On-slide synthesis of Schiff reagents

Abstract

The present disclosure relates to compositions, kits, and methods for in situ synthesis of Schiff reagents, i.e., directly on a substrate, for example, in a liquid pool disposed on the surface of the substrate. In some embodiments, the kit comprises at least a dye composition and a sulfite composition. In some embodiments, the dye composition comprises a compound having a 4-benzohydrylidene-2,5-cyclohexadien-1-imine core functionalized to include at least two substituted or unsubstituted amine groups, the compound having a molecular weight in the range of about 300 g / mol to about 600 g / mol. In some embodiments, the dye composition optionally includes an acid. In some embodiments, the sulfite composition includes a sulfite or a SO2 source.

Description

Related Applications

[0001] Cross - References to Related Applications This disclosure claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 366,091, filed on June 9, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

Technical Field

[0002] This disclosure relates to compositions and methods for staining biological specimens.

Background Art

[0003] The periodic acid - Schiff assay (the "PAS" was first reported in 1946 by McManus as a technique for visualizing mucin, glycogen, basement membrane, and fungal organisms by the combination of oxidation of polysaccharides by periodic acid and staining with Schiff reagent. PAS is a general - purpose carbohydrate stain that can be used to detect structures that contain a high proportion of carbohydrate macromolecules such as polysaccharides. The main polysaccharide identified by histological staining of human tissue sections is glycogen. PAS staining enables microscopic visualization and evaluation of glycogen levels in tissues. Disruption of the metabolic pathways that convert glucose to glycogen and vice versa can lead to glycogen accumulation. PAS staining is also used in the examination of basement membranes and is typically used to examine glomerular basement membranes in renal biopsies where glomerular disease is suspected. PAS staining can also be used to detect glycogen present in fungal cell walls as an aid in the diagnosis of fungal infections. The glycogen content of fungal cell walls is similar across many fungi, and thus PAS staining is a reliable method for confirming or ruling out the possibility of fungal infection.

[0004] The PAS assay is performed by reacting an analyte (e.g., a histological or cytological sample) with a predetermined amount of Schiff reagent, such as a commercially available Schiff reagent. The Schiff reagent can be derived from a single dye or a mixture of dyes such as pararosaniline, roaniline, magenta II, and new fuchsin (see Figure 1).

[0005] The solution containing the Schiff reagent chemically binds to the aldehyde in the sample to form a bright red, purple, or magenta-colored product. As shown in Figure 2A, the bisulfite molecule reacts with the pararosaniline dye to obtain a decolorized adduct with a sulfonated central carbon (see Figure 2B). The free and uncharged amine groups belonging to the aromatic ring react with the aldehyde group to form an aldimine. Since this aldimine group is electrophilic, it further reacts with the bisulfite ion. Finally, a red, purple, or magenta-colored bisulfite adduct is formed. Histologically, the aldehyde is either bound to or produced from the tissue structure. Therefore, the tissue structure itself is colored bright red. This mechanism is the same for all aldehydes in the tissue. The aldehyde condenses with the Schiff reagent to produce a new compound bound to the tissue. In this process, the chromophore is modified and a color is generated.

[0006] The Schiff reagent is an unstable reagent (see Figure 3). As described in [Kasten, F.H. (1960): The Chemistry of Schiff Reagent, Intl. Rev. Cytol. 10: 1-100.] in 1960, the stability of the Schiff reagent ranges from "several hours to over 6 months" depending on the components and storage method. Due to this instability, the storage and handling of the Schiff reagent are restricted, and the staining quality of the current PAS assay changes as the Schiff reagent ages. In fact, it has been found that when the vial of the Schiff reagent is opened, for example, after the vial has been open for one month, the staining quality deteriorates over time. In some embodiments, vials of commercially available Schiff reagent (opened and unopened) contain crystalline precipitates (see Figure 4), which can, in some cases, result in false positives when used for staining, especially when detecting fungi in biological samples. It is desirable to stain histological and / or cytological samples using a PAS Schiff reagent that does not contain crystalline precipitates. Summary of the Invention

[0007] The applicant has developed a composition, kit, and method for in-situ synthesis of Schiff reagents, i.e., directly on a substrate, for example, in a liquid pool placed on the surface of a substrate (e.g., a biological sample or a microscope slide). The term "in-situ synthesized Schiff reagent" etc. refers to an adduct formed from a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core and a dye or a mixture of dyes having a sulfite or SO2 source. The applicant has unexpectedly found that by synthesizing the Schiff reagent in-situ, consistent staining equivalent to that achieved using commercially available Schiff reagents is possible while enabling long-term stability of the reagent (e.g., having a stability of at least 1 year or at least 2 years). Further, by synthesizing the Schiff reagent in-situ, formation of precipitates is prevented, and thus, deposition of precipitates onto the substrate and / or the liquid pool placed on the substrate is avoided. Additionally, in-situ synthesis of the Schiff reagent enables "dialing in" the staining intensity by varying the components (or the concentration of components, e.g., dye concentration, acid concentration, and / or sulfite source concentration) used in the in-situ synthesis and / or the reaction conditions (time, temperature) of the synthesis. These and other advantages are described herein.

[0008] A first aspect of the present disclosure is represented by formulas (I) to (XII):

Chemical formula

Chemical formula

Chemical formula

[0009] (wherein,

[0010] R 1 、R 2 、R 3 、R 4 、R 5 and R 6is, independently, H, halogen, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), where R x and R y are, independently, H, or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or R 1 and R 2 together, or R 3 and R 4 together, or R 5 or R 6 together form a 5- or 6-membered cyclic or heterocyclic ring optionally substituted with one or more C1-C6 alkyl groups or one or more -N(R x )(R y ) groups, A is a counterion)

[0011] A dye composition comprising a compound having any one of the following, wherein the concentration of the compound having any one of the formulas (I)-(XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition.

[0012] In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are -N(R x )(R y ), and at least another two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are a branched or unbranched substituted or unsubstituted C1-C6 alkyl group or cycloalkyl group, and at least one of R x or R y is H. In some embodiments, R 1 , R 2, R 3 , R 4 , R 5 and R 6 At least two of x ), R y ) and at least two of 1 , R 2 , R 3 , R 4 , R 5 and R 6 are C1-C2 alkyl groups, and at least one of x or R y is H.

[0013] In some embodiments, the compound having any one of formulas (I)-(XII) is

Chemical formula

[0014] In some embodiments, the compound having any one of formulas (I)-(XII) has a molecular weight in the range of about 300 g / mol to about 600 g / mol. In some embodiments, the compound having any one of formulas (I)-(XII) has a molecular weight in the range of about 300 g / mol to about 425 g / mol. In some embodiments, the concentration is in the range of about 0.15% w / v to about 2.5% w / v based on the total volume of the dye composition. In some embodiments, the concentration is in the range of 0.15% w / v to about 1.8% w / v based on the total volume of the dye composition.

[0015] In some embodiments, the dye composition further comprises an acid. In some embodiments, the acid is a strong acid. In some embodiments, the strong acid is hydrochloric acid in the range of about 1M to about 1.3M. In some embodiments, the strong acid is nitric acid in the range of about 1M to about 1.3M. In some embodiments, the dye composition further comprises at least one of a solvent and / or an additive. In some embodiments, the dye composition further comprises a solvent and at least one additive. In some embodiments, the dye composition consists essentially of a compound having any one of formulas (I) to (XII) and a strong acid.

[0016] A second aspect of the present disclosure is a sulfite composition comprising a sulfite or a SO2 source selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid, wherein the concentration of the sulfite or the SO2 source ranges from about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition. In some embodiments, the concentration ranges from about 1% w / v to about 9% w / v based on the total weight of the sulfite composition. In some embodiments, the concentration ranges from about 1% w / v to about 4% w / v based on the total weight of the sulfite composition. In some embodiments, the sulfite or the SO2 source is not thiosulfate. In some embodiments, the sulfite composition further comprises an acid.

[0017] A third aspect of the present disclosure is a kit comprising (i) a dye composition and (ii) a sulfite composition, wherein the dye composition has formulas (I) to (XII):

Chemical formula

Chemical formula

Chemical formula

[0018] (wherein,

[0019] R 1 、R 2 、R3 , R 4 , R 5 and R 6 are, independently, H, halogen, a branched or unbranched substituted or unsubstituted C1-C6 alkyl or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), where R x and R y are, independently, H, or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or R 1 and R 2 together, or R 3 and R 4 together, or R 5 or R 6 together form a 5- or 6-membered cyclic or heterocyclic ring optionally substituted with one or more C1-C6 alkyl groups or one or more -N(R x )(R y ) groups, A is a counterion)

[0020] comprising a compound having any one of (I)-(XII), wherein the concentration of the compound having any one of formulas (I)-(XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition,

[0021] The sulfite composition comprises a sulfite or SO2 source selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid, and the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition, and is a kit.

[0022] A fourth aspect of the present disclosure is a kit comprising (i) an acid-free dye composition, (ii) a sulfite composition, and (iii) an acid, wherein the dye composition has the formulas (I)-(XII): [Chemical formula] [Chemical formula] [ka]

[0023] (In the formula,

[0024] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, a branched or unbranched substituted or unsubstituted C1-C6 alkyl or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ) and R x and R y are independently H or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or R 1 and R 2 together, or R 3 and R 4 together, or R 5 or R 6 are taken together to form one or more C1-C6 alkyl groups or one or more -N(R x )(R y ) groups, A is a counter ion.

[0025] The concentration of the compound having any one of formulas (I) to (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition;

[0026] The sulfite composition comprises a sulfite or SO2 source selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfite, and the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v, based on the total weight of the sulfite composition.

[0027] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of are -N(R x )(R y ), and at least another two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are a branched or unbranched, substituted or unsubstituted C1-C6 alkyl group or cycloalkyl group, and at least one of R x or R y is H. In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are -N(R x )(R y ), and at least another two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are C1-C2 alkyl groups, and at least one of R x or R y is H.

[0028] In some embodiments, the compound having any one of formulas (I)-(XII) is

Chemical formula

[0029] In some embodiments, a compound having any one of formulas (I) - (XII) has a molecular weight in the range of about 300 g / mol to about 600 g / mol. In some embodiments, a compound having any one of formulas (I) - (XII) has a molecular weight in the range of about 300 g / mol to about 425 g / mol. In some embodiments, the concentration is in the range of about 0.15% w / v to about 2.5% w / v, based on the total volume of the dye composition. In some embodiments, the concentration is in the range of 0.15% w / v to about 1.8% w / v, based on the total volume of the dye composition.

[0030] In some embodiments, the sulfite composition contains a sulfite or SO2 source selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid, and the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v, based on the total weight of the sulfite composition. In some embodiments, the concentration is in the range of about 1% w / v to about 9% w / v, based on the total weight of the sulfite composition. In some embodiments, the concentration is in the range of about 1% w / v to about 4% w / v, based on the total weight of the sulfite composition.

[0031] In some embodiments, the acid is a strong acid. In some embodiments, the acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, trifluoroacetic acid, hydroiodic acid, sulfuric acid, nitric acid, chloric acid, and perchloric acid.

[0032] A fifth aspect of the present disclosure is a method for staining a biological sample, comprising sequentially dispensing a dye composition and a sulfite composition onto the biological sample, wherein the dye composition has the formula (I) - (XII):

Chemical formula

Chemical formula

Chemical formula

[0033] (wherein

[0034] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are, independently, H, halogen, a branched or unbranched substituted or unsubstituted C1-C6 alkyl or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), and R x and R y are, independently, H, or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or R 1 and R 2 together, or R 3 and R 4 together, or R 5 or R 6 together form a 5- or 6-membered cyclic or heterocyclic ring optionally substituted with one or more C1-C6 alkyl groups or one or more -N(R x )(R y ) groups, A is a counterion)

[0035] comprising a compound having any one of

[0036] and the concentration of the compound having any one of formulas (I)-(XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition,

[0037] wherein the sulfite composition comprises a sulfite or SO2 source selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid, and the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition.In some embodiments, the dye composition further comprises an acid. In some embodiments, the acid is a strong acid. In some embodiments, the dye composition further comprises hydrochloric acid. In some embodiments, the dye composition further comprises nitric acid.

[0038] In some embodiments, the dye composition is dispensed before the sulfite composition. In other embodiments, the dye composition is dispensed after the sulfite composition is dispensed. In some embodiments, the temperature of the sample, the dye composition, and / or the sulfite composition is in the range of 30°C to about 50°C. In some embodiments, the temperature of the sample, the dye composition, and / or the sulfite composition is in the range of 30°C to about 40°C. In some embodiments, the sequentially dispensed dye composition and sulfite composition are maintained in contact with the sample for at least 4 minutes. In some embodiments, the sequentially dispensed dye composition and sulfite composition are maintained in contact with the sample for at least 8 minutes.

[0039] In some embodiments, the amount of the dye composition dispensed into the sample ranges from about 50 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample ranges from about 50 μL to about 200 μL. In some embodiments, the amount of the dye composition dispensed into the sample ranges from about 100 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample ranges from about 100 μL to about 200 μL. In some embodiments, the amount of the dye composition dispensed into the sample ranges from about 100 μL to about 150 μL, and the amount of the sulfite composition dispensed into the sample ranges from about 100 μL to about 150 μL.

[0040] In some embodiments, the sample is pretreated with a reagent that forms an aldehyde group for a predetermined time before dispensing the dye composition and / or the sulfite composition. In some embodiments, the sample is pretreated with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water) for a predetermined time, e.g., about 1 minute to about 10 minutes.

[0041] A sixth aspect of the present disclosure is a method for staining a biological sample, comprising simultaneously dispensing a dye composition and a sulfite composition into the biological sample, wherein the dye composition has the formulas (I)-(XII): [Chemical formula] [Chemical formula] [Chemical formula]

[0042] (wherein

[0043] R 1 R 2 R 3 R 4 R 5 and R 6 are independently H, halogen, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), and R x and R y are independently H, or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or R 1 and R 2 together, or R 3 and R 4 together, or R 5 or R 6 together form a 5- or 6-membered cyclic or heterocyclic ring optionally substituted with one or more C1-C6 alkyl groups or one or more -N(R x )(R y ) groups, A is a counterion)

[0044] and contains a compound having any one of them, and the concentration of the compound having any one of the formulas (I)-(XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition,

[0045] The sulfite composition contains a sulfite or SO2 source selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid, and the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition.

[0046] In some embodiments, the temperature of the sample, the dye composition, and / or the sulfite composition is in the range of 30°C to about 50°C. In some embodiments, the temperature of the sample, the dye composition, and / or the sulfite composition is in the range of 30°C to about 40°C. In some embodiments, the simultaneously dispensed dye composition and sulfite composition are maintained in contact with the sample for at least 4 minutes. In some embodiments, the simultaneously dispensed dye composition and sulfite composition are maintained in contact with the sample for at least 8 minutes.

[0047] In some embodiments, the amount of the dye composition dispensed into the sample is in the range of about 50 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 50 μL to about 200 μL. In some embodiments, the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 200 μL. In some embodiments, the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 150 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 150 μL.

[0048] In some embodiments, the sample is pretreated with a reagent that forms an aldehyde group for a predetermined time before dispensing the dye composition and / or the sulfite composition. In some embodiments, the sample is pretreated with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water) for a predetermined time, such as about 1 minute to about 10 minutes.

[0049] A seventh aspect of the present disclosure is a method of staining a biological sample, comprising sequentially dispensing a dye composition, a sulfite composition, and an acid onto the biological sample, wherein the dye composition has the formulas (I) to (XII):

Chem.

Chem.

Chem.

[0050] (wherein,

[0051] R 1 、R 2 、R 3 、R 4 、R 5 and R 6 are independently H, halogen, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), and R x and R y are independently H, or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or R 1 and R 2 together, or R 3 and R 4 together, or R 5 or R 6 together form a 5- or 6-membered cyclic ring or heterocyclic ring optionally substituted with one or more C1-C6 alkyl groups or one or more -N(R x )(R y ) groups, A is a counter ion)

[0052] and contains a compound having any one of them, and the concentration of the compound having any one of the formulas (I) to (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition,

[0053] The sulfite composition contains a sulfite or SO2 source selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid, and the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition.

[0054] In some embodiments, the dye composition is dispensed before the dispensing of the sulfite composition and / or the acid. In some embodiments, the temperature of the sample, the dye composition, the acid, and / or the sulfite composition is in the range of 30°C to about 50°C. In some embodiments, the temperature of the sample, the dye composition, the acid, and / or the sulfite composition is in the range of 30°C to about 40°C. In some embodiments, the successively dispensed dye composition, sulfite composition, and acid are maintained in contact with the sample for at least 4 minutes. In some embodiments, the successively dispensed dye composition, sulfite composition, and acid are maintained in contact with the sample for at least 8 minutes.

[0055] In some embodiments, the amount of the dye composition dispensed into the sample is in the range of about 50 μL to about 200 μL, the amount of the acid dispensed into the sample is in the range of about 50 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 50 μL to about 200 μL. In some embodiments, the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 200 μL, the amount of the acid dispensed into the sample is in the range of about 100 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 200 μL. In some embodiments, the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 150 μL, the amount of the acid dispensed into the sample is in the range of about 100 μL to about 150 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 150 μL.

[0056] In some environments, the acid is a strong acid. In some embodiments, the strong acid is hydrochloric acid. In some embodiments, the strong acid is nitric acid.

[0057] In some embodiments, the sample is pretreated with a reagent that forms aldehyde groups for a predetermined time before dispensing the dye composition, the acid, and / or the sulfite composition. In some embodiments, the sample is pretreated with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water) for a predetermined time, e.g., from about 1 minute to about 10 minutes.

[0058] An eighth aspect of the present disclosure is a method for staining a biological sample, comprising simultaneously dispensing a dye composition, a sulfite composition, and an acid onto the biological sample, wherein the dye composition has the formulas (I) to (XII):

Chemical formula

Chemical formula

Chemical formula

[0059] (wherein,

[0060] R 1 、R 2 、R 3 、R 4 、R 5 and R 6 are independently H, halogen, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), and R x and R y are independently H, or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or R 1 and R 2 together, or R 3 and R 4 together, or R 5 or R 6 together form one or more C1-C6 alkyl groups or one or more -N(Rx )(R y ) group forms an optionally substituted 5- or 6-membered cyclic or hetero-cyclic ring, A is a counter ion)

[0061] and includes a compound having any one of the following, and the concentration of the compound having any one of the formulas (I) to (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition,

[0062] The sulfite composition contains a sulfite or SO2 source selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid, and the concentration of the sulfite or SO2 source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition.

[0063] In some embodiments, the temperature of the sample, the dye composition, the acid, and / or the sulfite composition is in the range of 30°C to about 50°C. In some embodiments, the temperature of the sample, the dye composition, the acid, and / or the sulfite composition is in the range of 30°C to about 40°C. In some embodiments, the simultaneously dispensed dye composition, sulfite composition, and acid are maintained in contact with the sample for at least 4 minutes. In some embodiments, the simultaneously dispensed dye composition, sulfite composition, and acid are maintained in contact with the sample for at least 8 minutes.

[0064] In some embodiments, the amount of the dye composition dispensed into the sample ranges from about 50 μL to about 200 μL, the amount of the acid dispensed into the sample ranges from about 50 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample ranges from about 50 μL to about 200 μL. In some embodiments, the amount of the dye composition dispensed into the sample ranges from about 100 μL to about 200 μL, the amount of the acid dispensed into the sample ranges from about 100 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample ranges from about 100 μL to about 200 μL. In some embodiments, the amount of the dye composition dispensed into the sample ranges from about 100 μL to about 150 μL, the amount of the acid dispensed into the sample ranges from about 100 μL to about 150 μL, and the amount of the sulfite composition dispensed into the sample ranges from about 100 μL to about 150 μL.

[0065] In some environments, the acid is a strong acid. In some embodiments, the strong acid is hydrochloric acid. In some embodiments, the strong acid is nitric acid.

[0066] In some embodiments, the sample is pretreated with a reagent that forms an aldehyde group for a predetermined time before dispensing the dye composition, acid, and / or sulfite composition. In some embodiments, the sample is pretreated with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water) for a predetermined time, such as about 1 minute to about 10 minutes.

[0067] A ninth aspect of the present disclosure is a stained biological sample, comprising: (i) dispensing a dye composition to the biological sample, the dye composition comprising: (a) a compound having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core functionalized to contain at least two substituted or unsubstituted amine groups and having a molecular weight in the range of about 300 g / mol to about 600 g / mol, and (b) an acid, wherein the concentration of the compound is in the range of about 0.15% w / v to about 2.5% w / v based on the total volume of the dye composition; and (ii) dispensing a sulfite composition containing a sulfite or an SO2 source, wherein the concentration of the sulfite or the SO2 source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition. In some embodiments, the dye composition is dispensed to the biological sample first. In some embodiments, the dye composition and the sulfite composition are dispensed simultaneously. In some embodiments, the acid is a strong acid.

[0068] In some embodiments, the sample is pretreated with a reagent that forms aldehyde groups for a predetermined time before dispensing the dye composition and / or the sulfite composition. In some embodiments, the sample is pretreated with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water) for a predetermined time, such as about 1 minute to about 10 minutes.

[0069] In some embodiments, the acid is hydrochloric acid or nitric acid. In some embodiments, the compound is

Chemical formula

Brief Description of the Drawings

[0070] This patent or application file includes at least one drawing created in color. Copies of this patent or patent application publication with color drawings will be provided to the Office upon request and payment of the required fee.

[0071]

Figure 1

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Figure 2B

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Figure 6-2

Figure 7-1

Figure 7-2

Figure 8-1

Figure 8-2

DETAILED DESCRIPTION OF THE INVENTION

[0072] The present disclosure provides compositions, kits, and methods for staining biological samples such as biological samples disposed on microscope slides. In particular, the present disclosure provides in situ synthesized Schiff reagents that facilitate the staining of biological samples in a manner equivalent to staining using commercially available Schiff reagents.

[0073] Definitions

[0074] Conversely, unless explicitly indicated otherwise, in any method recited in the claims herein that includes two or more steps or acts, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0075] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly dictates otherwise. The term “comprising” is defined inclusively such that “comprising A or B” means including A, B, or both A and B.

[0076] As used in this specification and the claims, it should be understood that "or" has the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is to be construed as inclusive, i.e., including at least one of the elements or items in the list, but also including two or more, and optionally, items not recited in the additional list. Only terms such as "only one of" or "exactly one of", or when used in the claims, terms such as "consisting of", which clearly indicate the contrary, refer to including exactly one element out of several elements or a list of elements. Generally, the term "or" as used in this specification is to be construed as indicating an exclusive alternative (i.e., "one or the other but not both") only when preceded by an exclusive term such as "either", "only one of", "only one of only", or "exactly one of". "Consisting essentially of", when used in the claims, shall have the ordinary meaning as used in the field of patent law.

[0077] As used in this specification, terms such as "comprising", "including", "having", etc. are used interchangeably and have the same meaning. Similarly, "comprises", "includes", "has", etc. are used interchangeably and have the same meaning. Specifically, each term is defined in accordance with the general definition of U.S. patent law as "comprising", and thus is construed as an open term meaning "at least the following", and is also construed not to exclude additional features, limitations, aspects, etc. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus includes at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c. Further, although steps and processes may be outlined in a specific order in this specification, those skilled in the art will recognize that the ordering of steps and processes can vary.

[0078] As used in this specification and the claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of every element specifically listed within the list of elements, nor excluding any combinations of elements in the list of elements. This definition also allows for elements other than those specifically recited in the list of elements referred to by the phrase "at least one" to optionally exist, whether or not they are related to those specifically recited elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") can, in one embodiment, refer to at least one A, including optionally more than one, where B does not exist (and optionally includes elements other than B), in another embodiment, can refer to at least one B, including optionally more than one, where A does not exist (and optionally includes elements other than A), and in yet another embodiment, can refer to at least one A, including optionally more than one, and at least one B, including optionally more than one (and optionally includes other elements), and so on.

[0079] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0080] As used herein, the term "alkyl", by itself or as part of another substituent, unless otherwise specified, may be fully saturated, mono- or polyunsaturated, and may include straight-chain (i.e., unbranched), branched-chain, or combinations thereof, divalent and polyvalent radicals having the specified number of carbon atoms (i.e., C1-C 10 means 1 to 10 carbons). "Alkyl" is not cyclized. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers such as, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups have one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-butadienyl, 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkoxy is an alkyl bonded to the remainder of the molecule through an oxygen atom (-O-).

[0081] As used herein, "analog" or "derivative" is used in accordance with its plain ordinary meaning in chemistry and biology, and is structurally similar to another compound (i.e., the so-called "reference" compound), but has a different composition, for example, when an atom is replaced with an atom of a different element, or in the presence of a particular functional group, or when a particular functional group is replaced with another functional group, or in the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is similar or equivalent in function and appearance to the reference compound, but is not similar or equivalent in structure or origin.

[0082] As used herein, the term "antibody" (which may be abbreviated "Ab") refers to an immunoglobulin or immunoglobulin-like molecule, and includes, by way of example and not limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate (e.g., in mammals such as humans, goats, rabbits and mice), and antibody fragments that specifically bind to a molecule of interest (or a group of molecules that are very similar to the molecule of interest) to substantially eliminate binding to other molecules. Further, an antibody refers to a polypeptide ligand that specifically recognizes and binds to an epitope of an antigen and includes at least one light chain immunoglobulin variable region or heavy chain immunoglobulin variable region. An antibody may be composed of heavy and light chains, each of which has a variable region called the variable heavy (VH) region and the variable light (VL) region. The VH and VL regions together are responsible for binding to the antigen recognized by the antibody. The term antibody includes intact immunoglobulins, as well as variants and portions thereof well known in the art.

[0083] As used herein, the term "antigen" refers to a compound, composition or substance that can be specifically bound by the products of specific humoral or cellular immunity, such as antibody molecules or T cell receptors. Antigens can be any type of molecule, including, for example, haptens, simple intermediary metabolites, saccharides (e.g., oligosaccharides), lipids and hormones, and macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins.

[0084] As used herein, the term "biological sample" can be any solid or fluid sample obtained from, excreted by, or secreted by any organism, including but not limited to single-celled organisms such as bacteria, yeast, protozoa, and amoebae, and multicellular organisms (including samples from healthy or seemingly healthy human subjects or human patients suffering from a diagnosed or investigational condition or disease such as cancer, whether plant or animal). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor or vitreous humor, or any body secretion, effusion, or exudate (e.g., a body fluid obtained from an abscess or other site of infection or inflammation), or a body fluid obtained from a joint (e.g., a normal joint or an affected joint). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen such as a tumor biopsy), or can include cells (whether primary cells or cultured cells) or a medium conditioned by any cell, tissue, or organ. In some examples, the biological sample is a nuclear extract. In certain examples, the sample is a quality control sample, such as one of the cell pellet section samples of the present disclosure. In other examples, the sample is a test sample. The sample can be prepared using any method known to those of skill in the art. The sample can be obtained from a subject for regular screening or from a subject suspected of having a disorder such as a genetic abnormality, infection, or neoplasm. The described embodiments of the methods of the present disclosure can also be applied to samples that do not have a genetic abnormality, disease, disorder, etc., referred to as "normal" samples. The sample can contain a plurality of targets to which one or more detection probes can specifically bind.

[0085] As used herein, "C" where "a" and "b" are integers a ~C b"a" refers to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl, cycloalkynyl or aryl group, or the total number of carbon atoms and heteroatoms in a heteroalkyl, heterocyclyl, heteroaryl or heteroaricyclic group. That is, the ring of an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl or heteroaricyclic group can contain "a" to "b" carbon atoms. Thus, for example, a "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2, CH3CH2CH(CH3)- and (CH3)3C-. When "a" and "b" are not specified for an alkyl, alkenyl, alkynyl, cycloalkyl cycloalkenyl, cycloalkynyl, aryl, heteroaryl or heteroaricyclic group, the broadest scope described in these definitions is assumed.

[0086] As used herein, "cycloalkyl" or similar terms (e.g., cyclic alkyl group) refer to a completely saturated (no double or triple bonds) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings can be joined together in a fused manner. A cycloalkyl group can contain 3 to 10 atoms in the ring or 3 to 8 atoms in the ring. A cycloalkyl group can be unsubstituted or substituted. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0087] As used herein, the term "halogen atom" or "halogen" means any one of the radiation-stable atoms in Group 7 of the periodic table of elements such as fluorine, chlorine, bromine and iodine.

[0088] As used herein, the term "heteroatom" means including boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si). In some embodiments, the "heterocyclic ring" may contain one or more heteroatoms. In other embodiments, the aliphatic group may contain one or more heteroatoms or may be substituted with one or more heteroatoms.

[0089] As used herein, the term "reactive group" or "reactive functional group" refers to a functional group that can chemically associate, interact, hybridize, hydrogen bond, or couple with the functional groups of different moieties. In some embodiments, the "reaction" between two reactive groups or two reactive functional groups may mean that a covalent bond is formed between the two reactive groups or two reactive functional groups; or it may mean that the two reactive groups or two reactive functional groups associate with each other, interact with each other, hybridize with each other, hydrogen bond with each other, etc. Thus, in some embodiments, the "reaction" includes binding events such as the binding of a hapten to an anti-hapten antibody or the association of a supramolecular host molecule with a guest molecule.

[0090] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules characterized by binding to each other while substantially excluding binding to other molecules (e.g., a specific binding pair may have a binding constant that is at least 10-3 M greater, 10-4 M greater, or 10-5 M greater than the binding constant of either of the two components of the binding pair with other molecules in a biological sample). Specific examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidin such as streptavidin, and protein A). Specific binding moieties can also include molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0091] As used herein, terms such as "staining" and "stained" as used herein generally refer to any treatment of a biological specimen to detect and / or distinguish the presence, location, and / or amount (such as concentration) of a particular molecule (such as a lipid, protein, or nucleic acid) or a particular structure (such as a normal or malignant cell, cytoplasmic sol, nucleus, Golgi apparatus, or cytoskeleton) in the biological specimen. For example, staining can provide contrast between a particular molecule or a particular cell structure and the surrounding portion of the biological specimen, and the intensity of the staining can provide a measure of the amount of a particular molecule in the specimen. Staining can be used to assist in the observation of molecules, cell structures, and organisms using not only bright-field microscopy but also other observation tools such as phase-contrast microscopy, electron microscopy, and fluorescence microscopy. Some of the staining performed by System 2 can be used to visualize the outline of cells. Other staining performed by System 2 can depend on the particular cell component (such as a molecule or structure) being stained, without or with relatively little staining of other cell components. Examples of types of staining methods performed by System 2 include, but are not limited to, histochemical methods, immunohistochemical methods, and other methods based on intermolecular reactions (including non-covalent interactions) such as hybridization reactions between nucleic acid molecules. Particular staining methods include, but are not limited to, primary staining methods (such as H&E staining, Pap staining, etc.), enzyme-linked immunohistochemical methods, and in situ RNA and DNA hybridization methods such as fluorescence in situ hybridization (FISH).

[0092] Whenever a group or moiety is described as "substituted" or "optionally substituted" (or "optionally having" or "optionally containing"), the group can be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as "substituted or unsubstituted" in the case where the group is already substituted, if the group is substituted, the substituent can be selected from one or more of the indicated substituents. When no substituents are indicated, an indicated "optionally substituted" or "substituted" group can be substituted with one or more groups individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroaricyclic, aralkyl, heteroaralkyl, (heteroaricyclic)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, amino, ether, amino (e.g., a mono-substituted amino group or a di-substituted amino group), and their protected derivatives. Any of the above groups can contain one or more heteroatoms such as O, N, or S. For example, when a moiety is substituted with an alkyl group, the alkyl group can contain a heteroatom selected from O, N, or S (e.g., -(CH2-CH2-O-CH2-CH2)-).

[0093] As used herein, "% w / v" refers to the weight / volume percentage concentration in units of grams of solute per 100 mL of solution.

[0094] As used herein, the terms "about" or "substantially" mean up to + / - 10% of the indicated value, e.g., + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, or + / - 1% of the indicated value.

[0095] Summary

[0096] The present applicant has developed compositions, kits, and methods for in situ, i.e., on a substrate, e.g., on the surface of a substrate, e.g., in a liquid pool disposed on the surface of a biological sample, direct synthesis of Schiff reagents. A general method for in situ synthesis of Schiff reagents is shown in FIG. 5. The present applicant has also developed methods for staining biological samples, such as biological samples disposed on a substrate, using in situ synthesized Schiff reagents.

[0097] Compositions

[0098] The present disclosure provides a dye composition and a bisulfite or SO2 source composition. Each of these compositions can be dispensed simultaneously or sequentially onto a biological sample, such as a biological sample disposed on a slide. Further, each of these compositions may be included in one or more kits for use in an automated staining system.

[0099] Dye Composition

[0100] In some embodiments, the present disclosure provides a composition (hereinafter referred to as "dye composition") comprising one or more dyes having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core (hereinafter referred to as "dye" in this specification). In some embodiments, the dye composition comprises any acid, one or more optional solvents, and optionally one or more additives. In some embodiments, the dye composition comprises one or more dyes, an acid, and optionally one or more additives. In other embodiments, the dye composition comprises one or more dyes, one or more solvents, and optionally one or more additives. In still other embodiments, the dye composition comprises one or more dyes, an acid, one or more solvents, and optionally one or more additives. In some embodiments, the dye composition is stable over at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, etc. In some embodiments, the dye composition is free of precipitates over at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, etc. In some embodiments, the dye composition is substantially free of precipitates over at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, etc.

[0101] In some embodiments, one or more dyes having a 4-benzohydrilylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least one substituted or unsubstituted amine group. In other embodiments, one or more dyes having a 4-benzohydrilylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least one substituted or unsubstituted amine group and are further functionalized to include one or more C1-C4 alkyl groups. In other embodiments, one or more dyes having a 4-benzohydrilylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least two substituted or unsubstituted amine groups. In yet other embodiments, one or more dyes having a 4-benzohydrilylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least two substituted or unsubstituted amine groups and are further functionalized to include one or more C1-C4 alkyl groups. In further embodiments, one or more dyes having a 4-benzohydrilylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least two substituted or unsubstituted amine groups and are further functionalized to include two or more C1-C4 alkyl groups. In some embodiments, at least one substituted amine group is substituted with a C1-C4 alkyl group.

[0102] In some embodiments, one or more dyes having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least one unsubstituted amine group. In other embodiments, one or more dyes having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least one unsubstituted amine group and are further functionalized to include one or more C1-C4 alkyl groups. In other embodiments, one or more dyes having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least two unsubstituted amine groups. In yet other embodiments, one or more dyes having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core are functionalized to include at least two unsubstituted amine groups and are further functionalized to include one or more C1-C4 alkyl groups.

[0103] In further embodiments, one or more dyes having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core have a molecular weight in the range of about 300 g / mol to about 600 g / mol, about 300 g / mol to about 500 g / mol, about 300 g / mol to about 450 g / mol, about 300 g / mol to about 400 g / mol, about 300 g / mol to about 375 g / mol, about 300 g / mol to about 350 g / mol, about 310 g / mol to about 340 g / mol, or about 310 g / mol to about 330 g / mol.

[0104] In some embodiments, one or more dyes having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core are of formulas (I)-(XII):

Chem.

Chem.

Chem.

[0105] (wherein, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, halogen, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), R x and R y are independently H, or a branched or unbranched C1-C6 alkyl group optionally substituted with one or more halogen atoms, or R 1 and R 2 together, or R 3 and R 4 together, or R 5 or R 6 together form a 5- or 6-membered cyclic ring or heterocyclic ring optionally substituted with one or more C1-C6 alkyl groups or one or more -N(R x )(R y ) groups, and A is a counterion), and has any one of them.

[0106] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group or cycloalkyl group, -OH, -O-C1-C6 alkyl, or -N(R x )(R y ), R x and R y are independently H or a branched or unbranched C1-C6 alkyl group. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group, or -N(R x )(Ry ) and R x and R y are, independently, H or a C1-C2 alkyl group. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are, independently, H, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group, or -N(R x )(R y ), where R x and R y are, independently, H or -CH3. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are, independently, H, a branched or unbranched substituted or unsubstituted C1-C6 alkyl group, or -NH2.

[0107] In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are -N(R x )(R y ). In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are -N(R x )(R y ), and at least another two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are a branched or unbranched substituted or unsubstituted C1-C6 alkyl group or a cycloalkyl group. In some embodiments, R 1 , R 2 , R 3 , R 4, R 5 and R 6 At least two of them are -N(R x )(R y ), and at least another two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are branched or unbranched substituted or unsubstituted C1-C4 alkyl groups or cycloalkyl groups. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and at least another two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are branched or unbranched substituted or unsubstituted C1-C3 alkyl groups or cycloalkyl groups. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and at least another two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are C1-C2 alkyl groups.

[0108] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and R 1, R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are branched or unbranched, substituted or unsubstituted C1-C6 alkyl groups or cycloalkyl groups, and R x or R y At least one of them is H. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are branched or unbranched, substituted or unsubstituted C1-C4 alkyl groups or cycloalkyl groups, and R x or R y At least one of them is H. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are branched or unbranched, substituted or unsubstituted C1-C3 alkyl groups or cycloalkyl groups, and R x or R y At least one of them is H. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(Ry ) and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of which are C1-C2 alkyl groups, and R x or R y at least one of which is H.

[0109] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of which are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of the others are branched or unbranched substituted or unsubstituted C1-C6 alkyl groups or cycloalkyl groups. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of which are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of the others are branched or unbranched substituted or unsubstituted C1-C4 alkyl groups or cycloalkyl groups. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of which are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6At least two of the others are branched or unbranched substituted or unsubstituted C1-C3 alkyl groups or cycloalkyl groups. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Among them, at least two are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Among them, at least two of the others are C1-C2 alkyl groups.

[0110] In some embodiments, the compounds having formulas (I)-(III) have a molecular weight in the range of about 300 g / mol to about 600 g / mol, about 300 g / mol to about 550 g / mol, about 300 g / mol to about 525 g / mol, about 300 g / mol to about 500 g / mol, about 300 g / mol to about 475 g / mol, about 300 g / mol to about 450 g / mol, about 300 g / mol to about 425 g / mol, about 300 g / mol to about 400 g / mol, about 300 g / mol to about 390 g / mol, about 300 g / mol to about 380 g / mol, about 300 g / mol to about 370 g / mol, about 300 g / mol to about 360 g / mol, about 300 g / mol to about 365 g / mol, about 300 g / mol to about 350 g / mol, about 300 g / mol to about 345 g / mol, about 300 g / mol to about 340 g / mol, about 300 g / mol to about 335 g / mol, about 300 g / mol to about 330 g / mol.

[0111] In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are -N(R x )(R y ), and R 1 , R 2 , R 3 , R 4 , R 5and R 6 At least two of them are branched or unbranched, substituted or unsubstituted C1-C6 alkyl groups or cycloalkyl groups, and R x or R y At least one of them is H, and it has a molecular weight in the range of about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are branched or unbranched, substituted or unsubstituted C1-C4 alkyl groups or cycloalkyl groups, and R x or R y At least one of them is H, and it has a molecular weight in the range of about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -N(R x )(R y ), and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are branched or unbranched, substituted or unsubstituted C1-C3 alkyl groups or cycloalkyl groups, and R x or R y At least one of them is H, and it has a molecular weight in the range of about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6At least two of them are -N(R x )(R y ), and at least two other ones among R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are C1-C2 alkyl groups, and at least one of R x or R y is H, and it has a molecular weight in the range of about 300 g / mol to about 450 g / mol.

[0112] In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are -NH2, and at least two other ones among R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are branched or unbranched substituted or unsubstituted C1-C6 alkyl groups or cycloalkyl groups, and it has a molecular weight in the range of about 300 g / mol to about 450 g / mol. In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are -NH2, and at least two other ones among R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are branched or unbranched substituted or unsubstituted C1-C6 alkyl groups or cycloalkyl groups, and it has a molecular weight in the range of about 300 g / mol to about 400 g / mol. In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6At least two of them are -NH2, R 1 R 2 R 3 R 4 R 5 and R 6 At least another two of them are branched or unbranched substituted or unsubstituted C1-C6 alkyl groups or cycloalkyl groups and have a molecular weight in the range of about 300 g / mol to about 350 g / mol.

[0113] In some embodiments, R 1 R 2 R 3 R 4 R 5 and R 6 At least two of them are -NH2, R 1 R 2 R 3 R 4 R 5 and R 6 At least another two of them are branched or unbranched substituted or unsubstituted C1-C4 alkyl groups or cycloalkyl groups and have a molecular weight in the range of about 300 g / mol to about 450 g / mol. In some embodiments, R 1 R 2 R 3 R 4 R 5 and R 6 At least two of them are -NH2, R 1 R 2 R 3 R 4 R 5 and R 6 At least another two of them are branched or unbranched substituted or unsubstituted C1-C4 alkyl groups or cycloalkyl groups and have a molecular weight in the range of about 300 g / mol to about 400 g / mol. In some embodiments, R 1 R 2 R 3 R 4 R 5 and R 6 At least two of them are -NH2, R 1 R 2 R 3 R4 , R 5 and R 6 At least two of them are branched or unbranched substituted or unsubstituted C1-C4 alkyl groups or cycloalkyl groups and have a molecular weight in the range of about 300 g / mol to about 350 g / mol.

[0114] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the others are branched or unbranched substituted or unsubstituted C1-C3 alkyl groups or cycloalkyl groups and have a molecular weight in the range of about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of the others are branched or unbranched substituted or unsubstituted C1-C3 alkyl groups or cycloalkyl groups and have a molecular weight in the range of about 300 g / mol to about 400 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least two of them are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6At least two of them are branched or unbranched substituted or unsubstituted C1-C3 alkyl groups or cycloalkyl groups and have a molecular weight in the range of about 300 g / mol to about 350 g / mol.

[0115] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of which are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of the others are C1-C2 alkyl groups and have a molecular weight in the range of about 300 g / mol to about 450 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of which are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of the others are C1-C2 alkyl groups and have a molecular weight in the range of about 300 g / mol to about 400 g / mol. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of which are -NH2, and R 1 , R 2 , R 3 , R 4 , R 5 and R 6 at least two of the others are C1-C2 alkyl groups and have a molecular weight in the range of about 300 g / mol to about 350 g / mol.

[0116] In some embodiments, the dye is [Chemical formula] selected from

[0117] In some embodiments, the total concentration of one or more dyes having a 4-benzhydrylidene-2,5-cyclohexadien-1-imine core in the dye composition (including any of the dyes having any one of formulas (I) to (XII)) is from about 0.05% w / v to about 8% w / v, from about 0.1% w / v to about 6% w / v, from about 0.1% w / v to about 5% w / v, from about 0.1% w / v to about 4% w / v, from about 0.1% w / v to about 3.5% w / v, from about 0.1% w / v to about 3% w / v, from about 0.1% w / v to about 2.5% w / v, from about 0.1% w / v to about 2% w / v, from about 0.1% w / v to about 1.5% w / v, from about 0.1% w / v to about 1% w / v, from about 0.15% w / v to about 4% w / v, from about 0.15% w / v to about 3.5% w / v, from about 0.15% w / v to about 3% w / v, from about 0.15% w / v to about 2.5% w / v, from about 0.15% w / v to about 2% w / v, from about 0.15% w / v to about 1.5% w / v, from about 0.15% w / v to about 1% w / v, from about 0.2% w / v to about 4%, from about 0.2% w / v to about 3.5%, from about 0.2% w / v to about 3%, from about 0.2% w / v to about 2.5%, from about 0.2% w / v to about 2% w / v, from about 0.2% w / v to about 1.5% w / v, from about 0.2% w / v to about 1% w / v, from about 0.25% w / v to about 4%, from about 0.25% w / v to about 3.5%, from about 0.25% w / v to about 3%, from about 0.25% w / v to about 2.5%, from about 0.25% w / v to about 2% w / v, from about 0.25% w / v to about 1.5% w / v, from about 0.25% w / v to about 1% w / v, from about 0.3% w / v to about 4%, from about 0.3% w / v to about 3.5%, from about 0.3% w / v to about 3%, from about 0.3% w / v to about 2.5%, from about 0.3% w / v to about 2% w / v, from about 0.3% w / v to about 1.5% w / v, from about 0.3% w / v to about 1% w / v, from about 0.4% w / v to about 4%, from about 0.4% w / v to about 3.5%, from about 0.4% w / v to about 3%, from about 0.4% w / v to about 2.5%, from about 0.4% w / v to about 2% w / v, from about 0.4% w / v to about 1.5% w / v, from about 0.4% w / v to about 1% w / v, from about 0.5% w / v to about 4%, from about 0.5% w / v to about 3.5%, from about 0.5% w / v to about 3%, from about 0.5% w / v to about 2.5%, from about 0.5% w / v to about 2% w / v, from about 0.5% w / v to about 1.5% w / v, from about 0.5% w / v to about 1% w / v, from about 0.6% w / v to about 4%, from about 0.6% w / v to about 3.5%, from about 0.6% w / v to about 3%, from about 0.6% w / v to about 2.5%, from about 0.in the range of 6% w / v to about 2% w / v, about 0.6% w / v to about 1.5% w / v, about 0.6% w / v to about 1% w / v, about 0.8% w / v to about 4%, about 0.8% w / v to about 3.5%, about 0.8% w / v to about 3%, about 0.8% w / v to about 2.5%, about 0.8% w / v to about 2%, about 0.8% w / v to about 2%, about 1% w / v to about 4%, about 1% w / v to about 3.5%, about 1% w / v to about 3%, about 1% w / v to about 2.5%, about 1% w / v to about 2%, about 1.5% w / v to about 4%, about 1.5% w / v to about 3.5%, about 1.5% w / v to about 3%, about 1.5% w / v to about 2.5%, about 1.5% w / v to about 2%, about 2% w / v to about 4%, about 2% w / v to about 3.5%, about 2% w / v to about 3%, about 2% w / v to about 2.5%, about 2.5% w / v to about 4%, about 2.5% w / v to about 3.5%, or about 2.5% w / v to about 3%.

[0118] In some embodiments, the total concentration of one or more dyes is about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0.8% w / v, about 0.9% w / v, about 1% w / v, about 1.2% w / v, about 1.4% w / v, about 1.6% w / v, about 1.8% w / v, about 2% w / v, about 2.2% w / v, about 2.4% w / v, about 2.6% w / v, about 2.8% w / v, about 3% w / v, about 3.2% w / v, about 3.4% w / v, about 3.6% w / v, about 3.8% w / v or about 4% w / v.

[0119] In some embodiments, one or more dyes have a molecular weight in the range of about 300 g / mol to about 375 g / mol, and the total concentration of one or more dyes is in the range of about 0.15% w / v to about 1.8% w / v. In other embodiments, one or more dyes have a molecular weight in the range of about 300 g / mol to about 350 g / mol, and the total concentration of one or more dyes is in the range of about 0.3% w / v to about 1.2% w / v.

[0120] In some embodiments, one or more dyes are

Chemical formula

[0121] The total concentration of one or more dyes ranges from about 0.15% w / v to about 1.8% w / v, for example, from about 0.3% w / v to about 1.2% w / v.

[0122] acid

[0123] In some embodiments, the dye composition contains an acid. In some embodiments, the acid is a strong acid. In some embodiments, the strong acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, trifluoroacetic acid, hydroiodic acid, sulfuric acid, nitric acid, chloric acid, and perchloric acid. In some embodiments, the strong acid has a concentration in the range of about 0.8 M to about 2 M. In other embodiments, the strong acid has a concentration in the range of about 0.9 M to about 1.8 M. In still other embodiments, the strong acid has a concentration in the range of about 1 M to about 1.5 M. In some embodiments, the strong acid has a concentration of about 1 M or more, for example, about 0.9 M, about 0.95 M, about 1.0 M, about 1.05 M, about 1.1 M, about 1.15 M, about 1.2 M, about 1.25 M, about 1.3 M, about 1.35 M, about 1.4 M, about 1.45 M, about 1.5 M, about 1.55 M, about 1.6 M, about 1.65 M, etc. In some embodiments, the strong acid is hydrochloric acid, for example, 1.2 M hydrochloric acid. In some embodiments, the strong acid is nitric acid, for example, 1.25 M nitric acid.

[0124] In some embodiments, the acid is a weak acid. In some embodiments, the weak acids are formic acid, acetic acid, benzoic acid, oxalic acid, hydrofluoric acid, nitrous acid, sulfurous acid, and phosphoric acid.

[0125] In some embodiments, the pH of the dye composition ranges from about -0.5 to about 6.5. In other embodiments, the pH ranges from about -0.5 to about 6. In other embodiments, the pH ranges from about -0.5 to about 5.5. In other embodiments, the pH ranges from about -0.5 to about 5. In other embodiments, the pH ranges from about -0.5 to about 4.5. In other embodiments, the pH ranges from about -0.5 to about 4. In other embodiments, the pH ranges from about -0.5 to about 3.5. In other embodiments, the pH ranges from about -0.5 to about 3. In other embodiments, the pH ranges from about -0.5 to about 2.5. In other embodiments, the pH ranges from about -0.5 to about 2.

[0126] Solvent

[0127] In some embodiments, the dye composition comprises one or more solvents. In some embodiments, the solvent is an alcohol. In some embodiments, the solvent is a polyol.

[0128] Non-limiting examples of suitable solvents include methanol, ethanol, propanol, butanol, amyl alcohol, pentanol, fusel oil, hexanol, heptanol, octanol, cyclohexanol, benzyl alcohol, furfuryl alcohol, tetrahydrofurfuryl alcohol, hexane, heptane, octane, decane, petroleum ether, petroleum benzine, ligroin, gasoline, kerosene, cyclohexane, benzene, toluene, xylene, tetralin, decalin, terpene oil, chloroform, carbon tetrachloride, ethylene chloride, ethylidene chloride, trichloroethane, tetrachloroethane, trichloroethylene, tetrachloroethylene, trichloropropane, isopropyl chloride, dichloropropane, butyl chloride, amyl chloride, hexyl chloride, ethylene bromide, tetrabromoethane, chlorobenzene, dichlorobenzene, trichlorobenzene, bromobenzene, chlorotoluene, isopropyl ether, dibutyl ether, diisoamyl ether, hexyl ether, methyl phenyl ether, ethyl phenyl ether, butyl phenyl ether, ethyl benzyl ether, dioxane, 2-methylfuran, tetrahydrofuran, tetrahydropyran, cineole, acetone, methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, methyl amyl ketone, methyl hexyl ketone, diethyl ketone, ethyl butyl ketone, dipropyl ketone, diisobutyl ketone, diacetone alcohol, cholorone, isophorone, cyclohexanone, methyl cyclohexanone, acetophenone, ethyl formate, propyl formate, butyl formate, amyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, amyl acetate, methyl isoamyl acetate, methoxybutyl acetate, hexyl acetate, cyclohexyl acetate, benzyl acetate, methyl propionate, ethyl propionate, butyl propionate, amyl propionate, methyl butyrate, ethyl butyrate, butyl butyrate, amyl butyrate, methyl acetoacetate, ethyl acetoacetate, isoamyl isovalerate, methyl lactate, ethyl lactate, butyl lactate, amyl lactate, methyl benzoate, diethyl oxalate, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether, ethylene glycol diethyl ether,Ethylene glycol monoethyl ether acetate, ethylene glycol isopropyl ether, ethylene glycol monobutyl ether, ethylene glycol dibutyl ether, ethylene glycol monobutyl ether acetate, ethylene glycol monophenyl ether, methoxymethoxyethanol, ethylene glycol monoacetate, ethylene glycol diacetate, diethylene glycol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monoethyl ether acetate, diethylene glycol monobutyl ether, diethylene glycol monobutyl ether acetate, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethylene glycol acetate, propylene glycol, propylene glycol monoethyl ether, propylene glycol propyl ether, propylene glycol monobutyl ether, dipropylene glycol, dipropylene glycol monomethyl ether, dipropylene glycol monoethyl ether, trimethylene glycol, butanediol, hexylene glycol, formic acid, acetic acid, acetic anhydride, propionic acid, propionic anhydride, butyric acid, valeric acid, lactic acid, pyridine, picoline, quinoline, isoquinoline, dimethyl sulfoxide, triethyl phosphate, dimethylformamide, γ-butyrolactone, γ-valerolactone, 6-hexanolactone, methyl salicylate, ethyl salicylate, butyl salicylate, diethyl adipate, ethyl carbonate, butyl sulfide, and diacetone alcohol are included, but not limited thereto. In some embodiments, the solvent is DMSO.,

[0129] Additive

[0130] In some embodiments, the dye composition includes one or more additives, such as antioxidants, stabilizers, and / or biocides.

[0131] As used herein, the term "antioxidant" includes any compound or combination of compounds that prevents or slows the oxidation of components caused by harmful reactive oxygen species (ROS). Without limitation, phytochemical antioxidants such as tocopherol, phospholipids (PL), phytosterols, phycocyanin, vitamin E, A and C, beta-carotene, coenzyme Q10, fatty acids omega-3, omega-6 and w-9, polyphenols, terpenes such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, lecithin, sesamin, sesamol, sesamolin, alpha-tocopherol, gamma-tocopherol, salicylic acid, ascorbic acid, ascorbyl palmitate, fumaric acid, malic acid, sodium ascorbate and sodium metabisulfite, and chelating agents such as disodium EDTA, and any known antioxidants can be used. Pharmaceutically acceptable dietary supplements can also be used as antioxidants, such as plants, algae and lichens, and may include extracts of one or more of honey bee propolis, red clover, soybeans, capers, almonds, artichokes, green tea, pomegranates, orange red, grape seeds, bilberries, fotii root, ginseng, kidney beans, red algae, brown algae, green algae and lichens.

[0132] In some embodiments, the antioxidant is selected from the group consisting of ascorbic acid and its salts, tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate, and propyl gallate.

[0133] In other embodiments, the antioxidant is selected from the group consisting of ascorbic acid, thioglycerol, thiosorbitol, thiourea, sodium thiosulfate, thioacetic acid, cysteine, methionine, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), ascorbyl palmitate, hydroquinone, propyl gallate, nordihydroguaiaretic acid, vitamin E (α-tocopherol), and lecithin. Preferred antioxidants are micronized propyl gallate, micronized BHA, micronized BHT, vitamin E, ascorbic acid, sodium thiosulfate, and cysteine.

[0134] In yet other embodiments, the antioxidant is selected from the group consisting of hydroquinone, n-alkyl gallates (e.g., n-propyl gallate, n-octyl gallate, and n-dodecyl gallate); reducing sugars such as sorbitol and mannitol; benzoates and hydroxybenzoates; certain acids such as citric acid, tartaric acid, lactic acid, erythorbic acid ascorbic acid, uric acid, tannic acid, and salts of such acids (e.g., Mg2+, NH4+, Na+, K+, and Ca2+ salts); and chelating agents such as EDTA that remove metals that function as oxidizing agents.

[0135] In still further embodiments, the antioxidant is an oil-soluble antioxidant selected from the group consisting of ascorbyl palmitate, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA); propyl gallate, and alpha-tocopherol.

[0136] In a preferred embodiment, the antioxidant is ascorbic acid.

[0137] In some embodiments, the composition comprises a stabilizer. In other embodiments, the composition comprises a biocide or a preservative, such as an antibacterial agent. Suitable preservatives include, for example, isothiazolinones, glycols, azides, and combinations thereof. Exemplary preservatives include ProClin® 300 (2.30% 5-chloro-2-methyl-4-isothiazolin-3-one, 0.70% 2-methyl-4-isothiazolin-3-one, 2-3% alkyl carboxylate (stabilizer), and 93-95% modified glycol, available from Sigma-Aldrich, St. Louis, Missouri), ProClin® 950 (9.5-9.9% 2-methyl-4-isothiazolin-3-one, Sigma-Aldrich), and sodium azide.

[0138] Sulfide or SO2 source composition

[0139] The present disclosure also provides a composition comprising a sulfite or an SO2 source (hereinafter referred to as "sulfite or SO2 source composition" herein). In some embodiments, the sulfite or SO2 source composition comprises a sulfite or an SO2 source, any acid, and one or more optional additives. In some embodiments, the sulfite or SO2 source is selected from metabisulfite, bisulfite, dithionite, thiosulfate, sulfite, and sulfurous acid. In some embodiments, the sulfite or SO2 source composition is stable for at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, etc.

[0140] In some embodiments, the total concentration of sulfite or the sulfite or SO2 source in the sulfite or SO2 source composition is in the range of about 0.1% w / v to about 21.5% w / v, about 0.1% w / v to about 18% w / v, about 0.1% w / v to about 12% w / v, about 0.1% w / v to about 10% w / v, about 0.1% w / v to about 9% w / v, about 0.1% w / v to about 8% w / v, about 0.1% w / v to about 7% w / v, about 0.1% w / v to about 6% w / v, about 0.1% w / v to about 5% w / v, about 0.1% w / v to about 4% w / v, about 0.1% w / v to about 3% w / v, about 0.1% w / v to about 2% w / v, about 0.1% w / v to about 1% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.3% w / v, about 0.5% w / v to about 21.5% w / v, about 0.5% w / v to about 18% w / v, about 0.5% w / v to about 12% w / v, about 0.5% w / v to about 10% w / v, about 0.5% w / v to about 9% w / v, about 0.5% w / v to about 8% w / v, about 0.5% w / v to about 7% w / v, about 0.5% w / v to about 6% w / v, about 0.5% w / v to about 5% w / v, about 0.5% w / v to about 4% w / v, about 0.5% w / v to about 3% w / v, about 0.5% w / v to about 2% w / v, about 0.5% w / v to about 1% w / v, about 1% w / v to about 21.5% w / v, about 1% w / v to about 18% w / v, about 1% w / v to about 12% w / v, about 1% w / v to about 10% w / v, about 1% w / v to about 9% w / v, about 1% w / v to about 8% w / v, about 1% w / v to about 7% w / v, about 1% w / v to about 6% w / v, about 1% w / v to about 5% w / v, about 1% w / v to about 4% w / v, about 1% w / v to about 3% w / v, about 2.5% w / v to about 21.5% w / v, about 2.5% w / v to about 18% w / v, about 2.5% w / v to about 12% w / v, about 2.5% w / v to about 10% w / v, about 2.5% w / v to about 9% w / v, about 2.5% w / v to about 8% w / v, about 2.5% w / v to about 7% w / v, about 2.5% w / v to about 6% w / v, about 2.5% w / v to about 5% w / v, about 5% w / v to about 21.5% w / v, about 5% w / v to about 18% w / v, about 5% w / v to about 12% w / v, about 5% w / v to about 10% w / v, about 5% w / v to about 9% w / v, or about 5% w / v to about 8% w / v.

[0141] In embodiments where the source of the sulfite or SO2 source is not thiosulfate, the sulfite or SO2 source composition may include an acid (including those listed herein), and / or may have a pH of less than about 9, such as less than about 8.5, such as less than about 8, such as less than about 7.5, such as less than about 7. In some embodiments, the pH of the sulfite or SO2 source composition is from about 5 to 6.9. In some embodiments, the pH of the sulfite or SO2 source composition is from about 5 to 6.5. In some embodiments, the pH of the sulfite or SO2 source composition is from about 5.5 to 6.5.

[0142] In embodiments where the sulfite or SO2 source is thiosulfate, the sulfite or SO2 source composition has a pH greater than 7, such as greater than 7.5, such as greater than 8, such as greater than 8.5, or such as greater than 9. In some embodiments, where the sulfite or SO2 source is thiosulfate, the sulfite or SO2 source composition has a pH of about 10.

[0143] In some embodiments, the sulfite or SO2 source composition includes an antioxidant, including any of the antioxidants listed herein. In some embodiments, the sulfite or SO2 source composition includes a solvent, including any of the solvents listed herein. In some embodiments, the sulfite or SO2 source composition includes an alcohol or a glycol.

[0144] Kit

[0145] In some embodiments, the present disclosure provides a kit including at least two reservoirs, containers, and / or dispensers (collectively referred to herein as "containers"), wherein the first container contains a dye composition and the second container contains a sulfite or SO2 source composition. In some embodiments, the kit includes two containers, namely, a dye composition containing an acid such as any of a weak acid, a strong acid, an inorganic acid, or an organic acid, and a sulfite or SO2 source composition.

[0146] In other embodiments, the kit includes three containers, namely, a dye composition without acid, a sulfite or SO2 source composition, and an acid composition. In some embodiments, the acid composition includes any of the weak acids, strong acids, inorganic acids, or organic acids described herein as any of the weak acids, strong acids, inorganic acids, or organic acids.

[0147] Method for dyeing a sample

[0148] The present disclosure also provides a method for forming a Schiff reagent in situ, for example, on the surface of a substrate (e.g., a slide), on a sample disposed on the substrate, or directly in a liquid pool disposed on the substrate. Generally, the method includes applying or dispensing a dye composition, a sulfite or SO2 source composition, and / or an acid composition to the substrate, and reacting the various components of the composition at a predetermined time and a predetermined temperature. Thereafter, the Schiff reagent formed in situ can be removed from the substrate, such as by washing the substrate with a washing reagent or buffer. As described herein, the applicant has unexpectedly found that by using the Schiff reagent generated in situ to dye a sample, a staining quality and / or intensity at least equivalent to that obtained using a commercially available Schiff reagent can be obtained. Furthermore, the applicant has unexpectedly found that the staining intensity can be "dialed in" by changing the time during which any of the reagents is in contact with the sample, changing the temperature of the sample and / or the reagent disposed in contact with the sample, and / or changing the concentration of any of the reagents or components of the reagents (e.g., the concentration of the dye, the concentration of the sulfite source, and / or the concentration of the acid).

[0149] As used herein, the phrase "dispensed onto a substrate means" refers to dispensing a fluid or composition onto the surface of a substrate such as a microscope slide, a sample disposed on the substrate, or a pool of fluid or reagent disposed on the substrate and / or the sample. In some embodiments, the fluid or composition can be dispensed directly onto a sample disposed on the substrate. Alternatively, the fluid or composition can be dispensed adjacent to a sample disposed on the substrate, and the dispensed fluid or composition can then be moved towards the sample, for example, by tilting the substrate and / or using a jet of gas to extrude the fluid or composition from the surface of the substrate towards the sample. In another embodiment, the fluid or composition can be dispensed into a "pool" of fluid or reagent present on the surface of the substrate or sample. One of ordinary skill in the art will understand that subsequent dispensing of different compositions onto the same substrate and / or sample disposed on the substrate can be at the same or substantially the same location, such that the subsequently dispensed fluid or composition contacts an existing pool of fluid and the fluids and / or compositions can mix and / or react.

[0150] In some embodiments, a dye composition, a sulfite or SO2 source composition, and / or an acid composition can be applied to a sample using a manual process. In other embodiments, the dye compositions, sulfite or SO2 source compositions, and / or acid compositions described herein are applied or dispensed onto a biological sample, such as within an automated staining device. Non-limiting examples of suitable automated staining systems are described herein.

[0151] In some embodiments, the sample is treated with a reagent that forms aldehyde groups for a predetermined time before dispensing any of the dye composition, the bisulfite or SO2 source composition, and / or any acid composition. In some embodiments, the sample is treated with periodic acid (e.g., 1 gram of periodic acid in 100 mL of water) for about 1 to about 10 minutes (e.g., 5 minutes) before dispensing any of the dye composition, the bisulfite or SO2 source composition, and / or any acid composition. In some embodiments, periodic acid is dispensed onto the sample and maintained in contact with the sample for a predetermined time (e.g., about 1 to about 10 minutes). Subsequently, the periodic acid is rinsed or washed off the sample using, for example, a buffer solution, distilled water, or other washing reagent.

[0152] In some embodiments, the dye composition, the bisulfite or SO2 source composition, and / or any acid composition are sequentially dispensed onto the substrate such that the Schiff reagent can be synthesized in situ. In other embodiments, the dye composition, the bisulfite or SO2 source composition, and / or any acid composition are simultaneously dispensed onto the substrate such that the Schiff reagent can be synthesized in situ.

[0153] In some embodiments, the order in which the dye composition, the bisulfite or SO2 source composition, and / or any acid composition are dispensed depends on the components contained within the composition. For example, in some embodiments, if the dye composition contains an acid, the acid-containing dye composition can be dispensed onto the substrate first, followed by the sulfite or SO2 source composition onto the substrate. In other embodiments, the sulfite or SO2 source composition can be dispensed onto the substrate first, and then the acid-containing dye composition can be dispensed onto the substrate thereafter. In still other embodiments, the dye composition containing the acid and the sulfite or SO2 source composition are simultaneously dispensed onto the substrate.

[0154] In some embodiments, a dye composition that is acid-free (or has a pH greater than about 6.9) is first dispensed onto a substrate, and then an acid composition is dispensed onto the substrate (e.g., into a pool of fluid containing the acid-free dye composition) so as to lower the pH of the dye composition (e.g., such that the dye composition is acidified). In some embodiments, upon dispensing the acid composition, the pH of the pool is lowered to less than 6.9, such as less than 6.5, such as less than 6, such as less than 5.5, such as less than 5, such as less than 4.5, such as less than 4, such as less than 3.5, such as less than 3, such as less than 2.5, such as less than 2, etc. Subsequently, a sulfite or SO2 source composition is dispensed into the resulting acidified dye composition to obtain an in-situ synthesized Schiff reagent.

[0155] In other embodiments, the acid composition is first dispensed, and then an acid-free (or having a pH greater than 6.9) dye composition is then dispensed to form an acidified dye composition on the surface of the substrate (e.g., the acidified composition has a pH less than 6.9). Subsequently, a sulfite or SO2 source composition is dispensed into the resulting acidified dye composition to obtain an in-situ synthesized Schiff reagent.

[0156] In yet other embodiments, an acid-free (or having a pH greater than about 6.9) dye composition and an acid composition are simultaneously dispensed onto a substrate such that when the dispensed dye composition and acid composition are mixed on the surface of the substrate, the pH of the dye composition is lowered to obtain an acidified dye composition. Subsequently, a sulfite or SO2 source composition is dispensed into the resulting acidified dye composition to obtain an in-situ synthesized Schiff reagent.

[0157] In some embodiments, a sulfite or SO2 source composition is first dispensed onto a substrate, and then a dye composition that is free of acid (or has a pH greater than about 6.9) is dispensed onto the substrate. Subsequently, an acid composition is dispensed onto the substrate such that a Schiff reagent is synthesized in situ. In other embodiments, a sulfite or SO2 source composition is first dispensed onto a substrate, and then an acid composition is dispensed onto the substrate. Subsequently, a dye composition that is free of acid (or has a pH greater than about 6.9) is dispensed onto the substrate to synthesize a Schiff reagent in situ. In yet other embodiments, a sulfite or SO2 source composition is first dispensed onto a substrate, and then a dye composition free of acid and an acid composition are simultaneously applied to the substrate to synthesize a Schiff reagent in situ.

[0158] In some embodiments, the amounts of the dye composition, bisulfite or SO2 source composition, and optional acid composition dispensed onto the substrate are, independently, in the range of about 25 μL to about 500 μL, about 25 μL to about 400 μL, about 25 μL to about 300 μL, about 50 μL to about 300 μL, about 50 μL to about 250 μL, about 50 μL to about 225 μL, about 50 μL to about 200 μL, about 50 μL to about 175 μL, about 50 μL to about 150 μL, or about 100 μL to about 150 μL. By way of example only, a dye composition of about 50 μL to about 200 μL and a bisulfite or SO2 source composition of about 50 μL to about 200 μL can be dispensed onto the substrate simultaneously or sequentially.

[0159] In some embodiments, the in-situ synthesized Schiff reagent is allowed to remain in contact with the sample for a predetermined time. In this way, the staining intensity of the Schiff reagent can be "dialed in" taking into account differences in the components utilized in synthesizing the Schiff reagent to meet the preferences of a pathologist or medical professional, or differences in the concentrations of the components utilized in any of the compositions described herein (e.g., the concentration of the dye, the concentration of the acid, and / or the concentration of the sulfite source), taking into account differences in the type of sample or the fixation of the sample, and / or taking into account differences in the temperature of the sample and / or the composition components. In some embodiments, the in-situ synthesized Schiff reagent remains in contact with the sample for at least 1 minute, at least 2 minutes, at least 4 minutes, at least 8 minutes, at least 12 minutes, at least 16 minutes, at least 20 minutes, at least 24 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 80 minutes, at least 100 minutes, or at least 120 minutes.

[0160] In some embodiments, the sample and / or composition for forming the in-situ synthesized Schiff reagent is maintained at a predetermined temperature. For example, the predetermined temperature is a temperature above 25°C, above 30°C, or above 35°C. In other embodiments, the temperature of the sample and / or the composition for forming the in-situ synthesized Schiff reagent is in the range of about 30°C to about 90°C, about 30°C to about 50°C, about 30°C to about 40°C, or about 35°C to about 41°C. In still further embodiments, the temperature of the sample and / or the composition for forming the in-situ synthesized Schiff reagent is about 30°C. In still further embodiments, the temperature of the sample and / or the composition for forming the in-situ synthesized Schiff reagent is about 32°C. In still further embodiments, the temperature of the sample and / or the composition for forming the in-situ synthesized Schiff reagent is about 35°C. In still further embodiments, the temperature of the sample and / or the composition for forming the in-situ synthesized Schiff reagent is about 37°C. In still further embodiments, the temperature of the sample and / or the composition for forming the in-situ synthesized Schiff reagent is about 40°C.

[0161] In some embodiments, when the specimen is a sample embedded in paraffin, the sample can be deparaffinized using a suitable deparaffinization fluid. After the waste removal agent removes the deparaffinization liquid, any number of substances can be continuously applied to the specimen. The substances can be for pretreatment (e.g., protein crosslinking, exposing nucleic acids, etc.), denaturation, hybridization, washing (e.g., stringent washing), detection (e.g., visual or linking of marker molecules to probes), amplification (e.g., amplification of proteins, genes, etc.), counterstaining, cover glass, etc.

[0162] After the specimen has been processed, the user can transport the slide bearing the specimen to an imaging device for analysis or other downstream processing. For example, the imaging device may be a brightfield imager slide scanner. One brightfield imager is the iScan Coreo™ brightfield scanner sold by Ventana Medical Systems, Inc. In an automated embodiment, the imaging device is a digital pathology device disclosed in International Patent Application No. PCT / US2010 / 002772, published as International Publication No. WO 2011 / 049608, titled IMAGING SYSTEM AND TECHNIQUES, or in U.S. Patent Application No. 61 / 533,114, filed on September 9, 2011, titled IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME. International Patent Application No. PCT / US2010 / 002772 and U.S. Patent Application Publication No. 2014 / 0178169 are hereby incorporated by reference in their entirety. In other embodiments, the imaging device includes a digital camera coupled to a microscope.

[0163] The methods disclosed herein can be adapted for use in existing automated processing systems. For example, Ventana Medical Systems, Inc. is the assignee of numerous U.S. patents that disclose systems and methods for performing automated analysis, such as U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Patent Application Publication Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference. These systems can be adapted to be compatible with the present invention. Briefly, the automated slide processing systems described in the foregoing references are high-capacity slide processing systems that reciprocate a tray holding a plurality of slides (to minimize cross-contamination) in a substantially horizontal position between workstations that perform various slide processing operations on the slides. Fresh reagents can be applied to each slide during processing, and since the slides are processed separately at intervals within the tray, cross-contamination of the slides by the reagents can be substantially eliminated. In one configuration, the system includes a radiant heater, a combination deparaffinizer / stainer / solvent exchange workstation, a convection oven, and a coverslipper. In some embodiments, a tray of slides bearing paraffin-embedded tissue samples can be heated under the radiant heater of the system to spread and facilitate removal of the paraffin in the samples, and the samples can also be adhered to the slides. In some embodiments, the tray can then be transported to a multi-functional deparaffinizer / stainer / solvent exchange workstation where the slides can be deparaffinized, stained, and solvent-exchanged. In some embodiments, a tray of stained slides that are ready for coverslipping can then be reciprocated between the coverslipper of the system, which adds coverslips to the slides. Once the slides are coverslipped, the tray can be transported to the convection oven to cure the coverslips on the stained slides.In some embodiments, the high-capacity stainers described herein are commercially available from Ventana Medical Systems, Inc. located in Tucson, Arizona, USA.

[0164] Examples of other commercially available specimen processing systems that can apply the solutions and formulations described herein include the VENTANA SYMPHONY (individual slide stainer) and VENTANA HE600 (individual slide stainer) systems, as well as the Dako CoverStainer (batch stainer) from Agilent Technologies, the Leica ST4020 Small Linear Stainer (batch stainer), the Leica ST5020 Multistainer (batch stainer), and the Leica ST5010 Autostainer XL system (batch stainer), and the H&E stainers from Leica Biosystems Nussloch GmbH.

[0165] In some embodiments, the automated staining system includes one or more reservoirs, containers, and / or dispensers, each of which may contain any of the compositions disclosed herein. In some embodiments, the dye composition, the bisulfite or SO2 source composition, and any acid composition are dispensed separately onto the biological sample. In these embodiments, the solutions can be mixed by diffusion on the sample or mechanically, for example, by agitation with an air mixer or pipette.

[0166] In some embodiments, the automated specimen processing device can include a carousel that holds a plurality of substrates, such as microscope slides, each substrate containing a sample to be stained. In some embodiments, the automated staining device can also include a device for rotating the carousel at a predetermined speed and a mechanism for instructing and controlling the application of reagents, including the solutions and formulations described herein, to the substrates and samples during rotation of the carousel. In some embodiments, when the slides are loaded into the instrument, the test protocol determines which compositions (from which reservoirs, dispensers, or containers) are to be dispensed onto the substrates at specific times. In some embodiments, at the appropriate time, the dispenser rack rotates to align the correct compositions over the substrates, and the instrument dispenses a predetermined amount of one or more of the disclosed compositions onto the substrates.

[0167] In some embodiments, the system includes a slide tray that holds a plurality of slides in a substantially horizontal position (such as two rows where the slides are held at an angle between about 0.2 degrees and about 1.2 degrees from horizontal), and one or more workstations that receive the slide tray and perform one or more slide processing operations on the slides within the slide tray (e.g., arranged in a vertical stack). In some embodiments, the workstation can perform slide processing operations on one or more individual slides within the slide tray, such as at least two or four slides within the slide tray, or can perform slide processing operations on all of the slides within the slide tray simultaneously. In some embodiments, the one or more workstations dispense reagents to the slides within the slide tray with a minimal amount of reagent contacting the first slide that contacts the second slide, thereby minimizing cross-contamination between the slides. Such a workstation can include one or more directed nozzles for dispensing the compositions of the present disclosure onto the slides. For example, the one or more directed nozzles can include a pair of directed nozzles that dispense a dye composition, a bisulfite or SO2 source composition, and / or any acid composition in opposite directions across the surface of the slide. In further particular embodiments, the one or more directed nozzles can further include a directed nozzle that dispenses the composition towards the bottom surface of the slide. In other particular embodiments, the one or more workstations can simultaneously dispense a dye composition, a bisulfite or SO2 source composition, and / or any acid composition to at least two slides held in the slide tray within a given workstation, or the one or more workstations can simultaneously dispense the composition to all of the slides held in the slide tray within a given workstation. Additional system components and tray configurations (as well as control systems) are described in U.S. Patent Nos. 8,663,991, 7,468,161, and 9,528,918, the entire disclosures of which are incorporated herein by reference.

[0168] In some embodiments, the present disclosure is an apparatus for automatically processing biological specimens, comprising at least one slide tray that holds a plurality of slides in a substantially horizontal position, wherein the biological specimens are disposed on the slides; a slide tray; one or more workstations that receive the slide tray and perform one or more slide processing operations on the plurality of slides held by the slide tray; a transport unit that moves the slide tray in and out of the one or more workstations; a fluid module in fluid communication with the one or more workstations that supplies a dye composition, a sulfite or SO2 source composition, and / or any acid composition to the one or more workstations; a pneumatic module in fluid communication with the one or more workstations and the fluid module that supplies vacuum and / or pressurized gas to the one or more workstations and the fluid module; and a control module in electrical communication with the transport unit, the one or more workstations, the fluid module, and the pneumatic module, the control module adjusting the functions of the components of the apparatus during processing of the biological specimens. The apparatus may be adapted to deliver one or more of the dye composition, sulfite or SO2 source composition, and / or any acid composition described herein, sequentially or simultaneously.

[0169] Examples

[0170] Example 1 - Stability of Reagents for On-Slide Synthesis

[0171] The present applicants have found that an in-situ synthesized Schiff reagent promotes the staining of biological samples in a manner equivalent to that observed using a commercially available Schiff reagent, even after the dye used to synthesize the Schiff reagent has been stored at high temperature (e.g., 60 °C for 2 weeks) for a long period of time. For example, FIGS. 6A and 6B show the staining of tissue samples performed using an in-situ synthesized Schiff reagent prepared from pararosaniline dye stored at either 2 °C to 8 °C or 60 °C for 2 weeks. In particular, the in-situ synthesized Schiff reagent was equally stained regardless of the storage conditions of the precursor dye. In comparison, FIGS. 6C and 6D show the staining of tissue samples performed using a new commercially available Schiff reagent (FIG. 6C) and a commercially available Schiff reagent that has been opened for 2 weeks (FIG. 6D). In particular, the commercially available Schiff reagent that has been opened for 2 weeks showed a decrease in staining intensity compared to the newly opened commercially available Schiff reagent or the Schiff reagent prepared in-situ.

[0172] Example 2 - Reaction Time

[0173] Figures 7A and 7B show the staining intensity and contrast using in - situ synthesized Schiff reagent as a function of reaction time. It was demonstrated that the time and temperature during the incubation of the in - situ synthesized Schiff reagent on tissue samples can be changed simultaneously and individually to adjust the staining intensity to preference. Shorter incubation times of about 4 to about 16 minutes and / or lower temperatures, such as 37 °C, resulted in brighter staining intensity. Longer incubation times and temperatures, such as about 16 to about 32 minutes and / or about 40 to about 60 °C, resulted in darker staining intensity. In particular, when the in - situ synthesized Schiff reagent was maintained in contact with the sample for a longer period, the staining intensity and / or contrast increased. This demonstrated that the staining intensity and / or contrast can be "dialed in" by changing the time (and / or temperature) that the in - situ synthesized Schiff reagent remains in contact with the biological sample. Samples stained with the in - situ synthesized Schiff reagent had at least equivalent staining performance compared to control samples (Figure 7C) stained with a commercially available Schiff reagent.

[0174] Example 3 - Dye Concentration

[0175] Figures 8A and 8B show the staining intensity and contrast using in-situ synthesized Schiff reagents as a function of the concentration of the dye and the bisulfite / SO2 source. For thiosulfate, about 0.02 to about 0.8 M was tested. At 0.02 M, the staining was very weak because this condition was approximately equimolar with 0.6% pararosaniline. In particular, (while keeping the concentration of the sulfite or SO2 source constant), the staining intensity and / or contrast increased as the concentration of the dye increased. For the strong acid concentration, it was tested from 0.01 M to 4 M. Lower acid concentrations result in darker staining. This demonstrates that the staining intensity and / or contrast can be "dialed in" by varying the concentration of the dye utilized in the in-situ synthesis of the Schiff reagent. Compared to a control sample stained with a commercially available Schiff reagent (Figure 8C), the sample stained with the in-situ synthesized Schiff reagent had at least equivalent staining performance.

[0176] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified as necessary to provide further alternative embodiments using the concepts of various patents, applications, and publications.

[0177] Although the disclosure herein has been described with reference to specific embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. Accordingly, it is understood that many modifications can be made to the exemplary embodiments and other configurations can be devised without departing from the spirit and scope of the disclosure as defined by the appended claims.

Claims

1. Equations (I) to (XII): 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 (In the formula, R ２ , ６ , ｙ , ｙ , ｙ , ６ , ６ , ４ , ３ , １ , ｘ , １ , １ , ｘ , ５ , R ２ , R ３ , R ４ , R<0000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ A is the counterion.) A dye composition comprising a compound having any one of the following, wherein the concentration of the compound having any one of formulas (I) to (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition.

2. R １ , R ２ , R ３ , R ４ , R ５ and R ６ At least two of them are -N(R ｘ ) (Caution ｙ ) and R １ , R ２ , R ３ , R ４ , R ５ and R ６ At least two of these are branched or unbranched substitutions or unsubstituted C １ ~C ６ It is an alkyl group or cycloalkyl group, R ｘ or R ｙ The dye composition according to claim 1, wherein at least one of the elements is H.

3. R １ , R ２ , R ３ , R ４ , R ５ and R ６ At least two of them are -N(R ｘ ) (Caution ｙ ) and R １ , R ２ , R ３ , R ４ , R ５ and R ６ At least two other of these are C １ ~C ２ It is an alkyl group, R ｘ or R ｙ The dye composition according to claim 1, wherein at least one of the elements is H.

4. The compound having any one of formulas (I) to (XII) is 【Chemistry 4】 A dye composition according to claim 1, selected from the group consisting of the following.

5. The dye composition according to any one of claims 1 to 4, wherein the compound having any one of formulas (I) to (XII) has a molecular weight in the range of about 300 g / mol to about 600 g / mol.

6. The dye composition according to any one of claims 1 to 4, wherein the compound having any one of formulas (I) to (XII) has a molecular weight in the range of about 300 g / mol to about 425 g / mol.

7. The dye composition according to any one of claims 1 to 4, wherein the concentration is in the range of about 0.15% w / v to about 2.5% w / v, based on the total volume of the dye composition.

8. The dye composition according to any one of claims 1 to 4, wherein the concentration is in the range of about 0.15% w / v to about 1.8% w / v based on the total volume of the dye composition.

9. A dye composition according to any one of claims 1 to 4, further comprising an acid.

10. The dye composition according to claim 9, wherein the acid is a strong acid.

11. The dye composition according to claim 10, wherein the strong acid is 1 M to 1.3 M hydrochloric acid.

12. The dye composition according to claim 10, wherein the strong acid is 1 M to 1.3 M nitric acid.

13. A dye composition according to any one of claims 1 to 4, further comprising at least one of a solvent and / or an additive.

14. A dye composition according to any one of claims 1 to 4, further comprising a solvent and at least one additive.

15. The dye composition according to claim 1, comprising essentially the compound having any one of formulas (I) to (XII) and a strong acid.

16. A sulfite or SO selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfite, sulfite, and sulfite. ２ A sulfite composition containing a source, wherein the sulfite or SO ２ A sulfite composition in which the concentration of the source is in the range of about 0.1% w / v to about 18% w / v, based on the total weight of the sulfite composition.

17. A sulfite composition wherein the concentration is in the range of about 1% w / v to about 9% w / v based on the total weight of the sulfite composition.

18. A sulfite composition having a concentration in the range of about 1% w / v to about 4% w / v based on the total weight of the sulfite composition.

19. The aforementioned sulfite or SO ２ The sulfite composition according to claim 16, wherein the source is not a thiosulfate.

20. The sulfite composition according to claim 19, wherein the sulfite composition further comprises an acid.

21. (i) A kit comprising a dye composition according to any one of claims 1 to 4, and (ii) a sulfite composition according to any one of claims 16 to 20.

22. A kit comprising (i) an acid-free dye composition, (ii) a sulfite composition, and (iii) an acid, wherein the dye composition, the sulfite composition, and the acid are each contained in separate containers.

23. The dye composition is of formula (I) to (XII): 【Transformation 5】 【Transformation 6】 【Transformation 7】 (In the formula, R １ 、 R ２ 、 R ３ 、 R ４ 、 R ５ and R ６ are each independently H, halogen, a branched or unbranched substituted or unsubstituted C １ ~C ６ alkyl group or cycloalkyl group, -OH, -O-C １ ~C ６ alkyl, or -N(R ｘ )(R ｙ ), and R ｘ and R ｙ are each independently H, or a branched or unbranched C １ ~C ６ alkyl group optionally substituted with one or more halogen atoms, or R １ and R ２ together, or R ３ and R ４ together, or R ５ or R ６ together form a 5- or 6-membered cyclic or heterocyclic ring optionally substituted with one or more C １ ~C ６ alkyl groups or one or more -N(R ｘ )(R ｙ ), A is the counterion.) The kit according to claim 22, comprising a compound having any one of the following, wherein the concentration of the compound having any one of formulas (I) to (XII) is in the range of about 0.05% w / v to about 8% w / v based on the total volume of the dye composition.

24. R １ 、R ２ 、R ３ 、R ４ 、R ５ and R ６ at least two of which are -N(R ｘ )(R ｙ ), R １ 、R ２ 、R ３ 、R ４ 、R ５ and R ６ at least another two of which are branched or unbranched substituted or unsubstituted C １ ~C ６ alkyl group or cycloalkyl group, and at least one of R ｘ or R ｙ is H. The kit according to claim 23.

25. R １ , R ２ , R ３ , R ４ , R ５ and R ６ At least two of them are -N(R ｘ ) (Caution ｙ ) and R １ , R ２ , R ３ , R ４ , R ５ and R ６ At least two other of these are C １ ~C ２ It is an alkyl group, R ｘ or R ｙ The kit according to claim 23, wherein at least one of is H.

26. The compound having any one of formulas (I) to (XII) is 【Transformation 8】 A kit according to claim 23, selected from the group consisting of the following.

27. The kit according to any one of claims 23 to 26, wherein the compound having any one of formulas (I) to (XII) has a molecular weight in the range of about 300 g / mol to about 600 g / mol.

28. The kit according to any one of claims 23 to 26, wherein the compound having any one of formulas (I) to (XII) has a molecular weight in the range of about 300 g / mol to about 425 g / mol.

29. The kit according to any one of claims 23 to 26, wherein the concentration of the dye is in the range of about 0.15% w / v to about 2.5% w / v based on the total volume of the dye composition.

30. The kit according to any one of claims 23 to 26, wherein the concentration of the dye is in the range of about 0.15% w / v to about 1.8% w / v based on the total volume of the dye composition.

31. The sulfite composition is a sulfite or SO selected from the group consisting of metabisulfite, bisulfite, dithionite, thiosulfite, sulfite, and sulfite. ２ Including the source, the aforementioned sulfite or SO ２ The kit according to any one of claims 23 to 26, wherein the concentration of the source is in the range of about 0.1% w / v to about 18% w / v based on the total weight of the sulfite composition.

32. The kit according to claim 31, wherein the concentration of the sulfite source is in the range of about 1% w / v to about 9% w / v, based on the total weight of the sulfite composition.

33. The kit according to claim 31, wherein the concentration of the sulfite source is in the range of about 1% w / v to about 4% w / v, based on the total weight of the sulfite composition.

34. The kit according to any one of claims 23 to 26, wherein the acid is a strong acid.

35. The kit according to any one of claims 23 to 26, wherein the acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, trifluoroacetic acid, hydroiodic acid, sulfuric acid, nitric acid, chloric acid, and perchloric acid.

36. A method for staining a biological sample, comprising sequentially dispensing the dye composition and the sulfite composition of the kit described in claim 21 onto the biological sample.

37. The method according to claim 36, wherein the dye composition is dispensed before the sulfite composition.

38. The method according to claim 36, wherein the temperature of the sample, the dye composition, and / or the sulfite composition is in the range of 30°C to about 50°C.

39. The method according to claim 36, wherein the temperature of the sample, the dye composition, and / or the sulfite composition is in the range of 30°C to about 40°C.

40. The method according to claim 36, wherein the dye composition and the sulfite composition, which are dispensed sequentially, remain in contact with the sample for at least four minutes.

41. The method according to claim 36, wherein the dye composition and the sulfite composition, which are dispensed sequentially, remain in contact with the sample for at least eight minutes.

42. The method according to claim 36, wherein the amount of the dye composition dispensed into the sample is in the range of about 50 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 50 μL to about 200 μL.

43. The method according to claim 36, wherein the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 200 μL.

44. The method according to claim 36, wherein the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 150 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 150 μL.

45. A method for staining a biological sample, comprising simultaneously dispensing the dye composition and the sulfite composition of the kit described in claim 21 onto the biological sample.

46. The method according to claim 45, wherein the temperature of the sample, the dye composition, and / or the sulfite composition is in the range of 30°C to about 50°C.

47. The method according to claim 45, wherein the temperature of the sample, the dye composition, and / or the sulfite composition is in the range of 30°C to about 40°C.

48. The method according to claim 45, wherein the dye composition and the sulfite composition, dispensed simultaneously, remain in contact with the sample for at least four minutes.

49. The method according to claim 45, wherein the dye composition and the sulfite composition, dispensed simultaneously, remain in contact with the sample for at least eight minutes.

50. The method according to claim 45, wherein the amount of the dye composition dispensed into the sample is in the range of about 50 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 50 μL to about 200 μL.

51. The method according to claim 45, wherein the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 200 μL.

52. The method according to claim 45, wherein the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 150 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 150 μL.

53. A method for staining a biological sample, comprising sequentially dispensing the dye composition, the sulfite composition, and the acid of the kit described in any one of claims 23 to 26 onto the biological sample.

54. The method according to claim 53, wherein the dye composition is dispensed before the sulfite composition and / or the acid.

55. The method according to claim 53, wherein the temperature of the sample, the dye composition, the acid, and / or the sulfite composition is in the range of 30°C to about 50°C.

56. The method according to claim 53, wherein the temperature of the sample, the dye composition, the acid, and / or the sulfite composition is in the range of 30°C to about 40°C.

57. The method according to claim 53, wherein the dye composition, the acid, and the sulfite composition, which are dispensed sequentially, remain in contact with the sample for at least four minutes.

58. The method according to claim 53, wherein the dye composition, the acid, and the sulfite composition, which are dispensed sequentially, are kept in contact with the sample for at least eight minutes.

59. The method according to claim 53, wherein the amount of the dye composition dispensed to the sample is in the range of about 50 μL to about 200 μL, the amount of the acid dispensed to the sample is in the range of about 50 μL to about 200 μL, and the amount of the sulfite composition dispensed to the sample is in the range of about 50 μL to about 200 μL.

60. The method according to claim 53, wherein the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 200 μL, the amount of the acid dispensed into the sample is in the range of about 100 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 200 μL.

61. The method according to claim 53, wherein the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 150 μL, the amount of the acid dispensed into the sample is in the range of about 100 μL to about 150 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 150 μL.

62. A method for staining a biological sample, comprising simultaneously dispensing the dye composition, the sulfite composition, and the acid of the kit according to any one of claims 23 to 26 into the biological sample.

63. The method according to claim 62, wherein the temperature of the sample, the dye composition, the acid, and / or the sulfite composition is in the range of 30°C to about 50°C.

64. The method according to claim 62, wherein the temperature of the sample, the dye composition, the acid, and / or the sulfite composition is in the range of 30°C to about 40°C.

65. The method according to claim 62, wherein the dye composition, the acid, and the sulfite composition, which are dispensed simultaneously, remain in contact with the sample for at least four minutes.

66. The method according to claim 62, wherein the dye composition, the acid, and the sulfite composition, which are dispensed simultaneously, remain in contact with the sample for at least eight minutes.

67. The method according to claim 62, wherein the amount of the dye composition dispensed to the sample is in the range of about 50 μL to about 200 μL, the amount of the acid dispensed to the sample is in the range of about 50 μL to about 200 μL, and the amount of the sulfite composition dispensed to the sample is in the range of about 50 μL to about 200 μL.

68. The method according to claim 62, wherein the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 200 μL, the amount of the acid dispensed into the sample is in the range of about 100 μL to about 200 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 200 μL.

69. The method according to claim 62, wherein the amount of the dye composition dispensed into the sample is in the range of about 100 μL to about 150 μL, the amount of the acid dispensed into the sample is in the range of about 100 μL to about 150 μL, and the amount of the sulfite composition dispensed into the sample is in the range of about 100 μL to about 150 μL.

70. A stained biological sample, wherein (i) dispensing a dye composition into the biological sample, wherein the dye composition comprises (a) a compound having a 4-benzhydridene-2,5-cyclohexadiene-1-imine core functionalized to contain at least two substituted or unsubstituted amine groups, and having a molecular weight in the range of about 300 g / mol to about 600 g / mol, and (b) an acid, wherein the concentration of the compound is in the range of about 0.15% w / v to about 2.5% w / v based on the total volume of the dye composition, and (ii) sulfite or SO ２ Dispensing a sulfite composition containing the source, wherein the sulfite or SO ２ The source concentration is in the range of approximately 0.1% w / v to approximately 18% w / v, based on the total weight of the sulfite composition, and is a sulfite or SO ２ A stained biological sample prepared by dispensing a sulfite composition containing the source.

71. The stained biological sample according to claim 70, wherein the dye composition is first dispensed into the biological sample.

72. The stained biological sample according to claim 70, wherein the dye composition and the sulfite composition are dispensed simultaneously.

73. The stained biological sample according to claim 70, wherein the acid is a strong acid.

74. The aforementioned compound, 【Chemistry 9】 A stained biological sample according to claim 70, selected from the group consisting of the following.

75. The stained biological sample according to claim 74, wherein the acid is hydrochloric acid or nitric acid.

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