A fluorescent dye solution

Abstract

The present invention relates to the technical field of medical test supplies, and in particular to a fluorescent staining solution, which comprises the following components by mass: 1 to 5 parts of acridine orange, 1 to 3 parts of fluorescence enhancer, 0.3 to 1 part of digestion agent, 0.5 to 2 parts of digestion enhancer, 0.15 to 7 parts of buffer, 5 to 10 parts of permeation agent, 2.3 to 4 parts of osmotic pressure stabilizer, 5 to 10 parts of moisturizing agent, 0.1 to 1 part of ascorbic acid and 57 to 84.65 parts of deionized water. The fluorescent staining solution of the present invention can effectively digest and promote cell dispersion and tiling, is conducive to observing the interphase cell nuclear morphology, and can analyze chromosomes in metaphase.

Description

Technical Field

[0001] The invention relates to the technical field of medical test supplies, and in particular to a fluorescent dyeing liquid. Background Art

[0002] Vaginal secretion analysis is a routine item in gynecological examination. It has important clinical significance for the diagnosis of diseases such as inflammation and tumors of the female reproductive system, and is an important basis for the clinical diagnosis of vaginal diseases.

[0003] Fluorescent staining of vaginal secretions is a new method that overcomes many shortcomings of the traditional saline smear method and is gradually being widely used in laboratories. Its advantages are mainly reflected in the clear composition after staining, and different components present different colors for easy identification and distinction.

[0004] However, some fluorescent staining solutions on the market are not conducive to the dispersion and flattening of cell clusters, which affects the observation and analysis of cell nuclear morphology and makes it difficult to observe clear chromosome fluorescent bands. Summary of the invention

[0005] The purpose of the present invention is to solve the above-mentioned shortcomings. The present invention provides a fluorescent staining solution that can effectively digest and promote cell dispersion and tiling, is conducive to observing the interphase cell nuclear morphology, and can analyze chromosomes in metaphase.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A fluorescent dyeing liquid comprises the following components in parts by weight: 1 to 5 parts of acridine orange, 1 to 3 parts of a fluorescence enhancer, 0.3 to 1 part of a digestive agent, 0.5 to 2 parts of a digestive enhancer, 0.15 to 7 parts of a buffer, 5 to 10 parts of a penetrant, 2.3 to 4 parts of an osmotic pressure stabilizer, 5 to 10 parts of a humectant, 0.1 to 1 part of ascorbic acid and 57 to 84.65 parts of deionized water.

[0008] Preferably, the fluorescence enhancer is polyethylene glycol 1000, the digestive agent is protease, and the digestion enhancer is EDTA-2Na.

[0009] Preferably, the buffer is selected from at least one of Na2HPO4 and KH2PO4.

[0010] Preferably, the penetrant is Tween 20 and the humectant is glycerol.

[0011] Preferably, the osmotic pressure stabilizer is selected from at least one of NaCl and KCl.

[0012] Preferably, the method for preparing the fluorescent dye solution comprises the following steps:

[0013] S1: Weigh each component according to weight ratio;

[0014] S2: dissolving the moisturizer, osmotic agent, digestive agent, digestive enhancer, and osmotic pressure stabilizer in deionized water to form aqueous solution A, and cooling the solution to room temperature naturally for standby use;

[0015] S3: dissolving acridine orange in deionized water to form aqueous solution B for standby use;

[0016] S4: mixing the aqueous solution A in step S2 with the aqueous solution B in step S3 to form an aqueous solution C for standby use;

[0017] S5: mixing the buffer, ascorbic acid and fluorescence enhancer to form solution D for standby use;

[0018] S6: adding the solution D in step S5 dropwise to the aqueous solution C in step S4, stirring while adding dropwise, mixing and dissolving, cooling naturally and filtering to obtain the fluorescent dye solution.

[0019] Preferably, the filtration in step S6 is positive pressure filtration or negative pressure filtration.

[0020] Preferably, a cellulose membrane is used for the filtration, and the pore size of the cellulose membrane is 0.22 to 0.5 micrometers.

[0021] The present invention has the following beneficial effects:

[0022] (1) The different components are compounded to enhance each other's effects, thereby achieving a better fluorescent staining effect; among them, the interaction between the fluorescein acridine orange, the digestive agent protease and the fluorescence enhancer polyethylene glycol 1000 can enhance the fluorescence intensity of the sample being tested, making the test results more accurate; the interaction between the osmotic agent Tween 20, the osmotic pressure stabilizers NaCL and KCl, and the buffers Na2HPO4 and KH2PO4 can make the staining solution more stable; the digestive enhancer EDTA-2Na can complex metal cations and reduce the effect of metal cations on proteases, so that the fluorescently labeled proteins can be better detected and observed; at the same time, ascorbic acid can effectively prevent the oxidation of the slices, thereby making the effect of the entire formula more excellent.

[0023] (2) The preparation method of the fluorescent dyeing liquid of the present invention is simple to operate and can be prepared and used at any time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a fluorescence microscope image of epithelial cells stained with the fluorescent staining solution of Example 1;

[0025] Figure 2 is a fluorescence microscope image of epithelial cells stained with the fluorescent staining solution of Example 2;

[0026] Figure 3is a fluorescence microscope image of epithelial cells stained with the fluorescent staining solution of Comparative Example 1;

[0027] Figure 4 is a fluorescence microscope image of epithelial cells stained with the fluorescent staining solution of Comparative Example 2;

[0028] Figure 5 This is a fluorescence microscope image of the chromosome after being stained with the fluorescent staining solution of Example 1. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments.

[0030] Example 1

[0031] The present embodiment provides a fluorescent staining solution, which is composed of the following components: 2 parts of acridine orange, 2 parts of fluorescence enhancer, 0.6 parts of digestion agent, 1 part of digestion enhancer, 1.8 parts of buffer, 6 parts of permeabilizer, 2.9 parts of osmotic pressure stabilizer, 6 parts of humectant, 0.5 parts of ascorbic acid and 77.2 parts of deionized water.

[0032] Among them, the fluorescence enhancer is polyethylene glycol 1000, the digestive agent is protease, the digestion enhancer is EDTA-2Na, the buffer is composed of 1 part Na2HPO4 and 0.8 part KH2PO4, the osmotic agent is Tween 20, the humectant is glycerol, and the osmotic pressure stabilizer is composed of 0.9 parts NaCl and 2 parts KCl.

[0033] The preparation method comprises the following steps:

[0034] S1: Weigh each component according to the above ratio;

[0035] S2: dissolving the moisturizer, osmotic agent, digestive agent, digestive enhancer, and osmotic pressure stabilizer in deionized water to form aqueous solution A, and cooling the solution to room temperature naturally for standby use;

[0036] S3: dissolving acridine orange in deionized water to form aqueous solution B for standby use;

[0037] S4: mixing the aqueous solution A in step S2 with the aqueous solution B in step S3 to form aqueous solution C for standby use;

[0038] S5: mixing the buffer, ascorbic acid and fluorescence enhancer to form solution D for standby use;

[0039] S6: Add solution D in step S5 dropwise into aqueous solution C in step S4, stirring while adding, mixing and dissolving, cooling naturally, and filtering by positive pressure using a cellulose membrane with a pore size of 0.22 to 0.5 micrometers to obtain a fluorescent dye solution.

[0040] Example 2

[0041] The present embodiment provides a fluorescent staining solution, which is composed of the following components: 1 part of acridine orange, 1 part of fluorescence enhancer, 0.3 parts of digestion agent, 0.5 parts of digestion enhancer, 0.15 parts of buffer, 5 parts of permeabilizer, 2.3 parts of osmotic pressure stabilizer, 5 parts of humectant, 0.1 parts of ascorbic acid and 84.65 parts of deionized water.

[0042] Among them, the fluorescence enhancer is polyethylene glycol 1000, the digestive agent is protease, the digestion enhancer is EDTA-2Na, the buffer is composed of 0.05 parts of Na2HPO4 and 0.1 parts of KH2PO4, the osmotic agent is Tween 20, the humectant is glycerol, and the osmotic pressure stabilizer is composed of 0.8 parts of NaCl and 1.5 parts of KCl.

[0043] The preparation method comprises the following steps:

[0044] S1: Weigh each component according to the above ratio;

[0045] S2: dissolving the moisturizer, osmotic agent, digestive agent, digestive enhancer, and osmotic pressure stabilizer in deionized water to form aqueous solution A, and cooling the solution to room temperature naturally for standby use;

[0046] S3: dissolving acridine orange in deionized water to form aqueous solution B for standby use;

[0047] S4: mixing the aqueous solution A in step S2 with the aqueous solution B in step S3 to form an aqueous solution C for standby use;

[0048] S5: mixing the buffer, ascorbic acid and fluorescence enhancer to form solution D for standby use;

[0049] S6: Add solution D in step S5 dropwise into aqueous solution C in step S4, stirring while adding dropwise, mixing and dissolving, cooling naturally, and filtering under negative pressure using a cellulose membrane with a pore size of 0.22 to 0.5 micrometers to obtain a fluorescent dye solution.

[0050] Example 3

[0051] The present embodiment provides a fluorescent staining solution, which is composed of the following components: 5 parts of acridine orange, 3 parts of fluorescence enhancer, 1 part of digestion agent, 2 parts of digestion enhancer, 7 parts of buffer, 10 parts of permeabilizer, 4 parts of osmotic pressure stabilizer, 10 parts of humectant, 1 part of ascorbic acid and 57 parts of deionized water.

[0052] Among them, the fluorescence enhancer is polyethylene glycol 1000, the digestive agent is protease, the digestion enhancer is EDTA-2Na, the buffer is composed of 5 parts of Na2HPO4 and 2 parts of KH2PO4, the osmotic agent is Tween 20, the humectant is glycerol, and the osmotic pressure stabilizer is composed of 1 part of NaCl and 2 parts of KCl.

[0053] The preparation method comprises the following steps:

[0054] S1: Weigh each component according to the above ratio;

[0055] S2: dissolving the moisturizer, osmotic agent, digestive agent, digestive enhancer, and osmotic pressure stabilizer in deionized water to form aqueous solution A, and cooling the solution to room temperature naturally for standby use;

[0056] S3: dissolving acridine orange in deionized water to form aqueous solution B for standby use;

[0057] S4: mixing the aqueous solution A in step S2 with the aqueous solution B in step S3 to form aqueous solution C for standby use;

[0058] S5: mixing the buffer, ascorbic acid and fluorescence enhancer to form solution D for standby use;

[0059] S6: Add solution D in step S5 dropwise into aqueous solution C in step S4, stirring while adding dropwise, mixing and dissolving, cooling naturally, and filtering under negative pressure using a cellulose membrane with a pore size of 0.22 to 0.5 micrometers to obtain a fluorescent dye solution.

[0060] Comparative Example 1

[0061] Compared with Example 1, the difference is that an equal amount of deionized water is used to replace the digestion agent, and the other components and contents as well as the preparation method are consistent with those in Example 1.

[0062] Comparative Example 2

[0063] Compared with Example 1, the difference is that an equal amount of deionized water is used to replace the fluorescence enhancer, and the other components, contents and preparation method are consistent with those in Example 1.

[0064] The distribution ratios of the above embodiments and comparative examples are shown in Table 1 below:

[0065] Table 1: Composition ratio of each embodiment and comparative example (part)

[0066]

[0067]

[0068] Experimental testing

[0069] Vaginal secretions were collected, and cotton swabs soaked in physiological saline were used to evenly smear the vaginal secretions on a glass slide, and the secretions were dried by baking at 80±5°C for 1-3 minutes using a constant temperature baking machine to fix the vaginal secretions on the glass slide; 15 μl of the fluorescent staining solution prepared above was added dropwise to the vaginal secretions on the glass slide, and the slide was covered with a cover glass and observed under a fluorescence microscope. The results were as follows: Figure 1 to Figure 5 (In order to facilitate the observation of the staining effect and cell morphology, color drawings are provided here).

[0070] Observe under a fluorescence microscope. Figure 1-2 As shown, the cytoplasm of the epithelial cells in Examples 1 to 2 is light green, the nucleus is bright green, the fluorescence is strong and stable, the tiling effect is good, and each cell is fully presented; Figure 3 As shown, in Comparative Example 1, since no digestive agent was added, the epithelial cells were clumped and almost impossible to observe; Figure 4 As shown, in Comparative Example 2, since no fluorescence enhancer was added, the fluorescence was dim and almost no cell morphology could be observed; Figure 5 As shown, the observed chromosome bands are clear and the staining is obvious.

[0071] It can be seen that the scheme of the present invention enhances the effects of each other by compounding different components, so that the fluorescent staining effect is better; among them, the fluorescein acridine orange, the digestive agent protease and the fluorescence enhancer polyethylene glycol 1000 interact with each other, which can enhance the fluorescence intensity of the sample being tested and make the detection result more accurate; the osmotic agent Tween 20, the osmotic pressure stabilizers NaCL and KCl, and the buffers Na2HPO4 and KH2PO4 interact with each other, which can make the staining solution more stable; the digestive enhancer EDTA-2Na can complex metal cations, reduce the influence of metal cations on proteases, and enable the fluorescently labeled proteins to be better detected and observed; at the same time, ascorbic acid can effectively prevent the oxidation of the slices, thereby making the effect of the entire formula more excellent.

[0072] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A fluorescent dyeing solution, characterized in that: The invention comprises the following components in parts by weight: 1 to 5 parts of acridine orange, 1 to 3 parts of fluorescence enhancer, 0.3 to 1 parts of digestion agent, 0.5 to 2 parts of digestion enhancer, 0.15 to 7 parts of buffer, 5 to 10 parts of permeation agent, 2.3 to 4 parts of osmotic pressure stabilizer, 5 to 10 parts of humectant, 0.1 to 1 part of ascorbic acid and 57 to 84.65 parts of deionized water; The fluorescence enhancer is polyethylene glycol 1000, the digestive agent is protease, and the digestive enhancer is EDTA-2Na; the buffer is composed of Na2HPO4 and KH2PO4; the osmotic agent is Tween 20, and the moisturizing agent is glycerol; and the osmotic pressure stabilizer is composed of NaCl and KCl.

2. The fluorescent dyeing liquid according to claim 1, characterized in that: The preparation method of the fluorescent dye solution comprises the following steps: S1: Weigh each component according to weight ratio; S2: dissolving the moisturizer, osmotic agent, digestive agent, digestive enhancer, and osmotic pressure stabilizer in deionized water to form aqueous solution A, and cooling the solution to room temperature naturally for standby use; S3: dissolving acridine orange in deionized water to form aqueous solution B for standby use; S4: mixing the aqueous solution A in step S2 with the aqueous solution B in step S3 to form an aqueous solution C for standby use; S5: mixing the buffer, ascorbic acid and fluorescence enhancer to form solution D for standby use; S6: adding the solution D in step S5 dropwise to the aqueous solution C in step S4, stirring while adding dropwise, mixing and dissolving, cooling naturally and filtering to obtain the fluorescent dye solution.

3. The fluorescent dyeing liquid according to claim 2, characterized in that: The filtration in step S6 is positive pressure filtration or negative pressure filtration.

4. The fluorescent dyeing liquid according to claim 3, characterized in that: The filtration is performed using a cellulose membrane, the pore size of the cellulose membrane being 0.22 to 0.5 microns.

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