CN115683790A - Fluorescent staining agent, preparation method thereof and staining method - Google Patents
Fluorescent staining agent, preparation method thereof and staining method Download PDFInfo
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- CN115683790A CN115683790A CN202211376826.9A CN202211376826A CN115683790A CN 115683790 A CN115683790 A CN 115683790A CN 202211376826 A CN202211376826 A CN 202211376826A CN 115683790 A CN115683790 A CN 115683790A
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 136
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 238000007447 staining method Methods 0.000 title description 7
- 238000010186 staining Methods 0.000 claims abstract description 46
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 27
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 23
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- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 3
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- 238000004519 manufacturing process Methods 0.000 claims description 3
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Abstract
The invention relates to a fluorescent coloring agent, a preparation method thereof and a coloring method. The preparation method of the fluorescent staining agent comprises the following steps: mixing the agent A, the agent B, the cross-linking agent and the agent C to prepare a mixed solution; reacting the mixed solution at 30-60 ℃ for more than 4h to prepare a fluorescent coloring agent; the agent A comprises a nano material, the nano material comprises at least one of nano ferroferric oxide and nano silicon dioxide, the agent B comprises a fluorescent dye capable of being combined with nucleic acid, and the concentration of the nano material in the mixed solution is 0.05% (m/V) to 0.1% (m/V) and the concentration of the fluorescent dye in the mixed solution is 1% (m/V) to 50% (m/V) calculated by preparing 10 multiplied fluorescent staining agent; the pH value of the fluorescent staining agent is 7.4-7.6. The fluorescent staining agent has short staining time and good staining effect on vaginal secretion.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to a fluorescent staining agent and a preparation method and a staining method thereof.
Background
At present, the vaginal secretion detection method includes a wet sheet method, a gram staining method, a dry chemical enzyme method, a fluorescent staining method and the like. The wet sheet method and the gram staining method have high requirements on operators and require certain technical experience of the operators; the dry chemical enzyme method is easily interfered by various factors to cause high false positive; the fluorescent staining method can stain a plurality of components in vaginal secretion simultaneously to ensure that the forms of the components are easy to clearly identify, the staining time of a fluorescent staining reagent is about 1min generally, compared with other methods, the staining time of the fluorescent staining method is greatly improved, but the staining time still needs to be further improved for hospitals with large sample sizes.
Disclosure of Invention
Therefore, a fluorescent staining agent with short staining time and good staining effect on vaginal secretion and a preparation method thereof are needed.
In addition, the staining method has short staining time and good staining effect on vaginal secretion.
A method of preparing a fluorescent stain comprising the steps of:
mixing the agent A, the agent B, the cross-linking agent and the agent C to prepare a mixed solution; and
reacting the mixed solution at 30-60 ℃ for more than 4h to prepare a fluorescent coloring agent;
the agent A comprises a nano material, the nano material comprises at least one of nano ferroferric oxide and nano silicon dioxide, the agent B comprises a fluorescent dye capable of being combined with nucleic acid, the concentration of the nano material in the mixed solution is 0.05% (m/V) to 0.1% (m/V) and the concentration of the fluorescent dye in the mixed solution is 1% (m/V) to 50% (m/V) based on the preparation of 10 x fluorescent dye;
the agent C comprises 1% (m/V) to 20% (m/V) of glycerol, 0.1% (m/V) to 5% (m/V) of dimethyl sulfoxide and a buffer.
According to the preparation method of the fluorescent coloring agent, the cross-linking agent is adopted to enable the nano material and the fluorescent coloring agent to form a compound, so that the fluorescent coloring agent can enter cells more quickly under the driving of the nano material, the coloring time is shorter, and the coloring effect is good.
In one embodiment, the fluorescent stain has a pH of 7.4 to 7.6.
In one embodiment, when the reaction solution obtained by reacting the mixed solution at 30 to 60 ℃ for 4 hours or more is a concentrated solution of a fluorescent coloring agent, the method further includes a step of diluting the concentrated solution of the fluorescent coloring agent with the agent C to 1 × fluorescent coloring agent.
In one embodiment, the laser wavelength of the fluorescent dye is 460nm to 500nm, and the emission wavelength of the fluorescent dye is 500nm to 530nm.
In one embodiment, the fluorescent dye includes SYBRGreenI, SYBRGreenII, acridine orange, propidium iodide derivatives, hydroxyfluorescein diacetate succinate imidate derivatives, hoechst derivatives, 4, 6-diamidino-2-diphenylindole derivatives, and C 28 H 28 N 2 O 3 S 2 At least one of (1).
In one embodiment, the crosslinking agent comprises at least one of carbodiimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
In one embodiment, the buffer comprises one of phosphate buffer, tris buffer, and HEPES buffer.
In one embodiment, the agent C further comprises a preservative.
A fluorescent staining agent is prepared by the preparation method of the fluorescent staining agent.
A fluorescent stain comprising:
the agent A comprises a nano material, and the nano material comprises at least one of nano ferroferric oxide and nano silicon dioxide;
an agent B comprising a fluorescent dye capable of binding to a nucleic acid; and
an agent C comprising glycerol from 1% (m/V) to 20% (m/V), dimethyl sulfoxide from 0.1% (m/V) to 5% (m/V), and a buffer.
A method of dyeing comprising the steps of:
fixing the sample by using a fixing agent, and then drying; and
and dripping a staining agent on the dried sample for staining, wherein the raw material of the staining agent is the fluorescent staining agent prepared by the preparation method of the fluorescent staining agent.
In one embodiment, the fixing agent comprises at least one of methanol, ethanol, acetone, and acetaldehyde.
Drawings
FIG. 1 is the result of staining with the fluorescent stain of example 1;
FIG. 2 is the result of staining with the fluorescent stain of example 2;
FIG. 3 is the result of staining with the fluorescent stain of example 3;
FIG. 4 is the result of staining with the fluorescent stain of example 4;
FIG. 5 shows the result of staining with the fluorescent stain of example 5;
FIG. 6 shows the result of staining with the fluorescent stain of example 6.
Detailed Description
The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In the present specification, the term "10 × fluorescent staining reagent" refers to a 10-fold concentration of fluorescent staining reagent, and is used after being diluted 10-fold; "5 × fluorescent stain" means a fluorescent stain of 5 times concentration, and is used after being diluted 5 times; "1X fluorescent stain" means the concentration of the fluorescent stain used, as it is.
One embodiment of the present application provides a method for preparing a fluorescent coloring agent, which includes steps S10 and S20. Specifically, the method comprises the following steps:
step S10: the agent A, the agent B, the crosslinking agent and the agent C are mixed to prepare a mixed solution.
The agent A comprises a nano material, and the nano material comprises at least one of nano ferroferric oxide and nano silicon dioxide. The agent B includes a fluorescent dye capable of binding to the nucleic acid. The nanometer material is easy to be absorbed by cells, the nanometer material and the fluorescent dye capable of being combined with the nucleic acid form a compound after the action of the cross-linking agent, and the fluorescent dye can reach the cells more quickly with the assistance of the nanometer material, so that the fluorescent dye can be combined with the nucleic acid more quickly to realize dyeing. It is to be noted that the "nucleic acid" herein includes at least one of DNA and RNA.
In some embodiments, the nanomaterial has an average diameter of 1nm to 100nm. In an alternative specific example, the average diameter of the nanomaterial is 5nm, 10nm, 30nm, 50nm, or 80nm. Further, the average diameter of the nano material is 5 nm-70 nm. Further, the average diameter of the nano material is 5nm to 10nm.
In some embodiments, the nanomaterial has a particle size in a range from 1nm to 100nm. Further, the particle size range of the nano material is 5 nm-70 nm.
In an alternative specific example, the agent a is a solution of the nanomaterial.
In some embodiments, the fluorescent dye in the agent B has a laser wavelength of 460nm to 500nm and an emission wavelength of 500nm to 530nm.
In some embodiments, the fluorescent dye includes SYBR Green I, SYBR Green II, acridine orange, propidium iodide derivatives, hydroxyfluorescein diacetate succinate imide derivatives, hoechst derivatives, 4, 6-diamidino-2-diphenylindole, 4,6 diamidino-2-diphenylindole derivatives, and C 28 H 28 N 2 O 3 S 2 ((Z)-4-((3-methyl-benzo[d]thiamazol-2 (3H) -ylene) methyl) -1-propylquinolin-1-ium 4-methyllbenzenesulfonateAt least one of (a).
The cross-linking agent is used to complex the nanomaterial and the fluorescent dye. In some embodiments, the crosslinking agent comprises at least one of carbodiimide (EDC or EDAC), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
The agent C comprises 1% (m/V) to 20% (m/V) of glycerol, 0.1% (m/V) to 5% (m/V) of dimethyl sulfoxide and a buffer. In some embodiments, the buffer comprises one of a phosphate buffer, a Tris buffer, and a HEPES buffer. Further, the agent C comprises 1% (m/V) to 10% (m/V) of glycerol, 0.5% (m/V) to 3% (m/V) of dimethyl sulfoxide and a phosphate buffer. Further, the agent C comprises 1% (m/V) to 5% (m/V) of glycerol, 0.5% (m/V) to 2% (m/V) of dimethyl sulfoxide and 5mM to 50mM of a phosphate buffer.
In some embodiments, agent C further comprises a preservative. In an alternative specific example, the preservative is proclin 300. It is understood that in other embodiments, the preservative is not limited to proclin 300, but may be other substances.
Based on the preparation of 10 multiplied fluorescent staining agent, the concentration of the nano material in the mixed solution is 0.05 percent (m/V) to 0.1 percent (m/V), the concentration of the fluorescent dye in the mixed solution is 1 percent (m/V) to 50 percent (m/V), and the concentration of the cross-linking agent in the mixed solution is 0.5 percent to 50 percent. Furthermore, based on the preparation of 10 multiplied fluorescent dye, the concentration of the nano material in the mixed solution is 0.05 percent (m/V) to 0.09 percent (m/V), the concentration of the fluorescent dye in the mixed solution is 1 percent (m/V) to 15 percent (m/V), and the concentration of the cross-linking agent in the mixed solution is 1 percent to 15 percent. Furthermore, based on the preparation of 10 multiplied by fluorescent dye, the concentration of the nano material in the mixed solution is 0.06 percent (m/V) to 0.09 percent (m/V), the concentration of the fluorescent dye in the mixed solution is 5 percent (m/V) to 10 percent (m/V), and the concentration of the cross-linking agent in the mixed solution is 2 percent to 8 percent.
In some embodiments, the dye is present in a plurality, each fluorescent dye being present in the mixture at a concentration of from 1% (m/V) to 50% (m/V) based on the preparation of the 10 × fluorescent stain. Further, the dye is in a plurality of types, and the concentration of each fluorescent dye in the mixed solution is 1% (m/V) to 15% (m/V) based on the preparation of 10X fluorescent dye. Further, the dyes are plural, and each fluorescent dye is present in the mixed solution at a concentration of 5% (m/V) to 10% (m/V) in terms of preparation of 10X fluorescent dye.
In some embodiments, the step of preparing the mixed liquor comprises: mixing the agent A, the cross-linking agent and the agent C to prepare an AC mixed solution; and adding the agent B into the AC mixed solution to prepare a mixed solution. In other embodiments, the agent B and the crosslinking agent are mixed with the agent C to prepare a BC mixture; and adding the agent A to the BC mixed solution to prepare a mixed solution.
Step S20: and reacting the mixed solution at 30-60 ℃ for more than 4 hours to prepare the fluorescent staining agent.
In some embodiments, the reaction temperature in step S20 is 35 ℃ to 55 ℃ and the reaction time is 6h to 24h. In an alternative specific example, the time of the reaction in step S20 is 35 ℃, 40 ℃, 45 ℃ or 50 ℃; the reaction time in step S20 is 8h, 12h, 15h or 20h. Further, the reaction temperature in the step S20 is 45-55 ℃, and the reaction time is 10-15 h.
In some embodiments, after the mixed solution reacts under the water bath condition of 30-60 ℃, the method further comprises the step of adjusting the pH. The pH value of the fluorescent staining agent is 7.4-7.6.
It is understood that, in some embodiments, the reaction solution obtained by reacting the mixture at 30-60 ℃ for more than 4 hours is a concentrated solution of the fluorescent dye, i.e., the fluorescent dye obtained after the reaction is finished is a non-1 × fluorescent dye (e.g., 2 ×, 5 × or 10 ×). In this case, a reaction solution (i.e., a concentrated solution of a fluorescent dye) obtained by reacting the mixed solution at 30 to 60 ℃ for 4 hours or more may be used as the final product of the above-mentioned production method. So set up, can make the fluorescence stain of making convenient for store and transport. Of course, the method also includes the step of diluting the concentrated solution of the fluorescent staining reagent into 1 x fluorescent staining reagent by using the reagent C. It is understood that, in other embodiments, the reaction solution (i.e., the concentrated solution of the fluorescent dye) obtained by reacting the mixed solution at 30 to 60 ℃ for 4 hours or more may be diluted to obtain a 1 × fluorescent dye. By the arrangement, the water-soluble paint can be directly used without dilution when in use.
For example, in some embodiments, the 10 × fluorescent dye is prepared by weighing the A, B and cross-linking agents according to 10 × fluorescent dye, mixing with the C agent, and reacting at 30-60 deg.C for more than 4h. In use, the 10 × fluorescent stain is mixed with the agent C in a ratio of 1:9, to prepare a 1 × fluorescent dye for dyeing.
In addition, an embodiment of the present application further provides a fluorescent coloring agent, which is prepared by the preparation method of the fluorescent coloring agent according to any one of the above embodiments. The fluorescent dye can be used directly or after dilution without heating reaction.
In addition, another embodiment of the present application provides another fluorescent stain including: the agent A comprises a nano material, and the nano material comprises at least one of nano ferroferric oxide and nano silicon dioxide; the agent B comprises a fluorescent dye capable of binding to nucleic acid, and the agent C comprises 1% (m/V) to 20% (m/V) of glycerol, 0.1% (m/V) to 5% (m/V) of dimethyl sulfoxide and a buffer. More specifically, the compositions and amounts of the agent A, the agent B, the crosslinking agent and the agent C are as described above and will not be described herein again. When the fluorescent coloring agent is used, the agent A, the agent B, the cross-linking agent and the agent C are mixed to prepare a mixed solution, and then the mixed solution is reacted for more than 4 hours at the temperature of 30-60 ℃ to prepare the fluorescent coloring agent for use.
In some embodiments, agent a, agent B, cross-linking agent, and agent C are packaged separately. In other embodiments, the crosslinking agent is packaged in a single package and the agents A, B, and C are packaged together. In other embodiments, the cross-linking agent and the agent C are packaged separately, and the agent A and the agent B are packaged together after mixing. In other embodiments, the agent A, the agent B, the crosslinking agent, and the agent C are packaged together after mixing.
In addition, an embodiment of the present application also provides a dyeing method, which includes step S100 and step S200. Specifically, the method comprises the following steps:
step S100: after the sample is fixed with the fixative, it is dried.
After the cell is fixed, the pore diameter of the pores on the cell membrane is enlarged, and the complex formed by the nano material and the dye can enter the cell more easily. In some embodiments, the fixing agent comprises at least one of methanol, ethanol, acetone, and acetaldehyde. In an alternative embodiment, the fixing agent is anhydrous methanol.
In some embodiments, the sample is a vaginal secretion sample. The operation of fixing the sample with the fixing agent includes: and immersing the sample in the fixing agent for 1-30 min. Further, the fixed time is 2min to 15min.
Of course, before the sample is fixed by the fixative, the steps of smearing the sample and drying the sample are also included. Further, the air drying before the fixing agent treatment is natural air drying.
Step S200: and (4) dripping a coloring agent on the dried sample for dyeing.
Specifically, the raw material of the staining agent is the fluorescent staining agent prepared by the preparation method of the fluorescent staining agent of any embodiment. In some embodiments, the fluorescent stain prepared by the above method is a 1 × fluorescent stain. At this time, the fluorescent staining agent prepared by the above preparation method of the fluorescent staining agent is directly dropped on the dried sample for staining. In other embodiments, the fluorescent staining agent prepared by the above method is a concentrated solution of the fluorescent staining agent, and in this case, the fluorescent staining agent prepared by the above method is diluted with a C agent to be a 1 × fluorescent staining agent, and then the 1 × fluorescent staining agent is dropped on the dried sample for staining.
In some embodiments, the dyeing time is 10s to 30s. In an alternative embodiment, the time of staining is 10s, 15s, 20s, 25s or 30s.
It is understood that after the dyeing is finished, the method also comprises the step of washing the dyed sample by using water. Of course, after the stained sample is washed, the step of spin-drying or naturally drying the water on the sample is also included.
According to the dyeing method, the fluorescent dye prepared by the preparation method of the fluorescent dye in any embodiment is used as a raw material of the dye, so that the dyeing method is high in dyeing speed and good in dyeing effect.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except for inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures without specifying the specific conditions in the examples were carried out under the conventional conditions such as those described in the literature, in books, or as recommended by the manufacturer. "DMSO" herein refers to dimethyl sulfoxide; "mM" means mmol/L.
Example 1
Preparation of the agent C: according to the preparation amount of 1L, a certain amount of sodium dihydrogen phosphate, disodium hydrogen phosphate, glycerol, DMSO and proclin 300 are sequentially added into a 1L reactor, distilled water is added to a constant volume of 1L, and the mixture is fully stirred to completely dissolve all materials. The final solution (agent C) had the following concentrations of materials: PBS 50mM, glycerol 1% (m/V), DMSO 0.5% (m/V), proclin 300.5% (m/V). The pH was adjusted with 1.0M HCl to give a pH of 5.00 for component C. Storing at 4 deg.C for use.
2. Preparing a coloring agent:
(1) Diluting the agent A (the agent A is a nano silicon dioxide solution, the average diameter of the nano silicon dioxide is 7 nm) with the agent C, adding a crosslinking agent carbodiimide (EDAC) to enable the concentration of the nano silicon dioxide to be 0.08% (m/V) and the concentration of the EDAC to be 0.5% (m/V), and preparing the AC mixed solution. Then, acridine orange was added to the AC mixed solution to make the concentration of acridine orange 8% (m/V), followed by a water bath reaction at 50 ℃ for 12 hours, to obtain 10X fluorescent stain.
(2) Mixing the 10X fluorescent staining agent prepared in the step (1) with a C agent according to the ratio of 1:9 volume ratio, and the pH was adjusted to 7.5 with 0.1M hydrochloric acid to obtain the fluorescent dye of the present example.
Example 2
The preparation method of the fluorescent stain of the present example is substantially the same as that of example 1, except thatIn this embodiment, the raw material for preparing the fluorescent dye does not contain the nano-silica solution. The preparation method of the fluorescent staining agent of the embodiment comprises the following steps: diluting acridine orange with a C agent to obtain C 28 H 28 N 2 O 3 S 2 Was adjusted to pH 7.5 with 0.1M hydrochloric acid to give the fluorescent dye of the present example.
Example 3
Preparation of the agent C: same as in example 1.
2. Preparing a coloring agent:
(1) Diluting the agent A (the agent A is a nano ferroferric oxide solution, the average diameter of the nano ferroferric oxide is 7 nm) with the agent C, adding a crosslinking agent carbodiimide (EDAC), and enabling the concentration of the nano ferroferric oxide to be 0.08% (m/V) and the concentration of the EDAC to be 0.5% (m/V) to prepare the AC mixed solution. Then SYBRGreen I is added into the AC mixed solution to ensure that the concentration of SYBRGreen I is 1% (m/V), and then the mixed solution is subjected to water bath reaction at 50 ℃ for 12 hours to obtain a 10X fluorescent stain.
(2) Mixing the 10X fluorescent staining agent prepared in the step (1) with a C agent according to the ratio of 1:9 volume ratio, and the pH was adjusted to 7.5 with 0.1M hydrochloric acid to obtain the fluorescent dye of the present example.
Example 4
The preparation method of the fluorescent coloring agent in the present example is substantially the same as that in example 3, except that the raw materials for preparing the fluorescent coloring agent in the present example do not contain the nano ferroferric oxide solution. The preparation method of the fluorescent staining agent of the embodiment comprises the following steps: SYBRGreen I was diluted with C to a concentration of 0.1% (M/V) and adjusted to pH 7.5 with 0.1M hydrochloric acid to give the fluorescent stain of this example.
Example 5
Preparation of the agent C: same as in example 1.
2. Preparing a coloring agent: the difference from example 1 is that the amount of nanosilica used in the preparation of the fluorescent dye of this example is different from that of example 1. Specifically, the preparation steps of the staining agent of the embodiment include:
(1) Diluting the agent A (the agent A is a nano silicon dioxide solution, the average diameter of the nano silicon dioxide is 7 nm) with the agent C, adding crosslinking agent carbodiimide (EDAC) to ensure that the concentration of the nano silicon dioxide is 0.1 percent (m/V) and the concentration of the EDAC is 15 percent (m/V), and preparing the AC mixed solution. Then, acridine orange was added to the AC mixed solution to give a concentration of 50% (m/V) and then reacted in a water bath at 50 ℃ for 12 hours to give a 10X fluorescent stain.
(2) Mixing the 10X fluorescent staining agent prepared in the step (1) with a C agent according to the ratio of 1:9 volume ratio, and the pH was adjusted to 7.5 with 0.1M hydrochloric acid to obtain the fluorescent dye of the present example.
Example 6
Preparation of the agent C: same as in example 1.
2. Preparing a coloring agent: the fluorescent dye is substantially the same as in example 1, except that the amount of the nano silica used in the preparation process of the fluorescent dye in this example is different from that in example 1. Specifically, the preparation step of the staining agent of the embodiment includes:
(1) Diluting the agent A (the agent A is a nano silicon dioxide solution, the average diameter of the nano silicon dioxide is 7 nm) with the agent C, adding a crosslinking agent carbodiimide (EDAC), and enabling the concentration of the nano silicon dioxide to be 0.1% (m/V) and the concentration of the EDAC to be 5% (m/V) to prepare the AC mixed solution. Then, acridine orange was added to the AC mixed solution to give a concentration of 15% (m/V) and then reacted in a water bath at 50 ℃ for 12 hours to give a 10X fluorescent stain.
(2) Mixing the 10X fluorescent staining agent prepared in the step (1) with a C agent according to the ratio of 1:9 volume ratio, and the pH was adjusted to 7.5 with 0.1M hydrochloric acid to obtain the fluorescent dye of the present example.
Testing of
The vaginal discharge was stained with the fluorescent stains prepared in examples 1 to 6 to examine the staining effect of the fluorescent stains of the respective examples. The step of staining with the fluorescent staining agent of each example comprises:
(1) Preparing a vaginal secretion sample: the sample was smeared with a swab evenly into the central ring of the slide, with a thin coating.
(2) And (5) naturally airing.
(3) Immersing in anhydrous methanol for fixing for 2min.
(4) And (5) naturally airing.
(5) Dripping dyeing liquid according to different dyeing time for dyeing; the staining time of the fluorescent staining agent of example 1 was 10s, 20s and 30s; the staining times of the fluorescent stains of example 2 were 20s, 40s, and 60s; the staining times of the fluorescent stains of example 3 were 10s, 20s and 30s; the staining times of the fluorescent stains of example 4 were 20s, 40s, and 60s; the staining time of the fluorescent staining agent of example 5 was 10s, 20s and 30s; the staining times for the fluorescent stains of example 6 were 10s, 20s and 30s.
(6) Washing with water, and drying.
(7) Observing under a fluorescence microscope, taking pictures by using matched software, and making name records.
The dyeing results of the examples are shown in FIGS. 1 to 6, in which: FIGS. 1 to 6 are images taken under a condition of 40 times magnification by a fluorescence microscope; FIG. 1 is a result of staining with the fluorescent stain of example 1, FIG. 2 is a result of staining with the fluorescent stain of example 2, FIG. 3 is a result of staining with the fluorescent stain of example 3, FIG. 4 is a result of staining with the fluorescent stain of example 4, FIG. 5 is a result of staining with the fluorescent stain of example 5, and FIG. 6 is a result of staining with the fluorescent stain of example 6. It is to be noted that the vaginal secretions of example 1, example 2, example 5 and example 6 were from the same clinical specimen and the vaginal secretions of example 3 and example 4 were from another clinical specimen.
As is clear from FIGS. 1 and 3, when the staining was performed using the fluorescent staining agents of examples 1 and 3, high-quality images were obtained at a staining time of 20 seconds. After 20s of staining with the fluorescent stains of example 1 and example 3, the epithelial cells were yellow-green and the bacteria were red.
As can be seen from FIGS. 2 and 4, the fluorescent dyes of examples 2 and 4 were used to achieve a good dyeing effect for 40 to 60 seconds. After the fluorescent staining reagent of the embodiment 2 and the embodiment 4 is used for staining for 40 s-60 s, the epithelial cells are yellow green, and the bacteria are red. And compared with the fluorescent dyes of the embodiment 1 and the embodiment 3 added with the nano materials, the color of the sample dyed by the fluorescent dyes of the embodiment 2 and the embodiment 4 without the nano materials is darker; and the staining effect of the fluorescent stain of example 1 was better than that of the fluorescent stain of example 3.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments by those skilled in the art, are within the scope of the present invention as set forth in the appended claims. Therefore, the protection scope of the present patent shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.
Claims (10)
1. A method of preparing a fluorescent stain comprising the steps of:
mixing the agent A, the agent B, the cross-linking agent and the agent C to prepare a mixed solution; and
reacting the mixed solution at 30-60 ℃ for more than 4h to prepare a fluorescent coloring agent; wherein:
the agent A comprises a nano material, the nano material comprises at least one of nano ferroferric oxide and nano silicon dioxide, the agent B comprises a fluorescent dye capable of being combined with nucleic acid, and the concentration of the nano material in the mixed solution is 0.05% (m/V) to 0.1% (m/V) and the concentration of the fluorescent dye in the mixed solution is 1% (m/V) to 50% (m/V) based on the preparation of 10 multiplied fluorescent dye;
the agent C comprises 1% (m/V) to 20% (m/V) of glycerol, 0.1% (m/V) to 5% (m/V) of dimethyl sulfoxide and a buffer.
2. The method according to claim 1, wherein the fluorescent dye has a pH of 7.4 to 7.6.
3. The method according to claim 1, wherein the fluorescent dye has a laser wavelength of 460nm to 500nm and an emission wavelength of 500nm to 530nm.
4. The method of claim 1, wherein the fluorescent dye comprises SYBR Green I, SYBR Green II, acridine orange, propidium iodide derivatives, oxyfluorescein diacetate succinimide ester, oxyfluorescein diacetate succinate imide derivatives, hoechst derivatives, 4, 6-diamidino-2-diphenylindole derivatives, and C 28 H 28 N 2 O 3 S 2 At least one of (1).
5. The production method according to claim 1, wherein the crosslinking agent includes at least one of carbodiimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
6. The method of claim 1, wherein the buffer comprises one of a phosphate buffer, a Tris buffer, and a HEPES buffer.
7. The method according to any one of claims 1 to 6, wherein the agent C further comprises a preservative.
8. A fluorescent coloring agent obtained by the method for producing a fluorescent coloring agent according to any one of claims 1 to 7.
9. A fluorescent stain, comprising:
the agent A comprises a nano material, and the nano material comprises at least one of nano ferroferric oxide and nano silicon dioxide;
an agent B comprising a fluorescent dye capable of binding to a nucleic acid;
a crosslinking agent; and
an agent C comprising from 1% (m/V) to 20% (m/V) glycerol, from 0.1% (m/V) to 5% (m/V) dimethyl sulfoxide, and a buffer.
10. A dyeing method, characterized by comprising the steps of:
fixing the sample by using a fixing agent, and then drying; and
and (3) dripping a staining agent on the dried sample for staining, wherein the staining agent is prepared from the raw material of the fluorescent staining agent according to the preparation method of any one of claims 1-7.
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| CN111781177A (en) * | 2020-06-29 | 2020-10-16 | 安徽德莱康生物医疗科技有限公司 | Detection reagent and preparation method thereof |
| CN113049557A (en) * | 2021-03-12 | 2021-06-29 | 广州江元医疗科技有限公司 | Multiple fluorescent staining solution for genital secretion and preparation method and application thereof |
| CN114199653A (en) * | 2021-10-28 | 2022-03-18 | 济南德亨医学科技有限公司 | Microbial immunofluorescence staining solution for vagina |
| CN114813289A (en) * | 2022-04-20 | 2022-07-29 | 桂林优利特医疗电子有限公司 | Vaginal secretion fluorescent staining solution and staining method thereof |
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| US20080293584A1 (en) * | 2005-12-27 | 2008-11-27 | The Furukawa Electric Co., Ltd. | Fluorescent silica nano-particle, fluorescent nano-material, and biochip and assay using the same |
| CN110940646A (en) * | 2019-11-01 | 2020-03-31 | 江苏美克医学技术有限公司 | Double-fluorescence staining solution for vaginal microbial detection and application thereof |
| CN111781177A (en) * | 2020-06-29 | 2020-10-16 | 安徽德莱康生物医疗科技有限公司 | Detection reagent and preparation method thereof |
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