binding to it, wherein the term “indirectly” means that the binding agent does not have specific affinity to the target but has affinity to a substance which is associated with the target and is capable of specifically binding to this substance. The binding agent which is capable of direct binding to a target is sometimes referred to as a “primary binding agent”. A binding agent which is capable of indirect binding to a target is sometimes referred to as a “secondary binding agent”. The primary binding agent is typically used to contact the sample. It may be comprised of any molecule which will specifically bind to the target supposedly present in the sample. The secondary binding agent may be any molecule that binds the primary binding agent. In some embodiments, the primary or secondary binding agents may comprise an antibody, a fragment of an antibody capable of binding to a target or to an antibody bound to a target, or a nucleic acid which binds to a target or to a nucleic acid bound to a target. Detection systems employing the present conjugates for visualization of targets may comprise other binding agents, such as a tertiary or quaternary binding agents; the detection may include several primary binding agents directed to detect various targets in the sample, for example, two or more different molecules (such as two or more proteins, or a protein and a nucleic acid) or include several primary binding agents directed to detect the same target in the sample, for example multiple molecular probes detecting a gene of interest. The detection systems may also include several secondary binding agents, which may be molecules of same species, for example antibodies, or different species, for example antibody and nucleic acids.

[0108] In cases where the target does not inherently comprise peroxidase activity, at least one of the binding agents used to detect a target in the sample in the present visualization systems comprises a peroxidase activity in order to label the location of target in the sample with the peroxidase activity.

[0109] Use of the present conjugate molecules may be particularly advantageous in multiplexed methods where more than one target is to be stained or dyed. As described above, the present conjugate molecules have advantageous optical features that allow clear distinction between targets stained in different colors, both by observing the microscopic field of a stained sample, such as a histological sample, and analyzing a captured image of the stained sample. Another advantage is that the conjugates are substrates of the same enzyme, namely a peroxidase enzyme, such as HRP or SP. This significantly simplifies procedures for staining of multiple targets in samples, increases robustness of staining procedures, as one and the same protocol and the same reagents may be used for detection of the targets by employing different conjugates. It also makes the overall detection procedure less expensive and particularly suited for automated staining, imaging of stained samples and analyses of staining results.

[0110] Another advantage of the chromogenic conjugates of the present disclosure includes that the chromogenic conjugates utilize the same peroxidase substrate would avoid any potential differences in reactivities of different substrates and the chemical reactivity among the different chromogenic conjugates would not affect the staining result when two of more chromogenic conjugates are mixed. The final staining color would be determined mostly by the mixing ratio and the strength of the color, not by the different chemical reactivities of the substrate.

[0111] The present methods and kits can comprise any binding agents capable of detecting a target in a solid sample or a target immobilized onto or into a solid support. For example, the binding agent can form an immune-specific binding pair such as an antibody and antigen, or a non-immune specific binding pair such as a nucleic acid probe and a complementary sequence, or another type such as biotin and avidin. Binding agents of various types are common general knowledge in the field, and descriptions of binding agents can be found in Q. Ashton Acton, ed., “Antigens—Advances in Research and Application: 2013 Edition”, Scholarly Editions. or Ralph, ed, “The Nucleic Acid Protocols Handbook”, Humana Press.

[0112] In some embodiment, the binding agent in a kit is an antibody identifying a target antigen.

[0113] Typically, in the present target detection methods, a sample comprising a target is sequentially incubated in one or more incubation media. The term “incubation medium” meansin the present context an aqueous medium comprising particular compounds where a sample is maintained during a certain period of time (termed herein “incubation time”) in order to allow a desirable reaction between the particular compounds of the solution and the sample to take place. The incubation media may be the media in which the target is naturally found by a binding agent (“binding agent media”), or it may be one of the present chromogenic compositions (such as those described in the above sections).

[0114] In some embodiments, the time for maintaining and / or incubating the sample in an incubation medium, i.e. incubating time, may vary from approximately 3 seconds to overnight, for example, around 10 seconds, 20 seconds, 30 seconds, 1 minute, 2 minutes, etc., for example, 3-10 minutes, 10-20 minutes, 20-40 minutes, 40-60 minutes, 1-2 hours or longer, for example, overnight. In one embodiment, the incubating time at all steps of the detection procedure may have the same duration, i.e. every incubation may last 1 min, 2 min, 3 minutes, 5 minutes, 10 minutes, etc. The time may be selected based on the embodiment used. In another embodiment, the incubation time may vary from one step to another, for example, incubating the sample in a media comprising a binding agent may last from 1 minute to overnight, incubating the sample in a media comprising the chromogenic conjugate and a peroxide compound may last from 1 min to 15 minutes or longer.

[0115] Incubation may be performed in various temperature conditions, depending on the type of target, binding agent, conjugate, etc. The detection procedures are mainly temperature independent, however, if desired, the temperature may be used for regulating the duration of the incubation time, for example, lower temperatures may be used to prolong the incubation time, and, vice versa, higher temperatures may be used to shorten the time for incubation.

[0116] Basically, the binding agent media is a buffered aqueous solution of one or more binding agents that preferably has pH in the range from 4 to 9. In some embodiments the first incubation media may comprise an organic or inorganic salt. The inorganic salt may be selected from for example, sodium chloride, magnesium chloride, potassium chloride, calcium chloride, sodium phosphate, or ammonium sulfate. The organic salt may be selected from for example, sodium acetate, ammonium acetate or imidazole salts, for example, imidazole hydrochloride, etc.

[0117] The concentration of salt in binding agent media may range from approximately 10−3M to saturation, for example, from approximately 20 mM to approximately 200 mM, or from approximately 50 mM to approximately 500 mM. In some embodiments, the media may comprise salt in the concentration from approximately 10 mM to 500 mM. In other embodiments the medium may be free of salt.

[0118] As mentioned, typically, the pH value of binding agent media may vary from about 4 to about 9, such as between pH 3.5 and pH 9.5, for example, between pH 5 and pH 7, between pH 5.5 and pH 6.5 or between pH 6.5 and 7.5, or between pH 7 and pH 8, or between pH 7.5 and pH 8.5, or pH 8 and pH 9. Any buffer with a suitable buffer capacity may be used, for example, phosphate buffered sale (PBS), HEPES, EPPS and imidazole buffer. Other suitable buffers may be found in Good, N E., et al (1966) Hydrogen ion buffers for biological research. Biochem.5(2), 467-477. The pH value of the media may be essential for binding of binding agent to the target; it may be optimized depending on the nature of the binding agent and the target.

[0119] In some embodiments the binding agent media may comprise an organic modifier (by the term “organic modifier” is meant any non-water solvent), for example, N-methyl pyrrolidone (NMP), dimethylsulphoxide (DMSO), mono- and diethylene glycol, sulpholane, N,N-dimethylformamide (DMF), propanediol, polyethylene glycol (PEG), propylene glycol, etc. In some embodiments, the amount of the organic modifier may vary from around 1% to around 20% (v / v or w / v), or, in some embodiments, be higher than 20%.

[0120] In some embodiments the binding agent media may comprise a detergent, for example, polyethyleneglycol-p-isooctyphenyl ether (NP-40), ECOSURF or a surfactant for example, selected from the surfactants based on polyoxyethylene sorbitan monolaurate (Tween), or a surfactant based on block copolymers (PLURONIC etc.), etc. The amount of the detergent may vary from about 0.001% to about 5% v / v or w / v). In some embodiments the binding agent media may comprise a stabilizing agent for the binding agent, for example, bovine serum albumin or dextran. The amount of the stabilizing agent may vary from 0.01% to 20% (w / v).

[0121] In some embodiments the binding agent media may comprise an ion chelator (for example, ethylene diamine tetra acetic acid (EDTA) or ethylene diamine hydroxyl phenylacetic acid type chelator (EDHPA), etc.). In some embodiments, the concentration of the chelator may vary from about 10−9M to about 10−6M.

[0122] In some embodiments, the binding agent media may comprise one or more blocking agents for saturating non-specific binding sites, i.e. sites of the solid support that do not comprise the target. Some non-limiting examples of blocking agents suitable for different embodiments may be the Denhard's solution, bovine serum albumin, skimmed milk, etc.

[0123] Because a great variety of species of targets, binding agents and assay formats are contemplated, the composition of the binding agent media may vary and may be adjusted for particular embodiments using the knowledge of the art.

[0124] The present methods also comprise one or more wash steps before or after incubation of a sample using a washing media, for example, between the step of incubation of the sample with a binding agent and the step of staining the sample with one or more of the present chromogenic conjugates. Typically, a washing medium will be a medium of the same or similar composition as one that has been used for incubating or otherwise treating the sample in the step preceding the washing step, wherein the washing media lacks the active ingredient, i.e. a particular agent of the incubation step, for example, a binding agent, a conjugate molecule, etc.

[0125] The present methods may comprise one or more steps of incubating of the sample in a medium that would quench any undesirable peroxidase activity, such as endogeneous peroxidase activity in the sample or residual peroxidase activity associated with the target site. Typically, incubating the sample in a peroxidase activity quenching medium will precede a step of incubating the sample with a binding agent, or, in case of detection of several targets in a sample, it will take place before and after the first target is visualized with a first conjugate and before incubation of the sample with a second binding agent directed to a second target. A peroxidase activity quenching medium would typically comprise an amount of a peroxide compound, such as hydrogen peroxide. The amount of a peroxide compound in the medium may vary from 1% to 10% (v / v or w / v).

[0126] The present methods and uses include assays of various formats in which the present chromogenic conjugates are employed. In general, the assays where the conjugates may be used are any of those where DAB can be used. Some non-limiting embodiments of such assay formats are described below.

[0127] Targets or biomarkers may be present in cells or tissues, and they can be detected employing the methods described herein in any suitable assay format, for example in ImmunoHistoChemistry (IHC), or chromogenic in situ hybridization (CISH), or ELISA.

[0128] In some embodiments, the target may be a protein, e.g. a cellular membrane receptor or a cytoplasmic protein, in other embodiments, the target may be a nucleic acid, e.g. a cytoplasmic nucleic acid. Derivatives of any latter mentioned targets, e.g. fragments, precursors, mutants of target proteins or nucleic acids, etc. may also be targets in some embodiments.

[0129] Thus, in various embodiments, the target may be a biological or chemical target molecule, or a particle, or a molecular or cellular complex, or molecular or cellular structure, or a virus, or a microorganism, or a fragment of said target molecule, particle, complex, structure, virus or microorganism. Among targets contained in chemical and environmentalsamples may be different pollutants, toxins, warfare substances, members of molecular libraries, industrial noxious waste compounds, etc.

[0130] In some embodiments, the biological sample may be a suspension of cells or a tissue section. Target molecules or structures of cells in suspension may be detected using ELISA, IHC or CISH. When ELISA, IHC or ISH are used for the detection cells of the suspension are to be attached to a solid support, for example, ELISA plate or slide.

[0131] Preparation and process steps for cells, tissues or other samples in IHC or CISH are common general knowledge in the field. For descriptions of various steps, see for example “Immunohistochemical Staining Methods”, Dako IHC Guidebook, 6th Ed (2013); van der Loos, “User Protocol: Practical Guide to Multiple Staining”, Cambridge Research & Instrumentation, Inc. (2009); “Immunohistochemistry (IHC) an Application Guide”, Abcam, (2013); “Handbook of Practical Immunohistochemistry”, Lin & Prichard ed. (2015); Immunohistochemistry and In Situ Hybridization of Human Carcinomas”, Hayat ed. (2005), the disclosures of each of which are incorporated herein by reference in its entirety.

[0132] In CISH, a sample is taken and exposed to a nucleic acid binding agent which hybridizes by virtue of complementary base pairing to the target nucleic acid. The target nucleic acid in the sample is typically denatured to expose binding sites. In the present assays, the binding agent has or is linked to a peroxidase enzyme directly or indirectly, such as via an antibody, and one or more of the chromogenic conjugates of the present disclosure are thereafter contacted with the sample. The existence or amount of the target nucleic acid is detected by visual recognition of the chromogen manually or automatedly.

[0133] In IHC, a sample is taken and exposed to a binding agent such as an antibody or fragment thereof which specifically binds a target molecule such as a cell surface protein. In the present assays, the binding agent has or is linked to a peroxidase enzyme, such as via a secondary antibody, and one or more of the present chromogenic conjugates are thereafter contacted with the sample. The existence or amount of the target molecule is detected by visual recognition of the chromogen manually or automatedly.

[0134] In the present assays, a light microscope, often referred to as optical microscope, uses visible light and a system of lenses to magnify images of small samples containing the chromogenic conjugate. Bright-field microscopy is a simple optical microscopy illumination technique. The sample is illuminated by white light, and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample. The typical appearance of a bright-field microscopy image is a chromogenically colored sample on a bright background.

[0135] Automated staining and visualization devices may be used in various embodiments of the present methods, for example for the detection of multiple biological markers. Detection of multiple markers frequently requires balancing of the signals derived from the different chromogenic moieties. Automated staining devices are known in the field and the methods may be adapted for these devices.

[0136] In automated methods of analysis, computer-controlled automatic test equipment is used to evaluate the stained samples, using computations to derive quantitative measurements from an image. High-performance charge-coupled device (CCD) cameras can be used for visualizing the chromogenically stained samples having one or more colored precipitates. Image acquisition can be coupled with advanced widefield microscopes and various algorithms for image restoration. Color separation can be obtained using three-CCD devices (3CCD) and a dichroic beam splitter prism that splits the image into red, green and blue components. Each of the three CCDs is arranged to respond to a particular color.

[0137] The methods of analysis described herein can include some or all of the following steps: (a) collecting a first tissue or cell sample from an individual diagnosed with or suspected of having a cancer, particularly lung cancer cells, melanoma cells, or liver cancer cells; (b) applying a binding agent for a target, wherein the binding agent has peroxidase activity; (c) staining the first tissue or cell samples with one or more of the present chromogenic conjugates; (d) measuring the optical density of the stained tissue or cell samples from step (c), wherein the stained tissue or cell samples are illuminated with light having a wavelength absorbed by the one or more chromogenic conjugates.

[0138] In some embodiments, the present methods are for detecting a target, in a sample, for example, an object with peroxidase activity, biological marker, etc., wherein said target or sample is immobilized onto a solid support, wherein the methods comprise steps of (a) incubating a sample supposedly comprising target with one or more binding agents comprising peroxidase activity, wherein said one or more binding agents is / are capable of direct or indirect binding to the target and forming a complex comprising the target and one or more binding agents having peroxidase activity; (b) incubating the sample in a solution comprising one or more of the present chromogenic conjugates; (c) detecting the deposited chromogenic conjugate, and thereby detecting the target.

[0139] Yet another aspect of the present disclosure provides a method for staining a sample by mixing chromogenic conjugates of the present disclosure with other chromogens or mixing multiple chromogenic conjugates of the present disclosure. For example, Rhodamine 6G-L12- Cou can be mixed with a fluorescein chromogen to produce a yellow / orange color. Thiscombination is favorable because no single chromogen produces a suitable orange color. Additionally, rhodamine derivatives such as rhodamine 101 derivatives are expensive and chemically troublesome. A chromogen producing a yellow color will be considered weak to the human eye since yellow chromogens based on fluorescein have sharp upper absorbance peaks and absorb little green light, even in high concentration / intense stains. In other words, the yellow color does not change; they remain yellow. On the other hand, yellow dyes with broader absorbance extending weakly into the green absorbance range exhibit dichroism, meaning they produce two colors. In low concentration, these dyes appear yellow since they absorb blue light well; in higher concentrations, the color shifts towards red as green light is absorbed. Therefore, by mixing a green absorbing Rhodamine 6G with a yellow dye, the mixture can yield a designer dichromic dye, where hue changes slightly with intensity to yield an extended dynamic range of color contrast.

[0140] Although the present conjugate molecules are described with respect to their chromogenic properties, it is also contemplated that the one or more of the chromogenic conjugates of the present disclosure, optionally with other chromogenic conjugate known in the art, can be used as a fluorescent or other types of chromogenic molecules in assays and methods that detect fluorescence or other chromogens. Kit-of-Parts

[0141] A kit-of-parts comprising any of the present chromogenic conjugates or compositions comprising thereof is described herein for detecting a target in a sample. For example, a kit-of- parts can include one or more of the conjugates set forth, alternatively two, three, four or more of those conjugates, in a single composition or container or in separate compositions or containers.

[0142] Because the present methods are suitable for detection of a huge variety of targets in a variety samples in a variety assay formats, kits-of-parts may comprise many different items. However all kits-of-parts comprise a chromogenic conjugate, either in a solid form (powder, lyophilized, etc.) or as a composition comprising a chromogenic conjugate molecule or an incubation medium comprising a chromogenic conjugate molecule as described herein. The following are some non-limited exemplary embodiments of a kit-of-parts.

[0143] In one embodiment, the kit-of-parts may comprise; (i) a chromogenic conjugate as described herein, either in a solid form (powder, lyophilized, etc.) or in a liquid medium; and (ii) one or more binding agents capable of direct or indirect binding to a target, wherein binding agents may be any binding agents described herein.

[0144] In another embodiment the kit-of pats may comprise: (i) a solution of a first chromogenic conjugate as described in the present disclosure, wherein the first conjugate has first spectral characteristics; (ii) a solution of a second chromogenic conjugate as described in the present disclosure, wherein the second chromogenic conjugate has one or more spectral characteristics that are different from one or more spectral characteristics of the first chromogenic conjugate. Either of the first or the second conjugate can be one of the conjugates set forth below as embodiments in the Examples.

[0145] The present kit-of parts may comprise several parts (1, 2, 3, 4, 5, 6 or more) which are solutions of different chromogenic conjugates as any of the defined above, wherein each of the different conjugates has distinct spectral characteristics that are different from spectral characteristics of the other conjugates that are included in the kit.

[0146] In another embodiment the kit-of parts as any of the above may further comprise one or more binding agents capable of direct or indirect binding to a target, wherein binding agents may be any binding agents described herein.

[0147] In another embodiment the kit-of parts as any of the above may further comprise instructions for use of the chromogenic conjugate, staining interpretation and / or scoring guidelines.

[0148] In other embodiments the kit-of parts as any of the above may further comprise one or more of the following: an aqueous composition comprising DAB, ACHC or another peroxidase substrate; a binding agent capable of specifically binding to the chromogenic detectable label of the conjugate; protocols for staining, visualization and / or quantification of targets; one or more reference materials, e.g. a sample comprising a stained target; an additional stain, e.g. histological stain or a substrate solution for another than peroxidase enzyme; mounting media; incubation media, washing media, etc.

[0149] In another embodiment, the kit-of-parts may comprise (i) any of the items or all items of the above embodiments; (ii) means or components for visualization of targets and / or image capture, or a reference to such means; (iii) software for controlling the instruments; (iv) software for image analysts; (iv) locked image analysis algorithms.

[0150] Composition of the present kit-of-part may be designed to suit any of the applications described above of the present chromogenic conjugates.

[0151] Yet another aspect is a method to stain a tissue sample for the absence of a target. It has been recognized that if a certain portion of a tissue is stained with DAB, then the portion of the tissue cannot be stained with a different color, unless the first stain is very weak and the secondstain is very intense. It should be appreciated that it is possible to make a double stain of co- localized targets in mixed colors.

[0152] However, in such case of co-localized targets, the first chromogen reduces the number of antigens available for recognition by the second antibody and also reduces the number of the first primary antibodies (not recognized by HRP visualization in the first step) that might be visualized in a different color in the second step.

[0153] This effect can be performed to stain for the absence of a target such as a marker. For example, this was observed on in using two of the present chromogenic conjugates to stain colon carcinomas. Colon tissue is normally positive for Cytokeratin 18. Colon tissue was stained using a method comprising staining cytokeratin 18 with a chromogenic conjugate of a first color, then staining with a second chromogenic conjugate of a second color to stain all cytokeratins in the colon tissue. For example, the first and second chromogenic conjugate may precipitate at the same binding agent, and they may contact the colon tissue simultaneously or sequentially. Alternatively, this may be accomplished by having two different binding agents, one of which is specific for Cytokeratin 18, and the other is pan-specific for cytokeratin (both normal colon and carcinomas are positive for cytokeratins), wherein a first of the two chromogenic conjugates precipitates at a first binding agent and a second of the two chromogenic conjugates precipitates a second binding agent. After staining colon carcinoma tissue with first and second chromogenic conjugates of distinct colors, it was observed that in many carcinomas, small areas were identified by the second chromogenic conjugate, in that they were stained in the second color. If the carcionomas were stained in the first color only, these areas could easily be overlooked and the sample could incorrectly be deemed negative for Cytokeratin 18, even though the absence of a stain could have been caused by many factors, such as air bubbles during staining, necrotic tissue, bad fixation, or another cause. Thus, the technical effect of the second stain is that it positively confirms the absence of cytokeratin 18, indicating for example that the cells have been mutated and lost their ability to produce cytokeratin 18. It is known that colon carcinomas undergo such mutations. Therefore, this method of staining allows one to use first and second chromogenic conjugates to identify carcinomas that have such mutations. Specific Aspects

[0154] Below are specific aspects of the disclosure.

[0155] Aspect 1. A chromogenic conjugate comprising: (a) a chromogenic moiety, and(b) a peroxidase substrate moiety, wherein the chromogenic moiety and the peroxidase substrate moiety are linked together via a linker, wherein the conjugate is a compound of Formula Ia, Ib, Ic, or a salt thereof:c wherein, X is independently selected from –OH, -ORXor –NRXRXX; Y is independently selected from O or =N+RYRYY; Z, Z’, Z1, or Z1’are independently selected from O, S, or NRZ; wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R1’, R2’, R3’, R4’, R5’, R6’, R7’, R31, R32, R31’, R32’, RX, RXX, RY, RYY, and RZare independently selected fromhydrogen, OH and a substituent having less than 40 atoms, wherein three or more atoms can be connected to form a cyclic structure;m or p’ is an integer selected from 0 or 1; n or n’ is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is an integer selected from 0, 1, 2 or 3; L is a linker comprising 5 to 29 connected atoms; and PS is H, NH2, OH, or a peroxidase substrate moiety represented by the following Formula II, provide that at least one of PS is a peroxidase substrate moiety:Formula II wherein, L and PS are connected through R26; R21is -H -OR34, or -NR34R35; R22is -H, -OR34, or -NR34R35; R23is -OH; R24is -H, -OR34, or -NR34R35; R25is - H, -OR34, or -NR34R35; and R26is –C(=Z3)-(Z4)p-; wherein, each Z3and Z4is independently O, S, or NR36; each R34, R35, or R36is independently H, alkyl or aryl; andp is 0 or 1.

[0156] Aspect 2. The chromogenic conjugate of aspect 1, wherein R1is selected from hydrogen, OH, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, R1may be taken together with R2to form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; R2is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, R2may be taken together with R1, to form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; RX, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; RXX, when present, is selected from (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R3is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R4is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40)arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, when Y is –NRYRYY, R4may be taken together with RYYto form a 5- or 6-membered ring which is optionally substituted with one or more of the same or different R13or suitable R14groups; RYY, when present, is selected from (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively RYYmay be taken together with R4to form a 5- or 6-membered ring which is optionally substituted with one or more of the same or different R13or suitable R14groups; RY, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, RYmay be taken together with R5to form a 5- or 6-membered ring optionally substituted with one or more of the same or different R13or suitable R14groups; RZ, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R5is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, R5may be taken together with R6to form part of a benzo, naptho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, when Y is –NRYRYY, R5may be taken together with RYto form a 5- or 6-membered ring optionally substituted with one or more of the same or different R13or suitable R14groups; R6is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, R6together with R5may form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; R7, R8and R9are each, independently of one another, selected from hydrogen, R11, (C1- C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R10is selected from selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, halo, haloalkyl, –OR12, –SR12, –SOR12, – SO2R12, and nitrile; R11is selected from –NR15R15, –OR16, –SR16, halo, haloalkyl, –CN, –NC, –OCN, – SCN, –NO, –NO2, –N3, –S(O)R16, –S(O)2R16, –S(O)2OR16, –S(O)NR15R15, –S(O)2NR15R15– OS(O)R16, –OS(O)2R16, –OS(O)2NR15R15, –OP(O)2R16, –OP(O)3R16R16, –P(O)3R16R16, – C(O)R16, –C(O)OR16, –C(O)NR15R15, –C(NH)NR15R15, –OC(O)R16, –OC(O)OR16, – OC(O)NR15R15and –OC(NH)NR15R15; R12is selected from (C1-C20) alkyls or heteroalkyls optionally substituted with lipophilic substituents, (C5-C20) aryls or heteroaryls optionally substituted with lipophilic substituents and (C2-C26) arylalkyl or heteroarylalkyls optionally substituted with lipophilic substituents; R13is selected from hydrogen, (C1-C8) alkyl or heteroalkyl, (C5-C20) aryl or heteroaryl and (C6-C28) arylalkyl or heteroarylalkyl; R14is selected from –NR15R15, =O, –OR16, =S, –SR16, =NR16, =NOR16, halo, haloalkyl,S(O)NR15R15, –S(O)2NR15R15, –OS(O)R16, –OS(O)2R16, –OS(O)2NR15R15, –OS(O)2OR16, – OS(O)2NR15R15, –C(O)R16, –C(O)OR16, –C(O)NR15R15, –C(NH)NR15R15, –OC(O)R16, – OC(O)OR16, –OC(O)NR15R15and –OC(NH)NR15R15; each R15is independently hydrogen or R16, or alternatively, each R15is taken together with the nitrogen atom to which it is bonded to form a 5- to 8-membered saturated orunsaturated ring which may optionally include one or more of the same or different additional heteroatoms and which may optionally be substituted with one or more of the same or different R13or R16groups; each R16is independently R13or R13substituted with one or more of the same or different R13or R17groups; and each R17is selected from –NR13R13, –OR13, =S, –SR13, =NR13, =NOR13, halo, haloalkyl,OS(O)2NR13R13, –C(O)R13, –C(O)OR13, –C(O)NR13R13, –C(NH)NR15R13, –OC(O)R13, – OC(O)OR13, –OC(O)NR13R13and –OC(NH)NR13R13.

[0157] Aspect 3. The chromogenic conjugate of aspect 1, wherein the chromogenic moiety is a derivative of Cyanine, triarylmethane, fluorescein, or a salt thereof.

[0158] Aspect 4. The chromogenic conjugate of aspect 1, wherein the chromogenic moiety is a derivative of Cyanine or a salt thereof.

[0159] Aspect 5. The chromogenic conjugate of aspect 1, wherein the chromogenic moiety is a derivative of triarylmethane or a salt thereof.

[0160] Aspect 6. The chromogenic conjugate of aspect 1, wherein the chromogenic moiety is a derivative of fluorescein or a salt thereof.

[0161] Aspect 7. The chromogenic conjugate of aspect 1, wherein the chromogenic moiety is selected from the group consisting of Cy-3, Cy-5 , triarylmethanes, fluorescein, and O-carboxymethyl fluorescein.

[0162] Aspect 8. The chromogenic conjugate of aspect 1, wherein the chromogenic moiety is a 2'-piperazine amide derivative.

[0163] Aspect 9. The conjugate of aspect 1, wherein R23is –OH, and R24is –H.

[0164] Aspect 10. The conjugate of aspect 1, wherein either R21or R25is –OH, R22and R24are –H, and R23is –OH.

[0165] Aspect 11. The conjugate of aspect 1, wherein the peroxidase substrate is a residue of ferulic acid, cinnamic acid, caffeic acid, sinapinic acid, 2,4-dihydroxycinnamic acid or 4- hydroxycinnamic acid (coumaric acid).

[0166] Aspect 12. The conjugate of aspect 1, wherein L comprises 1 or 2 repeats of Formula IVa, IVb, IVc, IVd, or IVe:Formula Iva,wherein each R37is independently selected from methyl, ethyl, propyl, OCH2, CH2OCH2, (CH2OCH2)2, S, NH, NHCH2, NH(CH2)2, CH2NHCH2, cycloalkyl, alkyl-cycloalkyl, alkyl- cycloalkyl-alkyl, heterocycloalkyl, alkyl-heterocyclyl, alkyl-heterocyclyl-alkyl; each R38is independently NH or O; w is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each R39is independently CH2or CH2NH.

[0167] Aspect 13. The conjugate of aspect 1, wherein the linker is selected from:,.

[0168] Aspect 14. The chromogenic conjugate of aspect 1 selected from the group consisting of: ,,,.

[0169] Aspect 15. A composition comprising at least one chromogenic conjugate according to aspect 1 and a solvent, and optionally one or more of (i) an organic modifier; (ii) an enzyme enhancer; (iii) an iron chelator; (iv) a detergent; (v) an anti-microbial agent; (vi) organic or inorganic salt; or (vii) an enzyme substrate.

[0170] Aspect 16. A composition comprising Formula Ia of the chromogenic conjugate of aspect 1 for providing yellow color.

[0171] Aspect 17. A composition comprising Formula Ib of the chromogenic conjugate of aspect 1 for providing cyan or blue color.

[0172] Aspect 18. A composition comprising Formula Ic of the chromogenic conjugate of aspect 1 for providing yellow or turquoise color.

[0173] Aspect 19. A composition comprising more than one chromogenic conjugates of aspect 1 for detection of a target having peroxidase activity or linked to a peroxidase enzyme, in a sample.

[0174] Aspect 20. The composition of aspect 19, wherein the composition comprises a compound of Formula Ia, Ib, or a salt thereof.

[0175] Aspect 21. The composition of aspect 19, wherein the composition comprises a compound of Formula Ia, Ic, or a salt thereof:

[0176] Aspect 22. A composition comprising 5-50 wt % of magenta chromogenic conjugate and 50-95 wt% of the chromogenic conjugate of aspect 1.

[0177] Aspect 23. A method for preparing the chromogenic conjugate of aspect 1: reacting a peroxidase substrate moiety represented by Formula IIIa having a protected phenol and a carboxylic acid moiety with a linker moiety having two amino group with one of the amine with a protecting group P2represented by Formula IIIb to prepare P2-HN-L-PS- P1represented by Formula IIIc; removing the protecting group P2to prepare Formula IIId; reacting Formula Iaa, Formula Ibb, or Formula Icc with Formula IIId to prepare Formula IIIe, Formula IIIf, or Formula IIIg, respectively; and removing a protecting group P1to prepare the chromogenic conjugate of aspect 1 represented by Formula Ia, Ib or Ic:Formula IIIaFormula IIIbwherein, provided that at least one of RPSor RPS’isg.

[0178] Aspect 24. The method for preparing the chromogenic conjugate of aspect 1 comprising: reacting Formula Iaa, Formula Ibb, or Formula Icc with Formula IIIb; removing a protecting group P2to prepare Formula Idd, Iee, or Iff; reacting a peroxidase substrate moiety represented by Formula IIIa having a protected phenol and a carboxylic acid moiety with Idd, Iee, or Iff to prepare chemical compound represented by Formula IIIe, IIIf, or IIIg; and removing a protecting group P1to prepare the chromogenic conjugate of aspect 1 represented by Formula Ia, Ib or Ic,Formula IIIb, provided that at least one of RPSor RPSisFormula IIIg.

[0179] Aspect 25. The method of aspects 23 or 24, wherein large excess of H2N-L-NH2is used instead of P2-HN-L-NH2.

[0180] Aspect 26. A method for detection of a target in a sample by chromogenic detection comprising: incubating a sample comprising a target in an aqueous solution, wherein the target comprises peroxidase activity or the target is directly or indirectly linked to a peroxidase enzyme, wherein the aqueous solution comprises: a) at least one chromogenic conjugate according to aspect 1; b) a peroxide compound, at a time and temperature sufficient to form a colored precipitate of the chromogenic conjugate; detecting the colored precipitate of the chromogenic conjugate in the sample, thereby detecting the target in the sample.

[0181] Aspect 27. The method of aspect 22, further comprising incubating the sample two or more cycles with a chromogenic conjugate having Formula Va, Vb, Vc or Vd:Formula VaVcFormula Vd, wherein all variables are as defined in aspect 1, wherein each cycle comprises same or different chromogenic conjugates in same or different ratios.

[0182] Aspect 28. A method of performing chromogenic in situ hybridization comprising: contacting a nucleic acid target with a probe that hybridizes with the nucleic acid target under hybridization conditions, wherein the probe comprises (1) a nucleic acid sequence at least partially complementary to the nucleic acid target and (2) a peroxidase enzyme or a first member of a specific binding pair; wherein the target and probe form a complex;when the probe comprises (2), contacting the complex with a second member of the specific binding pair, wherein the second member of the specific binding pair, wherein the second member is directly or indirectly linked to a peroxidase enzyme, and specifically binds to the first member; incubating the complex with at least one of the chromogenic conjugates according to aspect 1; for a time and temperature sufficient to form a color precipitate at the target; detecting the color precipitate.

[0183] Aspect 29. A method of detecting two or more targets comprising: providing peroxidase activity at a first target in a sample; contacting the sample with a first chromogenic conjugate; forming a first colored precipitate at the first target; removing the peroxidase activity from the first target; removing non-precipitated first chromogenic conjugate from the sample; providing peroxidase activity at a second target in the sample; contacting the sample with a second chromogenic conjugate; forming a second colored precipitate at the second target; and detecting the first colored precipitate and the second colored precipitate, thereby detecting the first and second targets in the sample, wherein at least one of the first and second chromogenic conjugates is a conjugate according to aspect 1, and wherein the first chromogenic conjugate molecule and the second chromogenic conjugate have one or more spectral characteristics that are different from each other.

[0184] Aspect 30. The method of aspect 25, wherein the method further comprises: removing the peroxidase activity from the second target; removing non-precipitated second chromogenic conjugate from the sample; providing peroxidase activity at a third target in the sample; contacting the sample with a third chromogenic conjugate having one or more spectral characteristics that are different from those of the first and second chromogenic conjugates.

[0185] Aspect 31. A method for preparing a chromogenic conjugate of aspect 1 comprising: reacting a chromogenic moiety with a secondary amine compound containing an ester to prepare a secondary amide compound of the chromogenic moiety; converting the ester in the secondary amide compound of the chromogenic moiety to a carboxylic acid; andreacting the secondary amide compound of the chromogenic moiety having the carboxylic acid with a linker compound having an amine group to form the chromogenic conjugate.

[0186] Aspect 32. An immunohistochemical staining composition comprising two or more of chromogenic conjugates selected from the group consisting of a chromogenic conjugate of Formula Ia, a chromogenic conjugate of Formula Ib, a chromogenic conjugate of Formula Ic of aspect 1, and a chromogenic conjugate of Formula Id:Formula Id, wherein all variables are as defined in aspect 1.

[0187] Aspect 33. The immunohistochemical staining composition of aspect 32, wherein the composition provides red, orange, grass green, green, blue, or purple color.

[0188] Aspect 34. A immunohistochemical staining composition of aspect 32, wherein the color is determined by the ratio of the first, the second, the third and the fourth chromogenic conjugates.

[0189] Aspect 35. A kit for detection of a target having peroxidase activity or linked to a peroxidase enzyme in a sample comprising at least one chromogenic conjugate according to aspect 1.

[0190] Aspect 36. The kit of aspect 35, wherein the kit comprises more than one chromogenic conjugate according to aspect 1 to stain multiple targets in different colors.

[0191] Aspect 37. The kit of aspect 35, wherein the kit comprises a composition comprising 5-50 wt % of magenta chromogenic conjugate and 50-95 wt% of the chromogenic conjugate of aspect 1. EXAMPLES

[0192] The following Examples are non-limiting demonstration of synthetic methods for conjugates and intermediates used in the making of some selected chromogenic conjugates, and their practical applications in detecting of molecular targets in IHC and ISH assay formats. Example 1. Synthesis of Yellow Chromogen Conjugate Preparation of Peroxidase SubstrateExample 1-1: Preparation of compound 2, Coumaric acid ethyl ester

[0193] Coumaric acid (1, 101 g, 616 mmol) was refluxed for 72 hours in ethanol (600 mL 99.9%) and sulfuric acid, (6 mL, 96% purity). The reaction mixture was reduced to about 219 g on a rotary evaporator followed by addition of 400 mL of Dichloromethane (DCM) to yield a dark brownish solution. This DCM / Ethanol solution was then extracted with 216 mmol NaOH dissolved in 200 mL water and 100 mL saturated sodium hydrogen carbonate. The organic phase was separated and concentrated to produce a dark yellow oil, that was dissolved in 200 mL of DCM and the solvent was removed to dryness on a rotary evaporator at 95 °C. Upon cooling, the product was obtained as an off white waxy crystalline solid to yield 115 g of compound 2. Example 1-2: Preparation of Compound 3, O-tert-Butyl-Coumaric acid ethyl ester

[0194] Coumaric acid ethyl ester (2, 115 grams, 597 mmol) was dissolved in a mixture of 14 mL water and 331 mL of 99.9% EtOH. 345 mL of 2-Bromo-2-Methylpropane was added to the reaction mixture, followed by addition of 345 g of potassium carbonate. The mixture was stirred for 16 hours at 50 °C. The insoluble solids were filtered and washed with 350 mL of warm EtOH three times. The combined EtOH filtrates were evaporated to reduce to 134 grams of yellow oil. The steps were repeated, i.e. the 134 grams of oil was dissolved in 350 mL 96% EtOH and 350 mL 2-bromo-2-Methylpropane, followed by addition of 350 grams of K2CO3and the mixture was stirred for 16 hours at 50 °C. The insoluble solids were filtered and washed with 350 mL of warm EtOH three times. The combined EtOH filtrates were evaporated to reduce to yield 142 grams crude material. The crude material was dissolved in 300 mL DCM and extracted with 299 mmol sodium hydroxide dissolved in 299 mL of 20% EtOH in water. The organic layer was separated and evaporated to dryness to produce light brownish oil to yield 94 grams of compound 3. Example 1-3: Preparation of Compound 4, O-tert-Butyl-Coumaric acid.

[0195] O-tertButyl-Coumaric acid ethyl ester (compound 3, 94 g, 379 mmol) was dissolved in 470 mL of 96% EtOH, followed by addition of 10 M NaOH (76 mL 2 eq.) and the mixture was stirred for 1 hour at 40 °C. 470 mL of H2O was added and the mixture was stirred for another 30 minutes. Additional 470 mL of H2O was added to the reaction mixture, followed by cooling the mixture in an ice bath.80 mL of aqueous 4 M HCl was added to precipitate white solids. The solid was isolated by filtration, washed with H2O, and dried in vacuum over NaOH to yield, 79.5 grams of compound 4. Example 1-4: Preparation of Compound 6, Preparation of NHS Ester of O-tert-Butyl-Coumaric acid.

[0196] O-tert-Butyl-Coumaric acid (79.3 g, 360 mmol) and N-hydroxysuccinimide (NHS, 43.5 g, 378 mmol) were suspended in 450 mL of DCM with stirring for 15 minutes, followed by addition of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC⋅HCl, 72.5 g). The reaction mixture was stirred for 16 hours at room temperature to produce a clear, pale yellow NHS ester solution (compound 4a). Preparation of Compound 6.

[0197] 1,8-Diamino-3,6-dioxaoctane (compound 5, 263 mL, 1.8 mol) was cooled in an ice bath with stirring, followed by adding NHS ester (compound 4a, 360 mmol) solution dropwise over 45 minutes. The reaction mixture was stirred for an additional 5 minutes and the DCM phase was extracted with 450 mL of water, twice with 450 mL of aqueous 0.1 M NaCl, and with 432 mL of 1 M HCl in 20% Ethanol in water. The aqueous layers were combined and 50 mL of 10 M NaOH was added to the aqueous solution to separate an oil at the bottom of the aqueous phase. The oil was separated and evaporated to dryness on rotary evaporator at 95 °C. The oil was cooled and dissolved in 200 mL DCM, followed by removing DCM on a rotary evaporator at 95 °C to yield 94 grams (268 mmol, 75%) of compound 6.. Preparation of Yellow Chromogen ConjugateExample 1-5: Preparation of Compound 7, Fluorescein Proline tert-butyl ester.

[0198] Fluorescein (13.3 g) and NHS (9.15 g) was suspended in 200 mL of pyridine, followed by addition of N,N′-diisopropylcarbodiimide (DIPC, 8.7 mL) and the reaction mixture was heated to 90 °C. After stirring the mixture for about 1.5 hours, Proline butyl ester (13.57 g) was added and stirring continued for another 4 hours. The reaction mixture was cooled to room temperature. The solvent was removed and the residue was dissolved in 100 mL of ethanol, which was extracted with 440 mL of DCM. The DCM layer was washed with 300 mL of 0.25M TRIS twice, and with 300 mL of 1 M HCl twice. The organic phase was separated, and the solvent was removed by evaporation to yield 30,98 gram of crude compound 7. Example 1-6: Preparation of Compound 8, Fluorescein Proline-OH.

[0199] A solution of fluorescein Proline butyl ester (compound 7, 30.98 g) in DCM was cooled in an ice bath and 40 mL of TFA added dropwise, and the mixture was stirred at room temperature. The mixture was added to 1500 mL of t-Butylmethyl ether (MTBE) dropwise to form a precipitate. The precipitate was collected by filtration and washed with MTBE. Thewashed precipitate was redissolved in ethanol and concentrated to about 70 gram, followed by precipitating in 1500 mL of MTBE to yield compound 8 (13 g, 67% from Fluorescein). Example 1-7: Preparation of Compound 9.

[0200] Fluorescein Proline-OH (compound 8, 7.94 g, 18,5 mmol) was dissolved in 70 mL pyridine, followed by addition of 2.118 g of NHS and 3.64 g of EDC⋅HCl. The reaction mixture was stirred overnight and was added 5.18 g of compound 6. The resulting mixture was stirred and additional 1.49 g of EDC⋅HCl was added, if needed by in process monitoring, followed by continuous stirring overnight. 300 mL of DCM and 60 mL of ethanol were added to the mixture and the resulting mixture was extracted with 150 mL of sat. KHSO4 / water (1:4). 20 mL of EtOH was added and the extraction was repeated with 200 mL saturated sodium bicarbonate / water (1:1) twice. The organic phase was separated and evaporated to dryness. The residue was purified through a Silica-gel column chromatography to produce 2.09 g of product, compound 9. Example 1-8: Preparation of Compound 10.

[0201] Compound 9 (3.75 g) was dissolved in a mixture of 20 mL of N-methypyrrolidine (NMP) and 2 mL of diisopropylethylamine (DIPEA), followed by addition of t- butylbromoacetate (662 µL) and the resulting mixture was stirred at 65 °C for 3 hours. The mixture was allowed to cool to room temperature and 200 mL of DCM and 200 mL water were added to the mixture. The organic layer was separated and washed with 200 mL of sat. KHSO4 / water (1:4), and with 200 mL of saturated sodium bicarbonate. The organic phase is separated and evaporated to dryness. The residue was further purified using a silica-gel chromatography using a mixture of DCM and 0-10% ethanol as an eluent to yield 2.1 gram of compound 10. Example 1-9: Preparation of Compound 11.

[0202] A solution of compound 10 (2.1 g) in DCM was cooled in an ice bath and 21 mL TFA was slowly added, followed by stirring the mixture at room temperature for about 1 hour. The resulting solution was slowly added to 250 mL of ethyl ether to form precipitate, which was filtered and washed with ethyl ether. The solid was dried either under vacuum or in a desiccator over sodium hydroxide pellets to yield 1.77 g of compound 11.Example 2. Synthesis of Patent Blue Chromogen Conjugate, Compound 15.Example 2-1. Preparation of Compound 12, (Patent Blue V)-O-ethyl acetate.

[0203] Ethyl bromoacetate (21.3 g, 14.15 mL, 128 mmol) was dissolved in ethanol (EtOH, 99.9%, 125 mL), followed by addition of DIPEA (3.30 g, 4.49 mL, 25.5 mmol) and commercially available Patent Blue V sodium salt (5.00 g, 8.5 mmol), and the resulting solution was stirred at room temperature overnight. The reaction mixture was evaporated to a blue / black oil using a rotary evaporator at 50 °C. The oil was co-evaporated twice with 99.9% EtOH to yield 14.4 g of crude product mixture. Example 2-2. Preparation of Compound 13, (Patent Blue V)-O-acetic acid.

[0204] The crude (Patent Blue V)-O-acetic acid (compound 12, 14.4 g) was dissolved in 100 mL of 99.9% EtOH and 10 M sodium hydroxide (2.24 mL) was added. The reaction mixturewas stirred at 60 °C overnight, followed by addition of MilliQ water (MQ, 50 mL). The mixture was stirred for 3 hours at 60 °C, and the mixture was evaporated to dryness and co-evaporated twice with 99.9% EtOH on a rotary evaporator to yield 8.8 g of compound 13. Example 2-3. Preparation of Compound 14.

[0205] (Patent Blue V)-O-acetic acid (Compound 13, 1.99 g, 3.22 mmol) was dissolved in NMP (50 mL) with stirring and were added DIPEA (2.08 g, 2.80 mL, 16.1 mmol) and benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 3.35 g, 6.44 mmol). The mixture was stirred at room temperature for 30 minutes, followed by addition of 1,8-Diamino-3,6-dioxaoctane (compound 5, 0.50 g, 494 µL, 3.38 mmol) and the mixture was stirred overnight at 50 °C. A second portion of PyBOP (1.61 mmol, 0.84 g) was added to the mixture, as needed upon in process monitoring, and the mixture was stirred at 50 ° for 3 hours. A third portion of PyBOP (1.61 mmol, 0.84 g) was added and stirring continued at 50 °C overnight. The reaction mixture was poured into MQ (200 mL) and extracted with DCM (100 mL) five (5) times. The aqueous phase was evaporated to an oil using a rotary evaporator and redissolved in trifluoroacetic acid (TFA, 10 mL). The TFA solution was added to ethyl ether (about 280 mL) and the resulting precipitate was collected by centrifugation for 3 minutes at approximately 650 G. The precipitate is used without further purification in the next step. Example 2-4. Preparation of Compound 15.

[0206] Compound 14 (930 mg, 1.24 mmol) was dissolved in DMF (30 mL), followed by addition of O-tert-butyl-coumaric acid ethyl ester, 6 (441 mg, 2.00 mmol), triethylamine (0.61 mg, 834 µL, 6.0 mmol) and EDC⋅HCl (383 mg, 2.0 mmol) and the mixture was stirred at 75 °C overnight. The reaction mixture was evaporated to an oil using a rotary evaporator, redissolved in MQ (200 mL) and extracted with n-butanol (n-BuOH, 100 mL) three times. The organic phase (n-BuOH) was washed with 10% acetic acid (AcOH, 100 mL) three times and the combined aqueous phases was back-extracted with n-BuOH (100 mL). The combined n- BuOH phase was evaporated to dryness using a rotary evaporator and redissolved in TFA (10 mL). The solution was stirred for 30 minutes at room temperature before added to ethyl ether (280 mL) and the resulting precipitate was collected by centrifugation for 7.5 minutes at 1700 G. The precipitate was resuspended in ethyl ether (280 mL) and centrifuged again for 7.5 minutes at 1700 G. The precipitate was air-dried and redissolved in a minimum amount of 50% dimethyl sulfoxide (DMSO) in MQ. The solution was purified by semi-preparative High Performance Liquid Chromatography (C18 column, 2%-100% acetonitrile in MQ with 0.1%TFA over 26 minutes) and the combined fractions were evaporated to dryness and co- evaporated with EtOH more than three times to provide the product, compound 15 (160 mg). Example 3. Synthesis of Cyanine Chromogen Conjugate, Compound 18.

[0207] The Cyanine chromogenic conjugate (compound 18) is prepared using the procedure described in Example 1 starting from cyanine chromogen compound 16 instead of fluorescein and compound 6. Example 4. Staining of a tonsil sample with the Yellow Chromogen

[0208] Tonsil tissue sample was stained with the Yellow Chromogen, with DAB using the procedure provided in this disclosure. The result is shown in FIG.1. The yellow color is a bit dark / brown tint due to this being a Ki-67 stain (nuclei) and hematoxylin also stains the nuclei. Example 5. Mixed staining color of Yellow Chromogen with Magenta

[0209] Magenta was mixed into Yellow chromogen to see the change of color in the following ratio using the procedure provided in this disclosure:

[0210] The results are presented in FIG. 2. These pictures illustrate that two colocalized epitopes visualized with HRP Yellow and HRP Magenta can be expected to appear orange in a light microscope. Example 6. Staining with Turquoise Chromogen

[0211] Examples of tonsil tissue sample was stained with the turquoise chromogen using Cytokeratin AE1 / AE3 (GA053) as the primary antibody using the procedure provided in this disclosure. Hematoxylin stain (blue) was added to stain the nuclei. See FIGs.3-5. Even though blue and turquoise are very similar colors the contrast between hematoxylin and the turquoise chromogen is remarkable. Example 7. Staining with Yellow and Cyan Chromogen

[0212] Examples of tonsil and colon tissue samples were stained with S100 (yellow) and lambda light chain (cyan). Slides were stained on Dako Omnis. Formalin-fixed paraffin embedded (FFPE) normal tonsil and colon tissues were used. The double stain was performed according to the protocol shown in the table below.

[0213] All reagents were commercially available products, apart from the following: • Cyan chromogen: 2000 nM Cyan chromogen was diluted 1:2,35 in DM843 EnVision FLEX Substrate Buffer (Omnis) (final concentration 850 nM)• Yellow chromogen: 2000 nM Yellow chromogen was diluted 1:2,35 in DM843 EnVision FLEX Substrate Buffer (Omnis) (final concentration 850 nM) • Reducing buffer: 30 mM Tris(3-hydroxypropyl)phosphine (THPP), 50 mM Citrate, 0.5% SDS, pH 4.0.

[0214] Slides were dehydrated and mounted using a Sakura 4740 Coverslipper, and scanned on a Zeiss Axioscan Z1 in 20x magnification. See FIG.7 for Staining result of Tonsil tissue and FIG.8 for staining result of colon tissue.

[0215] It is expressly contemplated that any chromogenic conjugates in the present disclosure can be used in any of the methods of use, compositions, and kits-of-parts in the present disclosure.

[0216] In the present disclosure, numeric ranges are inclusive of the numbers defining the range. In the present disclosure, wherever the word “comprising” is found, it is contemplated that the words “consisting essentially of” or “consisting of” may be used in its place. It should be recognized that chemical structures and formula may be elongated or enlarged for illustrative purposes.

[0217] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those working in the fields to which this disclosure pertain.

[0218] All patents and publications referred to herein are expressly incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication. Further the dates of publication provided can be different from the actual publication dates which can be independently confirmed.

[0219] In view of this disclosure it is noted that the methods and apparatus can be implemented in keeping with the present teachings. Further, the various components, materials, structures and parameters are included by way of illustration and example only and not in any limiting sense. In view of this disclosure, the present teachings can be implemented in other applications and components, materials, structures and equipment to implement these applications can be determined, while remaining within the scope of the appended claims.

Claims

CLAIMS We claim:

1. A chromogenic conjugate comprising: (a) a chromogenic moiety, and (b) a peroxidase substrate moiety, wherein the chromogenic moiety and the peroxidase substrate moiety are linked together via a linker, wherein the conjugate is a compound of Formula Ia, Ib, Ic, or a salt thereof:c wherein, X is independently selected from –OH, -ORXor –NRXRXX; Y is independently selected from O or =N+RYRYY; Z, Z’, Z1, or Z1’are independently selected from O, S, or NRZ;wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R1’, R2’, R3’, R4’, R5’, R6’, R7’, R31, R32, R31’, R32’, RX, RXX, RY, RYY, and RZare independently selected from hydrogen, OH and a substituent having less than 40 atoms, wherein three or more atoms can be connected to form a cyclic structure;m or p’ is an integer selected from 0 or 1; n or n’ is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is an integer selected from 0, 1, 2 or 3; L is a linker comprising 5 to 29 connected atoms; and PS is H, NH2, OH, or a peroxidase substrate moiety represented by the following Formula II, provide that at least one of PS is a peroxidase substrate moiety:Formula II wherein, L and PS are connected through R26; R21is -H -OR34, or -NR34R35; R22is -H, -OR34, or -NR34R35; R23is -OH; R24is -H, -OR34, or -NR34R35; R25is - H, -OR34, or -NR34R35; and R26is –C(=Z3)-(Z4)p-;wherein, each Z3and Z4is independently O, S, or NR36; each R34, R35, or R36is independently H, alkyl or aryl; and p is 0 or 1.

2. The chromogenic conjugate of claim 1, wherein R1is selected from hydrogen, OH, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, R1may be taken together with R2to form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; R2is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, R2may be taken together with R1, to form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; RX, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; RXX, when present, is selected from (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R3is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryloptionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R4is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, when Y is – NRYRYY, R4may be taken together with RYYto form a 5- or 6-membered ring which is optionally substituted with one or more of the same or different R13or suitable R14groups; RYY, when present, is selected from (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively RYYmay be taken together with R4to form a 5- or 6-membered ring which is optionally substituted with one or more of the same or different R13or suitable R14groups; RY, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, RYmay be taken together with R5to form a 5- or 6-membered ring optionally substituted with one or more of the same or different R13or suitable R14groups; RZ, when present, is selected from hydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R5is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one ormore of the same or different R13or suitable R14groups, or, alternatively, R5may be taken together with R6to form part of a benzo, naptho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups, or alternatively, when Y is –NRYRYY, R5may be taken together with RYto form a 5- or 6-membered ring optionally substituted with one or more of the same or different R13or suitable R14groups; R6is selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, or, alternatively, R6together with R5may form part of a benzo, naphtho or polycyclic aryleno group which is optionally substituted with one or more of the same or different R13or suitable R14groups; R7, R8and R9are each, independently of one another, selected from hydrogen, R11, (C1- C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups; R10is selected from selected from hydrogen, R11, (C1-C20) alkyl or heteroalkyl optionally substituted with one or more of the same or different R14groups, (C5-C20) aryl or heteroaryl optionally substituted with one or more of the same or different R13or suitable R14groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with one or more of the same or different R13or suitable R14groups, halo, haloalkyl, – OR12, –SR12, –SOR12, –SO2R12, and nitrile; R11is selected from –NR15R15, –OR16, –SR16, halo, haloalkyl, –CN, –NC, –OCN, – SCN, –NO, –NO2, –N3, –S(O)R16, –S(O)2R16, –S(O)2OR16, –S(O)NR15R15, – S(O)2NR15R15–OS(O)R16, –OS(O)2R16, –OS(O)2NR15R15, –OP(O)2R16, – OP(O)3R16R16, –P(O)3R16R16, –C(O)R16, –C(O)OR16, –C(O)NR15R15, – C(NH)NR15R15, –OC(O)R16, –OC(O)OR16, –OC(O)NR15R15and –OC(NH)NR15R15; R12is selected from (C1-C20) alkyls or heteroalkyls optionally substituted with lipophilic substituents, (C5-C20) aryls or heteroaryls optionally substituted withlipophilic substituents and (C2-C26) arylalkyl or heteroarylalkyls optionally substituted with lipophilic substituents; R13is selected from hydrogen, (C1-C8) alkyl or heteroalkyl, (C5-C20) aryl or heteroaryl and (C6-C28) arylalkyl or heteroarylalkyl; R14is selected from –NR15R15, =O, –OR16, =S, –SR16, =NR16, =NOR16, halo, haloalkyl,C(NH)NR15R15, –OC(O)R16, –OC(O)OR16, –OC(O)NR15R15and –OC(NH)NR15R15; each R15is independently hydrogen or R16, or alternatively, each R15is taken together with the nitrogen atom to which it is bonded to form a 5- to 8-membered saturated or unsaturated ring which may optionally include one or more of the same or different additional heteroatoms and which may optionally be substituted with one or more of the same or different R13or R16groups; each R16is independently R13or R13substituted with one or more of the same or different R13or R17groups; and each R17is selected from –NR13R13, –OR13, =S, –SR13, =NR13, =NOR13, halo, haloalkyl, –CN, –NC, –OCN, –SCN, –NO, –NO2, =N2, –N3, –S(O)R13, –S(O)2R13, –S(O)2OR13, –S(O)NR13R13, –S(O)2NR13R13, –OS(O)R13, –OS(O)2R13, –OS(O)2NR13R13, – OS(O)2OR16, –OS(O)2NR13R13, –C(O)R13, –C(O)OR13, –C(O)NR13R13, – C(NH)NR15R13, –OC(O)R13, –OC(O)OR13, –OC(O)NR13R13and –OC(NH)NR13R13.

3. The chromogenic conjugate of claim 1, wherein the chromogenic moiety is a derivative of Cyanine, triarylmethane, fluorescein, or a salt thereof.

4. The chromogenic conjugate of claim 1, wherein the chromogenic moiety is selected from the group consisting of Cy-3, Cy-5 , triarylmethanes, fluorescein, O-carboxymethyl fluorescein, and a 2'-piperazine amide derivative.

5. The conjugate of claim 1, wherein R23is –OH, and R24is –H and / or either R21or R25is –OH, R22and R24are –H, and R23is –OH.

6. The conjugate of claim 1, wherein the peroxidase substrate is a residue of ferulic acid, cinnamic acid, caffeic acid, sinapinic acid, 2,4-dihydroxycinnamic acid or 4- hydroxycinnamic acid (coumaric acid).

7. The conjugate of claim 1, wherein L comprises 1 or 2 repeats of Formula IVa, IVb, IVc, IVd, or IVe:wherein each R37is independently selected from methyl, ethyl, propyl, OCH2, CH2OCH2, (CH2OCH2)2, S, NH, NHCH2, NH(CH2)2, CH2NHCH2, cycloalkyl, alkyl-cycloalkyl, alkyl- cycloalkyl-alkyl, heterocycloalkyl, alkyl-heterocyclyl, alkyl-heterocyclyl-alkyl; each R38is independently NH or O; w is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each R39is independently CH2or CH2NH.

8. The conjugate of claim 1, wherein the linker is selected from:,.

9. The chromogenic conjugate of claim 1 selected from the group consisting of:,,,.

10. A composition comprising at least one chromogenic conjugate according to claim 1 and a solvent, and optionally one or more of (i) an organic modifier; (ii) an enzyme enhancer; (iii) an iron chelator; (iv) a detergent; (v) an anti-microbial agent; (vi) organic or inorganic salt; or (vii) an enzyme substrate.

11. A composition comprising Formula Ia of the chromogenic conjugate of claim 1 for providing yellow color, a composition comprising Formula Ib of the chromogenic conjugate of claim 1 for providing cyan or blue color, or a composition comprising Formula Ic of the chromogenic conjugate of claim 1 for providing yellow or turquoise color.

12. A composition comprising more than one chromogenic conjugates of claim 1 for detection of a target having peroxidase activity or linked to a peroxidase enzyme, in a sample; optionally, wherein the composition comprises a compound of Formula Ia, Ib, or a salt thereof, optionally, wherein the composition comprises a compound of Formula Ia, Ic, or a salt thereof.

13. A composition comprising 5-50 wt % of magenta chromogenic conjugate and 50-95 wt% of the chromogenic conjugate of claim 1.

14. A method for preparing the chromogenic conjugate of claim 1: reacting a peroxidase substrate moiety represented by Formula IIIa having a protected phenol and a carboxylic acid moiety with a linker moiety having two amino group with one of the amine with a protecting group P2represented by Formula IIIb to prepare P2-HN-L-PS- P1represented by Formula IIIc; removing the protecting group P2to prepare Formula IIId; reacting Formula Iaa, Formula Ibb, or Formula Icc with Formula IIId to prepare Formula IIIe, Formula IIIf, or Formula IIIg, respectively; and removing a protecting group P1to prepare the chromogenic conjugate of claim 1 represented by Formula Ia, Ib or Ic:Formula IIIaFormula IIIbFormula IIIcwherein, provided that at least one of RPSor RPSisFormula IIIg.

15. The method for preparing the chromogenic conjugate of claim 1 comprising: reacting Formula Iaa, Formula Ibb, or Formula Icc with Formula IIIb; removing a protecting group P2to prepare Formula Idd, Iee, or Iff; reacting a peroxidase substrate moiety represented by Formula IIIa having a protected phenol and a carboxylic acid moiety with Idd, Iee, or Iff to prepare chemical compound represented by Formula IIIe, IIIf, or IIIg; and removing a protecting group P1to prepare the chromogenic conjugate of claim 1 represented by Formula Ia, Ib or Ic,wherein, provided that at least one of RPSor RPSisg.

16. The method of claims 14 or 15, wherein large excess of H2N-L-NH2 is used instead of P2-HN-L-NH2.

17. A method for detection of a target in a sample by chromogenic detection comprising: incubating a sample comprising a target in an aqueous solution, wherein the target comprises peroxidase activity or the target is directly or indirectly linked to a peroxidase enzyme, wherein the aqueous solution comprises: a) at least one chromogenic conjugate according to claim 1; b) a peroxide compound,at a time and temperature sufficient to form a colored precipitate of the chromogenic conjugate; detecting the colored precipitate of the chromogenic conjugate in the sample, thereby detecting the target in the sample.

18. The method of claim 17, further comprising incubating the sample two or more cycles with a chromogenic conjugate having Formula Va, Vb, Vc or Vd:Formula Vd, wherein all variables are as defined in claim 1, wherein each cycle comprises same or different chromogenic conjugates in same or different ratios.

19. A method of performing chromogenic in situ hybridization comprising: contacting a nucleic acid target with a probe that hybridizes with the nucleic acid target under hybridization conditions, wherein the probe comprises (1) a nucleic acid sequence at least partially complementary to the nucleic acid target and (2) a peroxidase enzyme or a first member of a specific binding pair; wherein the target and probe form a complex; when the probe comprises (2), contacting the complex with a second member of the specific binding pair, wherein the second member of the specific binding pair, wherein the second member is directly or indirectly linked to a peroxidase enzyme, and specifically binds to the first member; incubating the complex with at least one of the chromogenic conjugates according to claim 1; for a time and temperature sufficient to form a color precipitate at the target; detecting the color precipitate.

20. A method of detecting two or more targets comprising: providing peroxidase activity at a first target in a sample; contacting the sample with a first chromogenic conjugate; forming a first colored precipitate at the first target; removing the peroxidase activity from the first target; removing non-precipitated first chromogenic conjugate from the sample; providing peroxidase activity at a second target in the sample; contacting the sample with a second chromogenic conjugate;forming a second colored precipitate at the second target; and detecting the first colored precipitate and the second colored precipitate, thereby detecting the first and second targets in the sample, wherein at least one of the first and second chromogenic conjugates is a conjugate according to claim 1, and wherein the first chromogenic conjugate molecule and the second chromogenic conjugate have one or more spectral characteristics that are different from each other.

21. A method for preparing a chromogenic conjugate of claim 1 comprising: reacting a chromogenic moiety with a secondary amine compound containing an ester to prepare a secondary amide compound of the chromogenic moiety; converting the ester in the secondary amide compound of the chromogenic moiety to a carboxylic acid; and reacting the secondary amide compound of the chromogenic moiety having the carboxylic acid with a linker compound having an amine group to form the chromogenic conjugate.

22. An immunohistochemical staining composition comprising two or more of chromogenic conjugates selected from the group consisting of a chromogenic conjugate of Formula Ia, a chromogenic conjugate of Formula Ib, a chromogenic conjugate of Formula Ic of claim 1, and a chromogenic conjugate of Formula Id:wherein all variables are as defined in claim 1.

23. A kit for detection of a target having peroxidase activity or linked to a peroxidase enzyme in a sample comprising at least one chromogenic conjugate according to claim 1.

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