A color developing dye and preparation method and use thereof

Abstract

The invention relates to the field of biotechnology, and in particular to a color developing dye, a preparation method and uses thereof. The color developing dye provided by the invention comprises: 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogues or derivatives thereof. The invention finds through research that by combining 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogues or derivatives thereof, the color developing color of DAB dye can be changed from brown to yellow, thereby adding a new color option, and the sensitivity and stability of the color are not inferior to those of traditional DAB dyeing.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a color developing dye, a preparation method and application thereof. Background Art

[0002] DAB (3,3'-diaminobenzidine tetrahydrochloride) colorimetric reagent is a commonly used colorimetric reagent in the fields of research and detection such as immunology and immunohistochemistry, and is widely used in clinical and scientific research. The colorimetric principle is: peroxide undergoes a chemical reaction under the catalytic conditions of peroxidase, releasing oxidized oxygen, which then undergoes a redox reaction with the colorless hydrogen donor of the colorimetric reagent. The color-forming substance is oxidized to form a brown-yellow precipitate that is located at the site of peroxidase activity. After sufficient color development, the specimen is dehydrated and sealed in time, and the final product can be directly observed under a light microscope.

[0003] At present, the most commonly used color of DAB color developing reagent is still its traditional brown color, and the limited color options limit its use. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a color developing dye, a preparation method and a use thereof, wherein the color developing reagent realizes the color change of the DAB color developing reagent, so that the brown color of DAB changes to yellow, adding a new color option, and the sensitivity and stability of the color are not inferior to those of traditional DAB dyeing.

[0005] To this end, the present invention provides the following technical solutions:

[0006] A color-developing dye comprises: 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogue or derivative.

[0007] Optionally, naphthalene or its analogs or derivatives include:

[0008]

[0009] Optionally, the concentration range of 3,3'-diaminobenzidine tetrahydrochloride is 0.3-5 mg / mL, and the concentration range of naphthalene or its analogs or derivatives is 0.02-0.04 wt% by mass;

[0010] Optionally, also include DAB buffer.

[0011] A method for preparing a color developing dye comprises: preparing the dye using 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogs or derivatives.

[0012] Optional, including:

[0013] Prepare DAB buffer: the mass fraction of hydrogen peroxide is 0.02% to 0.04%, the molar concentration of imidazole is 30mM to 100mM, the molar concentration of surfactant is 30nM to 60nM, and the pH is 7.2 to 7.6;

[0014] Prepare DAB chromogen concentrate: dissolve 3,3'-diaminobenzidine tetrahydrochloride;

[0015] Preparation of concentrated solution of naphthalene or its analogues or its derivatives: dissolving naphthalene or its analogues or its derivatives;

[0016] The DAB chromogen concentrate and the naphthalene or its analogue or its derivative concentrate are diluted with DAB buffer to obtain a mixed solution in which the final concentration of 3,3'-diaminobenzidine tetrahydrochloride is 0.3-5 mg / mL and the mass fraction of naphthalene or its analogue or its derivative is 0.02-0.04 wt%.

[0017] Optionally, the surfactant is EDTA and / or Tween20;

[0018] Prepare DAB chromogen concentrate by dissolving 3,3'-diaminobenzidine tetrahydrochloride in dimethyl sulfoxide or Tris buffer; and / or

[0019] Preparation of concentrated solution of naphthalene or its analogues or its derivatives: dissolving naphthalene or its analogues or its derivatives in N,N-dimethylformamide.

[0020] The chromogenic dye is used in immunohistochemistry or immunoblotting staining.

[0021] Optionally, the immunohistochemical staining includes multiple immunohistochemical staining.

[0022] Optionally, the multiple immunohistochemical staining includes bright field immunohistochemical staining.

[0023] The technical solution of the present invention has the following advantages:

[0024] 1. The color-developing dye provided by the present invention comprises: 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogs or derivatives thereof; the present invention has found through research that the color-developing color of DAB dye can be changed from brown to yellow by combining 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogs or derivatives thereof, thereby adding a new color option, and the sensitivity and stability of the color are not inferior to those of traditional DAB dyeing;

[0025] Furthermore, by adjusting the color of the DAB dye from brown to yellow, it is possible to avoid or reduce the positive signal stained by the light-colored dye from being covered by the excessive color of the DAB dye. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is the effect of using the comparative example chromogenic dye and the example 3 chromogenic dye in the colon cancer panCK in the effect example of the present invention; in the figure, the left figure uses the comparative example chromogenic dye, and the right figure uses the example 3 chromogenic dye effect;

[0028] Figure 2 This is the effect of using the comparative example chromogenic dye and the embodiment 3 chromogenic dye for liver cancer PDL1 in the effect example of the present invention; in the figure, the left figure uses the comparative example chromogenic dye, and the right figure uses the embodiment 3 chromogenic dye effect;

[0029] Figure 3 This is the effect of using the comparative example chromogenic dye and the example 3 chromogenic dye in the gastric cancer panCK in the effect example of the present invention; in the figure, the left figure uses the comparative example chromogenic dye, and the right figure uses the example 3 chromogenic dye effect;

[0030] Figure 4 This is the effect of tonsil CD8 using the comparative example chromogenic dye and the chromogenic dye of Example 3 in the effect example of the present invention; in the figure, the left figure uses the comparative example chromogenic dye, and the right figure uses the chromogenic dye of Example 3;

[0031] Figure 5 It is the result of day 0 in the accelerated stability test in the effect example of the present invention;

[0032] Figure 6 It is the result of the 7th day in the accelerated stability test in the effect example of the present invention;

[0033] Figure 7 It is the result of the 14th day in the accelerated stability test in the effect example of the present invention;

[0034] Figure 8 It is the result of the working fluid stability at 0h in the effect example of the present invention;

[0035] Fig. 9 It is the result of the working fluid stability in the second hour in the effect example of the present invention;

[0036] Fig.10 This is the result of the working fluid stability at 4h in the effect example of the present invention;

[0037] Fig.11 This is the result of the working fluid stability at 8h in the effect example of the present invention;

[0038] Fig.12 This is the result of the stability of the working fluid in the effect example of the present invention at 24 hours. DETAILED DESCRIPTION

[0039] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0040] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0041] Example 1

[0042] This embodiment provides a color-developing dye, including: 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogues or derivatives, wherein the naphthalene or its analogues or derivatives are 2,3-dimethylnaphthalene, wherein the concentration of 3,3'-diaminobenzidine tetrahydrochloride is 0.3 mg / mL, and the concentration of 2,3-dimethylnaphthalene is 0.02 wt%.

[0043] Preparation method: Prepare DAB buffer: the mass fraction of hydrogen peroxide is 0.02%, the molar concentration of imidazole is 30mM, the molar concentration of EDTA is 30nM, and the pH is 7.2-7.6;

[0044] Prepare DAB chromogen concentrate: dissolve 3,3'-diaminobenzidine tetrahydrochloride in dimethyl sulfoxide (DMSO) (concentration: 6 mg / mL);

[0045] Preparation of 2,3-dimethylnaphthalene concentrated solution: dissolving 2,3-dimethylnaphthalene in N,N-dimethylformamide (DMF) (concentration of 2 wt%);

[0046] The DAB chromogen concentrate and the 2,3-dimethylnaphthalene concentrate were added to the DAB buffer for dilution, so that the final concentration of 3,3'-diaminobenzidine tetrahydrochloride in the obtained mixed solution was 0.3 mg / mL and the concentration of 2,3-dimethylnaphthalene was 0.02 wt%.

[0047] Example 2

[0048] This embodiment provides a color-developing dye, including: 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogues or derivatives, wherein the naphthalene or its analogues or derivatives are 2,6-dihydroxynaphthalene, the concentration of 3,3'-diaminobenzidine tetrahydrochloride is 5 mg / mL, and the concentration of 2,6-dihydroxynaphthalene is 0.04 wt%.

[0049] Preparation method: prepare DAB buffer: the mass fraction of hydrogen peroxide is 0.04%, the molar concentration of imidazole is 100mM, the molar concentration of EDTA is 60nM, and the pH is 7.2-7.6;

[0050] Prepare DAB chromogen concentrate: dissolve 3,3'-diaminobenzidine tetrahydrochloride in dimethyl sulfoxide (DMSO) (concentration: 100 mg / mL);

[0051] Preparation of 2,6-dihydroxynaphthalene concentrated solution: dissolving 2,6-dihydroxynaphthalene in N,N-dimethylformamide (DMF) (concentration of 4 wt%);

[0052] The DAB chromogen concentrate and the 2,6-dihydroxynaphthalene concentrate were added to the DAB buffer for dilution, so that the final concentration of 3,3'-diaminobenzidine tetrahydrochloride in the obtained mixed solution was 5 mg / mL and the concentration of 2,6-dihydroxynaphthalene was 0.04 wt%.

[0053] Example 3

[0054] This embodiment provides a color-developing dye, including: 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogues or derivatives, wherein the naphthalene or its analogues or derivatives are 2,6-dimethylnaphthalene, the concentration of 3,3'-diaminobenzidine tetrahydrochloride is 2.5 mg / mL, and the concentration of 2,6-dimethylnaphthalene is 0.03 wt%.

[0055] Preparation method: Prepare DAB buffer: the mass fraction of hydrogen peroxide is 0.03%, the molar concentration of imidazole is 60mM, the molar concentration of EDTA is 45nM, and the pH is 7.2-7.6;

[0056] Prepare DAB chromogen concentrate: dissolve 3,3'-diaminobenzidine tetrahydrochloride in dimethyl sulfoxide (DMSO) (concentration: 50 mg / mL);

[0057] Preparation of 2,6-dimethylnaphthalene concentrated solution: dissolving 2,6-dimethylnaphthalene in N,N-dimethylformamide (DMF) (concentration of 3 wt%);

[0058] The DAB chromogen concentrate and the 2,6-dimethylnaphthalene concentrate were added to the DAB buffer for dilution, so that the final concentration of 3,3'-diaminobenzidine tetrahydrochloride in the obtained mixed solution was 2.5 mg / mL and the concentration of 2,6-dimethylnaphthalene was 0.03 wt%.

[0059] Example 4

[0060] This embodiment provides a color-developing dye, including: 3,3'-diaminobenzidine tetrahydrochloride and naphthalene, the concentration of 3,3'-diaminobenzidine tetrahydrochloride is 3 mg / mL, and the concentration of naphthalene is 0.03 wt%.

[0061] Preparation method: Prepare DAB buffer: the mass fraction of hydrogen peroxide is 0.03%, the molar concentration of imidazole is 65mM, the molar concentration of EDTA is 45nM, and the pH is 7.2-7.6;

[0062] Prepare DAB chromogen concentrate: dissolve 3,3'-diaminobenzidine tetrahydrochloride in dimethyl sulfoxide (DMSO) (concentration: 60 mg / mL);

[0063] Preparation of naphthalene concentrate: dissolving naphthalene in N,N-dimethylformamide (DMF) (concentration of 3 wt%);

[0064] The DAB chromogen concentrate and the naphthalene concentrate were diluted with DAB buffer to obtain a mixed solution with a final concentration of 3,3'-diaminobenzidine tetrahydrochloride of 3 mg / mL and a concentration of naphthalene of 0.03 wt%.

[0065] Comparative Example

[0066] The difference between this comparative example and Example 3 is that 2,6-dimethylnaphthalene is not added to the prepared color-developing dye, and the final concentration of 3,3'-diaminobenzidine tetrahydrochloride in the final mixed solution is 2.5 mg / mL.

[0067] Effect example

[0068] 1. Dyeing was performed using the color-developing dye of Example 3 and the comparative example (DAB alone).

[0069] The staining method is:

[0070] 1. Dewaxing and hydration of paraffin sections: Take paraffin sections (colon cancer, liver cancer, gastric cancer, tonsil, etc.) and soak them in fresh xylene for 5 minutes, repeat 3 times; then soak the sections in graded ethanol, in order: 100% anhydrous ethanol for 5 minutes, 95% ethanol aqueous solution for 5 minutes, and 70% ethanol aqueous solution for 5 minutes; then wash the sections with sterile water for 1 minute, repeat 3 times;

[0071] 2. Antigen repair: Place the dewaxed and hydrated tissue sections in a repair cup, immerse them in 1× antigen repair solution (provided by Beno Panorama Biotechnology (Beijing) Co., Ltd.), place the repair cup in a microwave oven (microwave oven brand Midea, model M1-L213B), boil at high heat, and maintain at low heat for 10 min (pay attention to rehydration to prevent excessive evaporation and dry slices). Take out and cool naturally to room temperature, then soak in PBS for 1 min, then add 1% to 3% concentration of hydrogen peroxide for 10 min to remove endogenous peroxidase.

[0072] 3. Blocking: Take out the tissue sections, then wash them twice with PBS, remove the remaining washing solution, add blocking solution (provided by Benuo Panorama Biotechnology (Beijing) Co., Ltd.), and keep them moisturizing and shaking at room temperature for 10 minutes.

[0073] 3. Dyeing:

[0074] 3.1 Primary antibody incubation: Remove the blocking solution on the tissue sections, add diluted primary antibody solution (purchased from Cell signaling tecnology, catalog number panCK (CST4545), PDL1 (CST13684), CD8 (CST70306)) with a pipette, immerse the sample area, incubate at room temperature with moisturizing and shaking for 1 hour, wash the tissue sections with 1×TBST buffer for 2 minutes, and repeat 3 times.

[0075] 3.2HRP labeled secondary antibody incubation: remove the remaining washing solution on the tissue section, directly add HRP secondary antibody working solution, immerse the sample area, incubate at room temperature for 30 minutes, soak the tissue section in 1×TBST buffer for 2 minutes, and repeat 3 times.

[0076] 3.3 Color rendering

[0077] Use the color developing dye of the comparative example to develop color: incubate the tissue sections for 2-10 minutes (the staining time is determined by the color depth). After the color development is completed, rinse the tissue sections with purified water to terminate the reaction. Or

[0078] The color development dye of Example 3 was used for color development: the tissue sections were incubated for 2-10 minutes (the staining time was determined according to the color depth). After the color development was completed, the tissue sections were rinsed with purified water to terminate the reaction.

[0079] 3.4 Hematoxylin staining: Incubate with hematoxylin (purchased from Solebol) for 5-20 min (the staining time is determined by the color depth), incubate with differentiation solution (purchased from Solebol) for 5-10 s, and wash the slides with running water.

[0080] 3.5 Sealing: When the tissue sections are slightly dry, use a pipette to add water-soluble sealing agent on the glass slide and cover it with a glass slide.

[0081] 3.6 Image acquisition: The imaging device acquires the staining result image.

[0082] The results are as follows Figure 1-Figure 4 As shown in the figure, it can be seen that the solution of the present invention can change the color of the DAB dye from brown to yellow, adding a new color option, and the sensitivity of the color is not inferior to that of traditional DAB dyeing.

[0083] 2. Stability test

[0084] The coloring dye of Example 3 and the comparative example (DAB alone) were used for dyeing to detect stability. The stability detection method was as follows:

[0085] Stability: Two tests were conducted: accelerated stability and working fluid stability;

[0086] Accelerated stability: Reagent A (DAB chromogen concentrate, prepared in Example 3), reagent B (2,6-dihydroxynaphthalene concentrate, prepared in Example 3), and reagent C (DAB buffer, prepared in Example 3) were placed in a 37°C incubator, and the chromogenic dye prepared in accordance with Example 3 was used for staining test on days 0, 7, and 14 (according to the above staining method, tonsil CD8), and the staining intensity, expression pattern, and positive rate were compared with those on day 0 to see if there were significant differences. The results are as follows Figure 5-7 , and see the table below.

[0087] Table 1

[0088] Performance indicators / days Day 0 Day 7 Day 14 CD8 target staining intensity *** *** *** CD8 target expression pattern *** *** *** CD8 target positive rate *** *** ***

[0089] * indicates the level. The more * there are, the higher the level is and the better the corresponding effect is.

[0090] Working fluid stability:

[0091] After the staining working solution (Example 3) was prepared in proportion, it was placed in a 4°C refrigerator and staining tests were performed at 0h, 2h, 4h, 8h, and 24h (according to the above staining method, tonsil CD8) to compare whether there was a significant difference in staining intensity, expression pattern, and positive rate compared with the 0h. The results are as follows Figure 8-12 and the table below.

[0092] Table 2

[0093] Performance index / hour 0h 2h 4h 8h 24h CD8 target staining intensity *** *** *** *** *** CD8 target expression pattern *** *** *** *** *** CD8 target positive rate *** *** *** *** ***

[0094] * indicates the level. The more * there are, the higher the level is and the better the corresponding effect is.

[0095] From the above results, it can be concluded that the stability of the color-developing dye of the present invention is not inferior to that of the traditional DAB dyeing.

[0096] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A color-developing dye, characterized in that: include: 3,3'-Diaminobenzidine tetrahydrochloride and naphthalene or its analogues or derivatives; The naphthalene or its analogs or derivatives include:

2. The color-developing dye according to claim 1, characterized in that The concentration range of 3,3'-diaminobenzidine tetrahydrochloride is 0.3-5 mg / mL, and the concentration range of naphthalene or its analogue or derivative is 0.02-0.04 wt %.

3. The color-developing dye according to claim 2, characterized in that DAB buffer is also included.

4. The method for preparing the chromogenic dye according to any one of claims 1 to 3, characterized in that: include: The invention is prepared by using 3,3'-diaminobenzidine tetrahydrochloride and naphthalene or its analogue or derivative.

5. The method for preparing a chromogenic dye according to claim 4, characterized in that: include: Prepare DAB buffer: the mass fraction of hydrogen peroxide is 0.02% to 0.04%, the molar concentration of imidazole is 30mM to 100mM, the molar concentration of surfactant is 30nM to 60nM, and the pH is 7.2 to 7.6; Prepare DAB chromogen concentrate: dissolve 3,3'-diaminobenzidine tetrahydrochloride; Preparation of concentrated solution of naphthalene or its analogues or its derivatives: dissolving naphthalene or its analogues or its derivatives; The DAB chromogen concentrate and the naphthalene or its analogue or its derivative concentrate are diluted with DAB buffer to obtain a mixed solution in which the final concentration of 3,3'-diaminobenzidine tetrahydrochloride is 0.3-5 mg / mL and the mass fraction of naphthalene or its analogue or its derivative is 0.02-0.04 wt%.

6. The method for preparing a chromogenic dye according to claim 5, characterized in that: The surfactant is EDTA and / or Tween20; Prepare DAB chromogen concentrate by dissolving 3,3'-diaminobenzidine tetrahydrochloride in dimethyl sulfoxide or Tris buffer; and / or Preparation of concentrated solution of naphthalene or its analogues or its derivatives: dissolving naphthalene or its analogues or its derivatives in N,N-dimethylformamide.

7. Use of the chromogenic dye according to any one of claims 1 to 3 in immunohistochemistry or immunoblotting staining.

8. The use according to claim 7, characterized in that The immunohistochemical staining includes multiple immunohistochemical staining.

9. The use according to claim 8, characterized in that The multiplex immunohistochemical staining includes bright field immunohistochemical staining.

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