CN115683790B - Fluorescent dye, preparation method thereof and dyeing method - Google Patents
Fluorescent dye, preparation method thereof and dyeing method Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 153
- 238000004043 dyeing Methods 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 109
- 239000011259 mixed solution Substances 0.000 claims abstract description 41
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000002086 nanomaterial Substances 0.000 claims abstract description 30
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 27
- 239000003086 colorant Substances 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 11
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 11
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 11
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000005543 nano-size silicon particle Substances 0.000 claims abstract description 5
- 235000012239 silicon dioxide Nutrition 0.000 claims abstract description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 29
- 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 24
- 239000000243 solution Substances 0.000 claims description 21
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 claims description 11
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims description 11
- 239000000377 silicon dioxide Substances 0.000 claims description 11
- -1 SYBR GreenII Chemical compound 0.000 claims description 8
- 150000001718 carbodiimides Chemical class 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 7
- 238000004378 air conditioning Methods 0.000 claims description 6
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 239000003755 preservative agent Substances 0.000 claims description 5
- 230000002335 preservative effect Effects 0.000 claims description 5
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical class [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 claims description 5
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 4
- 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 4
- 239000007995 HEPES buffer Substances 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 claims description 2
- 230000005284 excitation Effects 0.000 claims 1
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- 210000003756 cervix mucus Anatomy 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 8
- 238000010186 staining Methods 0.000 description 26
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 235000011187 glycerol Nutrition 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000000975 dye Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 239000000463 material Substances 0.000 description 5
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- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
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- 239000000834 fixative Substances 0.000 description 4
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a fluorescent dye, a preparation method thereof and a dyeing method. The preparation method of the fluorescent dye 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 for more than 4 hours at the temperature of 30-60 ℃ 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 a 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 a 10X fluorescent dye; the pH of the fluorescent dye is 7.4-7.6. The fluorescent dye has short dyeing time and good dyeing effect on vaginal secretion.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to a fluorescent dye, a preparation method thereof and a dyeing method.
Background
Currently, vaginal secretion is detected by wet-patch method, gram staining method, dry chemical enzyme method, fluorescent staining method, and the like. Wet-sheet and gram-dyeing methods have high requirements for operators and require certain technical experience of operators; the dry chemical enzyme method is easy to be interfered by various factors, so that the false positive is high; the fluorescent staining method can simultaneously stain various components in vaginal secretion to ensure that the morphology of the vaginal secretion is easier to clearly recognize, and the staining time of a fluorescent staining reagent is generally about 1 min.
Disclosure of Invention
Based on this, it is necessary to provide a fluorescent dye which has a short dyeing time and good dyeing effect on vaginal secretions, and a preparation method thereof.
In addition, a dyeing method which has short dyeing time and good dyeing effect on vaginal secretion is also provided.
A preparation method of a fluorescent dye comprises the following steps:
mixing the agent A, the agent B, the cross-linking agent and the agent C to prepare a mixed solution; a kind of electronic device with high-pressure air-conditioning system
Reacting the mixed solution for more than 4 hours at the temperature of 30-60 ℃ to prepare a fluorescent coloring agent;
the agent A comprises a nanomaterial comprising 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 nanomaterial 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) in terms of preparing a 10X 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 solution.
According to the preparation method of the fluorescent dye, the cross-linking agent is adopted to form a composite of the nano material and the fluorescent dye, so that the fluorescent dye can enter cells more rapidly under the drive of the nano material, the dyeing time is shorter, and the dyeing 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 more than 4 hours is a concentrated solution of a fluorescent dye, the method further comprises the step of diluting the concentrated solution of the fluorescent dye into 1 x fluorescent dye by using the agent C.
In one embodiment, the laser wavelength of the fluorescent dye is 460 nm-500 nm, and the emission wavelength of the fluorescent dye is 500-530 nm.
In one embodiment, the fluorescent dye comprises SYBRGreenI, SYBRGreenII, acridine orange, propidium iodide, a propidium iodide derivative, hydroxyfluorescein diacetate succinimidyl ester, a hydroxyfluorescein diacetate succinimidyl ester derivative, hoechst, hoechst derivative, 4, 6-diamidino-2-diphenylindole, 4,6-Diamidino-2-diphenylindole derivatives and C 28 H 28 N 2 O 3 S 2 At least one of them.
In one embodiment, the crosslinking agent includes 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 dye is prepared by the preparation method of the fluorescent dye.
A fluorescent stain comprising:
a reagent a comprising a nanomaterial comprising at least one of nano-ferroferric oxide and nano-silica;
an agent B comprising a fluorescent dye capable of binding to nucleic acid; a kind of electronic device with high-pressure air-conditioning system
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 solution.
A dyeing method comprising the steps of:
fixing the sample by using a fixing agent, and airing; a kind of electronic device with high-pressure air-conditioning system
And (3) dropwise adding a coloring agent to the dried sample for dyeing, wherein the raw material of the coloring agent is the fluorescent coloring agent prepared by the preparation method of the fluorescent coloring agent.
In one embodiment, the fixative includes at least one of methanol, ethanol, acetone, and acetaldehyde.
Drawings
FIG. 1 is a graph showing the result of staining with the fluorescent stain of example 1;
FIG. 2 is a graph showing the result of staining with the fluorescent stain of example 2;
FIG. 3 is a graph showing the result of staining with the fluorescent stain of example 3;
FIG. 4 is a graph showing the result of staining with the fluorescent stain of example 4;
FIG. 5 is a graph showing the result of staining with the fluorescent stain of example 5;
FIG. 6 shows the result of the fluorescent dye of example 6.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the invention, which may be embodied in many different forms and is not limited to the embodiments described 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 herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The "10 x fluorescent dye" herein means a 10-fold concentration of fluorescent dye, and is used after 10-fold dilution; "5 Xfluorescent stain" refers to a 5-fold concentration of fluorescent stain that is used after 5-fold dilution; "1X fluorescent stain" refers to the concentration of fluorescent stain used, directly.
An embodiment of the present application provides a method for preparing a fluorescent dye, where the method includes step S10 and step S20. Specifically:
step S10: and mixing the agent A, the agent B, the crosslinking agent and the agent C 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. Agent B includes a fluorescent dye capable of binding to nucleic acid. The nanometer material is easy to be absorbed by cells, and a compound is formed after the nanometer material and fluorescent dye capable of being combined with nucleic acid are acted by a cross-linking agent, and the fluorescent dye can reach the inside of the cells more quickly with the help 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 "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 nanomaterial is 5nm to 70nm. Further, the average diameter of the nanomaterial is 5nm to 10nm.
In some embodiments, the nanomaterial has a particle size in the range of 1nm to 100nm. Further, the particle size of the nanomaterial is in the range of 5nm to 70nm.
In an alternative specific example, agent a is a solution of nanomaterial.
In some embodiments, the laser wavelength of the fluorescent dye in agent B is 460nm to 500nm and the emission wavelength of the fluorescent dye is 500 to 530nm.
In some embodiments, the fluorescent dye comprises SYBR Green I, SYBR Green II, acridine orange, propidium iodide derivatives, hydroxyfluorescein diacetate succinimidyl ester derivatives, hoechst, hoechst derivatives, 4, 6-diamidino-2-diphenylindole derivatives, and C 28 H 28 N 2 O 3 S 2 ((Z)-4-((3-methyl-benzo[d]thiazol-2 (3H) -ylethylene) -1-propylquinone-1-ium 4-methyl-sulfonyl).
The cross-linking agent is used to form a complex of the nanomaterial and the fluorescent dye. In some embodiments, the crosslinking agent includes 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 solution. In some embodiments, the buffer comprises one of phosphate buffer, tris buffer, and 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 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 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.
The concentration of the nano material in the mixed solution is 0.05-0.1% (m/V), the concentration of the fluorescent dye in the mixed solution is 1-50% (m/V), and the concentration of the cross-linking agent in the mixed solution is 0.5-50%, calculated by preparing 10X fluorescent dye. Further, the concentration of the nano material in the mixed solution is 0.05% (m/V) to 0.09% (m/V), the concentration of the fluorescent dye in the mixed solution is 1% (m/V) to 15% (m/V), and the concentration of the cross-linking agent in the mixed solution is 1% to 15%, based on the preparation of the 10X fluorescent dye. Further, in order to prepare the 10X fluorescent dye, the concentration of the nano material in the mixed solution is 0.06% (m/V) to 0.09% (m/V), the concentration of the fluorescent dye in the mixed solution is 5% (m/V) to 10% (m/V), and the concentration of the cross-linking agent in the mixed solution is 2% -8%.
In some embodiments, the dye is present in a plurality of types, each fluorescent dye being present in the mixture at a concentration of 1% (m/V) to 50% (m/V) based on the preparation of 10 x fluorescent stain. Further, the number of dyes is varied, and the concentration of each fluorescent dye in the mixed solution is 1% (m/V) to 15% (m/V) in terms of preparing 10X fluorescent dye. Further, the number of the dyes is varied, and the concentration of each fluorescent dye in the mixed solution is 5% (m/V) to 10% (m/V) in terms of preparing 10X fluorescent dye.
In some embodiments, the step of preparing the mixed liquor comprises: mixing the agent A and the cross-linking agent with 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 cross-linking agent are mixed with the agent C to prepare a BC blend; and adding the agent A into the BC mixed solution to prepare the mixed solution.
Step S20: and (3) reacting the mixed solution for more than 4 hours at the temperature of 30-60 ℃ to prepare the fluorescent coloring agent.
In some embodiments, the reaction temperature in step S20 is 35℃to 55℃and the reaction time is 6 hours to 24 hours. 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, the step of adjusting the pH is further included after the reaction of the mixed liquor is completed under water bath conditions of 30-60 ℃. The pH of the fluorescent dye is 7.4-7.6.
It will be appreciated that in some embodiments, the reaction mixture is reacted at 30℃to 60℃for more than 4 hours to obtain a reaction mixture which is a concentrated solution of fluorescent dye, i.e., the fluorescent dye obtained at the end of the reaction is a non-1X fluorescent dye (e.g., 2X, 5X, or 10X). In this case, 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 used as the final product of the above-mentioned production method. By the arrangement, the prepared fluorescent dye is convenient to store and transport. Of course, the method further comprises the step of diluting the concentrated solution of the fluorescent dye into 1X fluorescent dye by using the agent C. It will be appreciated that in other embodiments, the reaction mixture may be diluted to obtain a 1 x fluorescent dye by reacting the mixture at 30-60 ℃ for more than 4 hours. The device is arranged in such a way that the device is directly used without dilution.
For example, in some embodiments, the 10X fluorescent stain is prepared by weighing the amounts of agent A, agent B and cross-linking agent, and then mixing with agent C and reacting at 30℃to 60℃for more than 4 hours. In use, 10 x fluorescent stain is mixed with agent C according to 1:9, thereby preparing the 1X fluorescent dye for dyeing.
In addition, an embodiment of the present application further provides a fluorescent dye, which is prepared by the preparation method of the fluorescent dye of any one of the above examples. The fluorescent dye is not required to be used after heating reaction, and can be directly used or used after dilution.
In addition, another embodiment of the present application also provides another fluorescent stain, which includes: the agent A comprises a nano material, wherein 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) glycerol, 0.1% (m/V) to 5% (m/V) dimethyl sulfoxide and a buffer solution. More specifically, the compositions and amounts of agent A, agent B, crosslinking agent and agent C are as described above and are not described in detail herein. When the fluorescent dye is used, the agent A, the agent B, the cross-linking agent and the agent C are required to be 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 dye for use.
In some embodiments, the agent a, the agent B, the cross-linking agent, and the agent C are each packaged separately. In other embodiments, the cross-linking agent is packaged in one package, and the agent A, agent B, and agent C are packaged together. In other embodiments, the cross-linking agent and agent C are packaged separately, and the agent A and agent B are packaged together after mixing. In other embodiments, the agent A, the agent B, the cross-linking agent, and the agent C are packaged together after being mixed.
In addition, an embodiment of the present application also provides a dyeing method, which includes step S100 and step S200. Specifically:
step S100: and fixing the sample by using a fixing agent, and airing.
After the cells are immobilized, the pore size of the pores on the cell membrane becomes larger, and the complex formed by the nanomaterial and the dye is easier to enter the cells. In some embodiments, the fixative includes at least one of methanol, ethanol, acetone, and acetaldehyde. In an alternative embodiment, the fixative is anhydrous methanol.
In some embodiments, the sample is a vaginal secretion sample. The operation of fixing the sample with the fixing agent includes: immersing the sample in a fixing agent for 1-30 min. Further, the fixing time is 2 min-15 min.
Of course, the step of smearing and air-drying the sample is also included before the sample is fixed with the fixative. Further, the air drying before the fixing agent treatment is natural air drying.
Step S200: and (5) dropwise adding a coloring agent to the dried sample for dyeing.
Specifically, the raw material of the fluorescent dye is the fluorescent dye prepared by the preparation method of the fluorescent dye in any embodiment. In some embodiments, the fluorescent stain produced by the above method of producing a fluorescent stain is a 1 x fluorescent stain. At this time, the fluorescent dye prepared by the preparation method of the fluorescent dye is directly dripped on the dried sample for dyeing. In other embodiments, the fluorescent dye prepared by the method for preparing a fluorescent dye is a concentrated solution of the fluorescent dye, at this time, the fluorescent dye prepared by the method for preparing a fluorescent dye is diluted into 1×fluorescent dye by using the C agent, and then the 1×fluorescent dye is dropped on the dried sample for dyeing.
In some embodiments, the time of staining is between 10s and 30s. In an alternative embodiment, the time of staining is 10s, 15s, 20s, 25s or 30s.
It will be appreciated that after the staining is completed, the step of washing the stained sample with water is also included. Of course, after washing the stained sample, the step of drying the sample with water or naturally drying the sample is further 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 is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer. Herein, "DMSO" refers to dimethyl sulfoxide; "mM" means mmol/L.
Example 1
Preparation of agent C: according to the preparation amount of 1L, a certain amount of sodium dihydrogen phosphate, disodium hydrogen phosphate, glycerin, DMSO and proclin 300 are sequentially fed into a 1L reactor, distilled water is added to a constant volume of 1L, and the materials are fully stirred to be completely dissolved. The final solution (agent C) had the following material concentrations: 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 bring the pH of component C to 5.00. Preserving at 4 ℃ for standby.
2. Preparing a coloring agent:
(1) The agent A (agent A is a nano silica solution, the average diameter of nano silica is 7 nm) was diluted with the agent C, and a cross-linking agent carbodiimide (EDAC) was added to make the concentration of nano silica 0.08% (m/V) and the concentration of EDAC 0.5% (m/V) to prepare an AC mixed solution. Then, acridine orange was added to the AC mixed solution to give an acridine orange concentration of 8% (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 the C agent according to the weight ratio of 1:9 volume ratio, and adjusting pH to 7.5 with 0.1M hydrochloric acid to obtain the fluorescent dye of the example.
Example 2
The preparation method of the fluorescent dye of this example is substantially the same as that of example 1, except that the raw material for preparing the fluorescent dye of this example does not contain a nanosilica solution. The preparation method of the fluorescent dye comprises the following steps: diluting acridine orange with C agent to give C 28 H 28 N 2 O 3 S 2 The concentration of (C) was 0.8% (M/V), and the pH was adjusted to 7.5 with 0.1M hydrochloric acid to obtain a fluorescent dye of this example.
Example 3
Preparation of agent C: the same as in example 1.
2. Preparing a coloring agent:
(1) Diluting agent A (agent A is nano ferroferric oxide solution, the average diameter of nano ferroferric oxide is 7 nm) with agent C, adding cross-linking agent carbodiimide (EDAC) to make the concentration of nano ferroferric oxide be 0.08% (m/V), and EDAC concentration be 0.5% (m/V), thus obtaining the AC mixed solution. SYBRGreen I was then added to the AC mixture to a SYBRGreen I concentration of 1% (m/V), and reacted in a water bath at 50℃for 12 hours to give a 10 Xfluorescent stain.
(2) Mixing the 10X fluorescent staining agent prepared in the step (1) with the C agent according to the weight ratio of 1:9 volume ratio, and adjusting pH to 7.5 with 0.1M hydrochloric acid to obtain the fluorescent dye of the example.
Example 4
The preparation method of the fluorescent dye of this example is substantially the same as that of example 3, except that the preparation raw material of the fluorescent dye of this example does not contain a nano ferroferric oxide solution. The preparation method of the fluorescent dye comprises the following steps: SYBRGreen I is diluted with C to a concentration of SYBRGreen I of 0.1% (M/V), and pH is adjusted to 7.5 with 0.1M hydrochloric acid to give the fluorescent stain of this example.
Example 5
Preparation of agent C: the same as in example 1.
2. Preparing a coloring agent: the difference is that the amount of nanosilica used in the preparation of the fluorescent dye of this example is different from that of example 1, which is substantially the same as that of example 1. Specifically, the preparation steps of the coloring agent of this embodiment include:
(1) The agent A (agent A is a nano silica solution, the average diameter of nano silica is 7 nm) is diluted with the agent C, and a cross-linking agent carbodiimide (EDAC) is added to make the concentration of nano silica 0.1% (m/V) and the concentration of EDAC 15% (m/V) to prepare an AC mixed solution. Then, acridine orange was added to the AC mixed solution to give an acridine orange 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 the C agent according to the weight ratio of 1:9 volume ratio, and adjusting pH to 7.5 with 0.1M hydrochloric acid to obtain the fluorescent dye of the example.
Example 6
Preparation of agent C: the same as in example 1.
2. Preparing a coloring agent: the difference is that the amount of nanosilica used in the preparation of the fluorescent dye of this example is different from that of example 1, which is substantially the same as that of example 1. Specifically, the preparation steps of the coloring agent of this embodiment include:
(1) The agent A (agent A is a nano silica solution, the average diameter of nano silica is 7 nm) is diluted with the agent C, and a cross-linking agent carbodiimide (EDAC) is added to make the concentration of nano silica 0.1% (m/V) and the concentration of EDAC 5% (m/V) to prepare an AC mixed solution. Then, acridine orange was added to the AC mixed solution to give an acridine orange 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 the C agent according to the weight ratio of 1:9 volume ratio, and adjusting pH to 7.5 with 0.1M hydrochloric acid to obtain the fluorescent dye of the example.
Testing
Vaginal secretions were 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 steps of dyeing with the fluorescent dyes of the respective embodiments include:
(1) Vaginal secretion sample preparation: the sample was uniformly smeared with a swab in the central ring of the slide with a thin coating.
(2) Naturally airing.
(3) Immersed in absolute methanol for 2min.
(4) Naturally airing.
(5) Dripping the dyeing liquid according to different dyeing time to dye; the staining times of the fluorescent staining agent of example 1 were 10s, 20s and 30s; the staining times of the fluorescent staining agent of example 2 were 20s, 40s and 60s; the staining times of the fluorescent staining agent of example 3 were 10s, 20s and 30s; the staining times of the fluorescent staining agent of example 4 were 20s, 40s and 60s; the staining times of the fluorescent staining agent of example 5 were 10s, 20s and 30s; the staining times of the fluorescent staining agent of example 6 were 10s, 20s and 30s.
(6) Washing with water, and spin-drying.
(7) Observing under a fluorescence microscope, taking pictures by using matched software, and recording names.
The results of the staining of each example are shown in fig. 1 to 6, wherein: fig. 1 to 6 are each an image taken under a condition of 40 times of 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. The vaginal secretions of examples 1, 2, 5 and 6 were derived from the same clinical sample, and the vaginal secretions of examples 3 and 4 were derived from another clinical sample.
As can be seen from FIGS. 1 and 3, when the fluorescent dyes of example 1 and example 3 were used for dyeing, high-quality fluorescent imaging patterns were obtained at a dyeing time of 20 s. After 20s staining with the fluorescent stains of example 1 and example 3, the epithelial cells were yellowish green and the bacteria were red.
As is clear from FIGS. 2 and 4, the fluorescent dyes of example 2 and example 4 provide a good dyeing effect when used for dyeing for 40 to 60 seconds. After staining for 40s to 60s with the fluorescent staining agents of example 2 and example 4, the epithelial cells appeared yellowish green and the bacteria appeared red. And the color of the samples dyed with the fluorescent stains of examples 2 and 4 without addition of the nanomaterial was darker than the fluorescent stains of examples 1 and 3 with addition of the nanomaterial; and the staining effect of the fluorescent staining agent of example 1 was better than that of the fluorescent staining agent of example 3.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
Claims (7)
1. The preparation method of the fluorescent dye is characterized by comprising the following steps of:
mixing the agent A, the agent B, the cross-linking agent and the agent C to prepare a mixed solution; a kind of electronic device with high-pressure air-conditioning system
Reacting the mixed solution for more than 4 hours at the temperature of 30-60 ℃ to prepare a fluorescent coloring agent; wherein:
the agent A comprises a nano material, wherein 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, the fluorescent dye comprising SYBR Green I, SYBR GreenII, acridine orange, propidium iodide derivatives, hydroxyfluorescein diacetate succinimidyl ester derivatives, hoechst, hoechst derivatives, 4, 6-diamidino-2-diphenylindole derivatives and C 28 H 28 N 2 O 3 S 2 At least one of (a) and (b);
the crosslinking agent comprises at least one of carbodiimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide;
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 10X 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 solution; the buffer includes one of phosphate buffer, tris buffer and HEPES 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 excitation wavelength of the fluorescent dye is 460nm to 500nm, and the emission wavelength of the fluorescent dye is 500nm to 530nm.
4. A method of preparation according to any one of claims 1 to 3 wherein the agent C further comprises a preservative.
5. A fluorescent dye, characterized by being prepared by the method for preparing a fluorescent dye according to any one of claims 1 to 4.
6. The fluorescent dye is characterized by comprising the following preparation raw materials:
a reagent a comprising a nanomaterial comprising at least one of nano-ferroferric oxide and nano-silica;
an agent B comprising a fluorescent dye capable of binding to nucleic acid; the agent B comprises a fluorescent dye capable of binding to nucleic acid, the fluorescent dye comprising SYBR GreenI, SYBR GreenII, acridine orange, propidium iodide derivative, hydroxyfluorescein diacetate succinimidyl ester derivative, hoechst, hoechst derivative, 4, 6-diamidino-2-diphenylindole derivative and C 28 H 28 N 2 O 3 S 2 At least one of (a) and (b);
a crosslinking agent comprising at least one of carbodiimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide; a kind of electronic device with high-pressure air-conditioning system
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 solution, wherein the buffer solution comprises one of phosphate buffer solution, tris buffer solution and HEPES buffer solution.
7. A dyeing method, characterized by comprising the steps of:
fixing the sample by using a fixing agent, and airing; a kind of electronic device with high-pressure air-conditioning system
And (3) dropwise adding a coloring agent to the dried sample for dyeing, wherein the raw material of the coloring agent is the fluorescent coloring agent prepared by the preparation method of the fluorescent coloring agent as claimed in any one of claims 1 to 4.
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| 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 |
| 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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| JP5400297B2 (en) * | 2005-12-27 | 2014-01-29 | 古河電気工業株式会社 | Fluorescent nanosilica particles, nanofluorescent material, biochip using the same, and assay method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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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