CN116813654B - Fluorescent dye, magnetic fluorescent coding microsphere, and preparation method and application thereof - Google Patents
Fluorescent dye, magnetic fluorescent coding microsphere, and preparation method and application thereof Download PDFInfo
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- CN116813654B CN116813654B CN202311076433.0A CN202311076433A CN116813654B CN 116813654 B CN116813654 B CN 116813654B CN 202311076433 A CN202311076433 A CN 202311076433A CN 116813654 B CN116813654 B CN 116813654B
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- fluorescent dye
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- 239000004005 microsphere Substances 0.000 title claims abstract description 146
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 113
- 238000002360 preparation method Methods 0.000 title claims abstract description 38
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 33
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- 238000007385 chemical modification Methods 0.000 description 1
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- 238000000151 deposition Methods 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- RWFKYBVNHRKZSN-UHFFFAOYSA-N ethyl 4-methyl-1h-pyrrole-2-carboxylate Chemical compound CCOC(=O)C1=CC(C)=CN1 RWFKYBVNHRKZSN-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 229920001002 functional polymer Polymers 0.000 description 1
- ZTYYDUBWJTUMHW-UHFFFAOYSA-N furo[3,2-b]furan Chemical compound O1C=CC2=C1C=CO2 ZTYYDUBWJTUMHW-UHFFFAOYSA-N 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- WCPAKWJPBJAGKN-UHFFFAOYSA-N oxadiazole Chemical compound C1=CON=N1 WCPAKWJPBJAGKN-UHFFFAOYSA-N 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 125000003538 pentan-3-yl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- PBMFSQRYOILNGV-UHFFFAOYSA-N pyridazine Chemical compound C1=CC=NN=C1 PBMFSQRYOILNGV-UHFFFAOYSA-N 0.000 description 1
- MHOZZUICEDXVGD-UHFFFAOYSA-N pyrrolo[2,3-d]imidazole Chemical compound C1=NC2=CC=NC2=N1 MHOZZUICEDXVGD-UHFFFAOYSA-N 0.000 description 1
- RQGPLDBZHMVWCH-UHFFFAOYSA-N pyrrolo[3,2-b]pyrrole Chemical compound C1=NC2=CC=NC2=C1 RQGPLDBZHMVWCH-UHFFFAOYSA-N 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- JWVCLYRUEFBMGU-UHFFFAOYSA-N quinazoline Chemical compound N1=CN=CC2=CC=CC=C21 JWVCLYRUEFBMGU-UHFFFAOYSA-N 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 150000004666 short chain fatty acids Chemical class 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- ONCNIMLKGZSAJT-UHFFFAOYSA-N thieno[3,2-b]furan Chemical compound S1C=CC2=C1C=CO2 ONCNIMLKGZSAJT-UHFFFAOYSA-N 0.000 description 1
- VJYJJHQEVLEOFL-UHFFFAOYSA-N thieno[3,2-b]thiophene Chemical compound S1C=CC2=C1C=CS2 VJYJJHQEVLEOFL-UHFFFAOYSA-N 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1059—Heterocyclic compounds characterised by ligands containing three nitrogen atoms as heteroatoms
- C09K2211/107—Heterocyclic compounds characterised by ligands containing three nitrogen atoms as heteroatoms with other heteroatoms
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom
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- Chemical & Material Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a fluorescent dye, a magnetic fluorescent coding microsphere, and a preparation method and application thereof. The fluorescent dye has the following structural general formula:wherein R is 1 R is R 2 Each independently comprises phenyl, thienyl, C 1 ~C 6 Alkyl-substituted thienyl and C 3 ~C 10 Any one or a combination of a plurality of heteroaryl substituted thienyl, R 3 Comprising carbon-carbon double bonds, R 4 Comprises C 1 ~C 6 An alkyl group. The fluorescent dye has good stability and proper wavelength, and can be applied to the preparation of magnetic fluorescent coding microspheres.
Description
Technical Field
The invention relates to the field of organic synthesis, in particular to a fluorescent dye, a magnetic fluorescent coding microsphere, a preparation method and application thereof.
Background
The core of the coding technology is the technology of coding microspheres, namely the technology of embedding one or more fluorescent dyes into the microspheres by adjusting the proportion of the dyes, thereby achieving the coding. The magnetic fluorescent coded microsphere is a fluorescent microsphere with magnetic property, and compared with the traditional nonmagnetic coded microsphere, the magnetic coded microsphere has the characteristics of magnetic sorting, low cost, simple operation, easier automation and the like.
One important substance in magnetic fluorescent encoded microspheres is a fluorescent dye, which is selected at a suitable wavelength to match the encoded channel. The organic small molecular dye with suitable wavelength is difficult to find in the market, so that research and development personnel adopt fluorescent quantum dots, but the synthesis condition of the fluorescent quantum dots is harsh, and the emission wavelength is changed according to the size of the adjustment particles, so that the batch stability of the fluorescent quantum dots is poor.
Disclosure of Invention
Based on the above, some embodiments of the present invention provide a fluorescent dye with good stability and suitable wavelength, which can be applied to magnetic fluorescent coding microspheres, and a preparation method thereof.
In addition, the invention also provides a magnetic fluorescent coding microsphere and a preparation method and application thereof.
A fluorescent dye having the general structural formula:，
wherein R is 1 And R2 each independently comprises phenyl, thienyl, C 1 ~C 6 Alkyl-substituted thienyl and C 3 ~C 10 In heteroaryl-substituted thienylAny one or a combination of a plurality of R 3 Comprising carbon-carbon double bonds, R 4 Comprises C 1 ~C 6 An alkyl group.
In some of these embodiments, the fluorescent dye satisfies at least one of the following conditions:
（1）R 1 R is R 2 Each independently includes any one or a combination of a plurality of phenyl groups, thienyl groups, methylthiophene groups and bithiophene groups;
（2）R 3 including methacrylate groups.
In some embodiments, the fluorescent dye is selected from one or more of the following structures:
、/>、
、/>。
the preparation method of the fluorescent dye comprises the following steps:
reacting the compound A with sodium nitrite to prepare a compound B;
reacting the compound B with a compound C to prepare a compound D;
reacting the compound D with triethylamine and boron trifluoride to prepare the fluorescent dye;
the structural formulas of the compound A, the compound B, the compound C and the compound D are respectively、/>、/>And->。
In some of these embodiments, the step of reacting compound a with sodium nitrite comprises: dissolving the compound A in a first alcohol solvent, adding part of the acid reagent, then dropwise adding an aqueous solution containing sodium nitrite, adding the rest part of the acid reagent after the dropwise adding is finished, and reacting for 2-5 h, wherein the molar ratio of the compound A to the sodium nitrite is 1: (1.15-3).
In some of these embodiments, the step of reacting the compound B with the compound C satisfies at least one of the following conditions:
(1) The molar ratio of the compound B to the compound C is 1: (1-1.5);
(2) The reaction temperature is 100-150 ℃ and the reaction time is 1-3 hours;
(3) Acetic acid and acetic anhydride are also added during the reaction.
In some of these embodiments, the step of reacting the compound D with triethylamine, boron trifluoride satisfies at least one of the following conditions:
(1) The molar ratio of the compound D to the triethylamine to the boron trifluoride is 1 (5-10): 5-10;
(2) The reaction temperature is 25-30 ℃ and the reaction time is 12-17 h.
In some of these embodiments, the R 1 R is R 2 Each independently comprises thienyl, C 1 ~C 6 Alkyl-substituted thienyl and C 3 ~C 10 Any one or a combination of several heteroaryl substituted thienyl, wherein the preparation steps of the compound A comprise:
r is R 1 -CHO and R 2 -C(=O)-CH 3 Carrying out dehydration condensation reaction to prepare a compound E;
reacting the compound E with nitromethane to prepare a compound F;
reacting the compound F with concentrated sulfuric acid to prepare a compound G;
reacting the compound G with ammonium acetate to prepare the compound A;
wherein, the structural formulas of the compound E, the compound F and the compound G are respectively as follows: 、/>And->。
In some of these embodiments, the R 3 Comprising methacrylate groups, and the preparation step of the compound C comprises the following steps: reacting a compound H with a reducing agent to prepare a compound J, and reacting the compound J with methacryloyl chloride to prepare a compound C, wherein the structural formulas of the compound H and the compound J are respectivelyAnd。
a magnetically fluorescent encoded microsphere comprising:
a magnetic microsphere matrix; a kind of electronic device with high-pressure air-conditioning system
The coating layer is coated inside the magnetic microsphere and comprises a coating monomer and a copolymer of fluorescent dye, wherein the fluorescent dye is the fluorescent dye;
the coating monomer comprises one or more of styrene, divinylbenzene, methacrylic acid, hydroxyethyl methacrylate and glycidyl methacrylate.
In some of these embodiments, the magnetically fluorescent encoded microspheres satisfy at least one of the following conditions:
(1) In the preparation raw materials of the coating layer, the mass ratio of the coating monomer to the fluorescent dye is 100: (1-40);
(2) The mass ratio of the magnetic microspheres to the coating layer is (1-3) 1;
(3) The average particle diameter of the magnetic fluorescent coding microsphere is 6-6.5 mu m.
In some of these embodiments, the fluorescent dye comprises a first dye and a second dye, the first dye comprisingThe second dye comprises +>、Is->One or more of them.
In some embodiments, the mass ratio of the first dye to the second dye is (1-16): 1.
A preparation method of magnetic fluorescent coding microspheres comprises the following steps:
the magnetic microsphere, the coating monomer and the fluorescent dye are mixed and subjected to polymerization reaction under the action of an initiator to prepare the magnetic fluorescent coding microsphere, wherein the fluorescent dye is the fluorescent dye, and the coating monomer comprises one or more of styrene, divinylbenzene, methacrylic acid, hydroxyethyl methacrylate and glycidyl methacrylate.
In some of these embodiments, the method of preparation satisfies at least one of the following conditions:
(1) The initiator comprises any one or more of azodiisobutyronitrile, azodiisoheptonitrile, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate and potassium persulfate;
(2) The temperature of the polymerization reaction is 75-80 ℃ and the time is 24-28 h;
(3) The magnetic microsphere, the coating monomer and the fluorescent dye are mixed, and the steps of polymerization reaction under the action of an initiator comprise:
Mixing the magnetic microsphere with part of coating monomer and part of initiator for first polymerization to prepare a first polymer;
and polymerizing the first polymer with the rest of the coating monomers, the rest of the initiator and the fluorescent dye for the second time.
The magnetic fluorescent coding microsphere is applied to the preparation of a diagnostic kit.
A diagnostic kit comprising the magnetic fluorescent coding microsphere described above.
Compared with the traditional BODIPY dye, the fluorescent dye has longer emission wavelength, can reach a near infrared region, and can increase the overall pi conjugation by introducing phenyl, substituted or unsubstituted thiophene structure, thereby further red shifting the emission wavelength, obtaining the fluorescent dye with larger emission wavelength and being capable of matching with a coding channel. Compared with fluorescent quantum dots, the fluorescent dye has good stability. In addition, the fluorescent dye also has double bond groups, and can be coated in the magnetic microsphere in a polymerization mode. Therefore, the fluorescent dye has good stability and proper wavelength, and can be applied to the preparation of magnetic fluorescent coding microspheres.
Drawings
FIG. 1 is a scanning electron microscope image of magnetic microspheres used in some embodiments;
FIG. 2 is an emission wavelength spectrum of the compound I prepared in example 1;
FIG. 3 is an emission wavelength spectrum of the compounds II, III and IV prepared in examples 2-4;
FIG. 4 is a graph showing the results of the detection of the magnetic fluorescent encoded microspheres prepared in example 5 using a flow cytometer;
FIG. 5 is a graph showing the results of the detection of the magnetic fluorescent encoded microspheres prepared in example 6 using a flow cytometer;
FIG. 6 is a graph showing the results of the detection of the magnetic fluorescent encoded microspheres prepared in example 7 using a flow cytometer;
FIG. 7 is a graph showing the results of the detection of the magnetic fluorescent encoded microspheres prepared in example 8 using a flow cytometer;
FIG. 8 is a graph showing the results of the detection of the magnetic fluorescent encoded microspheres prepared in example 9 using a flow cytometer;
FIG. 9 is an emission wavelength spectrum of the compound V and the compound VI prepared in comparative example 1 and comparative example 2.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to specific embodiments that are now described. Preferred embodiments of the invention are given in the detailed description. This invention may, however, 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 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.
Unless otherwise indicated or contradicted, terms or phrases used in the present invention have the following meanings:
in the present invention, "first," "second," etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "first," "second," etc. can include at least one such feature, either explicitly or implicitly.
In the present invention, "one or more" means any one, any two or more of the listed items. Wherein "several" means any two or more.
In the present invention, the percentage concentrations referred to refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system after the component is added.
The word "preferably" or the like in the present invention refers to embodiments of the invention that may provide certain benefits in certain circumstances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed in the present invention, the range is considered to be continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
The terms "comprising" and "having" and any variations thereof in embodiments of the present invention are intended to cover a non-exclusive inclusion. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those listed steps or elements but may alternatively include other steps or elements not listed or inherent to such process, method, article, or apparatus.
Reference in the specification to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the described embodiments of the application may be combined with other embodiments.
The term "alkyl"refers to saturated hydrocarbons containing primary (positive) carbon atoms, or secondary carbon atoms, or tertiary carbon atoms, or quaternary carbon atoms, or combinations thereof. Phrases containing this term, e.g., "C 1 ~C 6 Alkyl "means an alkyl group containing 1 to 6 carbon atoms, and each occurrence may be, independently of the other, C 1 Alkyl, C 2 Alkyl, C 3 Alkyl, C 4 Alkyl, C 5 Alkyl, C 6 An alkyl group. Suitable examples include, but are not limited to: methyl (Me, -CH) 3 ) Ethyl (Et, -CH) 2 CH 3 ) 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ) 2-propyl (i-Pr, i-propyl, -CH (CH) 3 ) 2 ) 1-butyl (n-Bu, n-butyl, -CH) 2 CH 2 CH 2 CH 3 ) 2-methyl-1-propyl (i-Bu, i-butyl, -CH) 2 CH(CH 3 ) 2 ) 2-butyl (s-Bu, s-butyl, -CH (CH) 3 )CH 2 CH 3 ) 2-methyl-2-propyl (t-Bu, t-butyl, -C (CH) 3 ) 3 ) 1-pentyl (n-pentyl, -CH) 2 CH 2 CH 2 CH 2 CH 3 ) 2-pentyl (-CH (CH 3) CH2CH2CH 3), 3-pentyl (-CH (CH) 2 CH 3 ) 2 ) 2-methyl-2-butyl (-C (CH) 3 ) 2 CH 2 CH 3 ) 3-methyl-2-butyl (-CH (CH) 3 )CH(CH 3 ) 2 ) 3-methyl-1-butyl (-CH) 2 CH 2 CH(CH 3 ) 2 ) 2-methyl-1-butyl (-CH) 2 CH(CH 3 )CH 2 CH 3 ) 1-hexyl (-CH) 2 CH 2 CH 2 CH 2 CH 2 CH 3 ) 2-hexyl (-CH (CH) 3 )CH 2 CH 2 CH 2 CH 3 ) 3-hexyl (-CH (CH) 2 CH 3 )(CH 2 CH 2 CH 3 ) 2-methyl-2-pentyl (-C (CH) 3 ) 2 CH 2 CH 2 CH 3 ) 3-methyl-2-pentyl (-CH (CH) 3 )CH(CH 3 )CH 2 CH 3 ) 4-methyl-2-pentyl (-CH (CH) 3 )CH 2 CH(CH 3 ) 2 ) 3-methyl-3-pentyl (-C (CH) 3 )(CH 2 CH 3 ) 2 ) 2-methyl-3-pentyl (-CH (CH) 2 CH 3 )CH(CH 3 ) 2 ) 2, 3-dimethyl-2-butyl (-C (CH) 3 ) 2 CH(CH 3 ) 2 ) 3, 3-dimethyl-2-butyl (-CH (CH) 3 )C(CH 3 ) 3 。
"heteroaryl" means that at least one carbon atom is replaced by a non-carbon atom on the basis of an aryl group, which may be an N atom, an O atom, an S atom, etc. For example, "C 3 ~C 10 Heteroaryl "means heteroaryl groups containing 3 to 10 carbon atoms, each occurrence of which may be independently C 3 Heteroaryl, C 4 Heteroaryl, C 5 Heteroaryl, C 6 Heteroaryl, C 7 Heteroaryl or C 8 Heteroaryl groups. Suitable examples include, but are not limited to: furan, benzofuran, thiophene, benzothiophene, pyrrole, pyrazole, triazole, imidazole, oxazole, oxadiazole, thiazole, tetrazole, indole, carbazole, pyrroloimidazole, pyrrolopyrrole, thienopyrrole, thienothiophene, furopyrrole, furofuran, thienofuran, benzisoxazole, benzisothiazole, benzimidazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, quinoline, isoquinoline, naphthyridine, quinoxaline, phenanthridine, primary pyridine, quinazoline, and quinazolinone.
The first aspect of the invention provides a fluorescent dye, which has the following structural general formula:，
wherein R is 1 R is R 2 Each independently comprises phenyl, thienyl, C 1 ~C 6 Alkyl-substituted thienyl and C 3 ~C 10 Any one or a combination of a plurality of heteroaryl substituted thienyl, R 3 Comprising carbon-carbon double bonds, R 4 Comprises C 1 ~C 6 An alkyl group.
Compared with the traditional BODIPY dye, the fluorescent dye adopts the aza-boron dipyrrole as a parent structure, has longer emission wavelength, can reach a near infrared region, and has good light stability. In addition, the dye has the unique advantages of small Stokes shift (Stokes shift), narrow spectrum and small interference among dyes; the dye molecules are uncharged and neutral. In addition, the introduction of phenyl, substituted or unsubstituted thiophene structure can increase the overall pi conjugation, thereby red shifting the emission wavelength and obtaining fluorescent dye with larger emission wavelength. In addition, the fluorescent dye also has double bond groups, and can be coated in the magnetic microsphere in a polymerization mode.
The magnetic fluorescent coding microsphere prepared by the fluorescent dye can be obviously separated in an APC channel, has no overlapping phenomenon, and has small fluorescence intensity CV and uniform dyeing.
In some embodiments, R 1 R is R 2 Each independently includes any one or a combination of a plurality of phenyl, thienyl, methylthiophene and bithiophene. R is R 3 Including methacrylate groups. R is R 4 Including methyl, ethyl, propyl, isopropyl, and the like.
In some of these embodiments, the fluorescent dye comprises one or more of the following structures:
、/>、
、/>。
the second aspect of the present invention provides a method for preparing a fluorescent dye, which is a method for preparing the fluorescent dye, comprising the following steps:
step S110: reacting the compound A with sodium nitrite to prepare a compound B;
step S120: reacting compound B with compound C to prepare compound D;
step S130: reacting the compound D with triethylamine and boron trifluoride to prepare fluorescent dye;
the structural formulas of the compound A, the compound B, the compound C and the compound D are respectively、、/>And->。
Specifically, the synthetic route of the fluorescent dye is as follows:
。
in some of these embodiments, step S110 includes: dissolving a compound A in a first alcohol solvent, adding a part of acidic reagent, dropwise adding an aqueous solution containing sodium nitrite, adding the rest of acidic reagent after the dropwise adding is finished, and reacting for 2-5 hours, wherein the molar ratio of the compound A to the sodium nitrite is 1: (1.15-3).
Specifically, in step S110, the molar ratio of the compound a to the sodium nitrite may be, but is not limited to, 1:1.15, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3 or a range consisting of any two of these values.
Optionally, the volume of the first alcohol solvent is 50 ml-100 ml. For example, the volume of the first alcohol solvent may be, but is not limited to, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, or a range consisting of any two of these values.
Alternatively, the acidic reagent includes, but is not limited to, hydrochloric acid. Optionally, the volume of the partial acidic reagent is 0.2ml to 1ml. For example, the volume of the partially acidic reagent may be, but is not limited to, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1mL, or a range consisting of any two of these values. Optionally, the volume of the remaining acidic reagent is 1ml to 2ml. For example, the volume of remaining acidic reagent may be, but is not limited to, 1.2mL, 1.3mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL, 2mL, or a range consisting of any two of these values.
Optionally, dropwise adding the aqueous solution containing sodium nitrite for 0.5-1 h. For example, the time of the dropping may be, but not limited to, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h or a range consisting of any two of these values.
In some of these embodiments, in step S120, the molar ratio of compound B to compound C is 1: (1-1.5). For example, the molar ratio of compound B to compound C may be, but is not limited to, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or a range consisting of any two of these values.
In some embodiments, in step S120, the reaction temperature is 100-150 ℃. For example, the reaction temperature may be, but is not limited to, 100 ℃, 105 ℃, 110 ℃, 115 ℃, 120 ℃, 125 ℃, 130 ℃, 135 ℃, 140 ℃, 145 ℃, 150 ℃ or a range composed of any two of these values.
Optionally, in step S120, the reaction time is 1h to 3h. For example, the reaction time may be, but is not limited to, 1h, 1.5h, 2h, 2.5h, 3h, or a range consisting of any two of these values.
Optionally, acetic acid and acetic anhydride are also added in step S120. Specifically, the volume of acetic acid is 5 mL-10 mL. For example, the volume of acetic acid may be, but is not limited to, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, or a range consisting of any two of these values. Specifically, the volume of acetic anhydride is 0.5 mL-1 mL. For example, the volume of acetic anhydride may be, but is not limited to, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1mL, or a range consisting of any two of these values.
In some embodiments, in step S130, the molar ratio of the compound D, triethylamine and boron trifluoride is 1 (5-10): 5-10.
Alternatively, the molar ratio of compound D to triethylamine is 1: (5-10). For example, the molar ratio of compound D to triethylamine may be, but is not limited to, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a range consisting of any two of these values.
Alternatively, the molar ratio of compound D to boron trifluoride is 1: (5-10). For example, the molar ratio of compound D to boron trifluoride may be, but is not limited to, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or a range consisting of any two of these values.
Optionally, in step S130, the reaction temperature is 25 ℃ to 30 ℃. For example, the reaction temperature may be, but is not limited to, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ or a range composed of any two of these values.
Optionally, in step S130, the reaction time is 12h to 17h. For example, the reaction time may be, but is not limited to, 12h, 13h, 14h, 15h, 16h, 17h or a range consisting of any two of these values.
In some embodiments, R 1 And R is 2 Are all phenyl, the compound A is 2, 4-diphenyl pyrrole, and the structural formula is . It will be appreciated that compound a is commercially available as such.
In other embodiments, R 1 R is R 2 Each independently comprises thienyl, C 1 ~C 6 Alkyl-substituted thienyl and C 3 ~C 10 Any one or a combination of a plurality of heteroaryl substituted thienyl, and the preparation steps of the compound A are as follows:
step S210: r is R 1 -CHO and R 2 -C(=O)-CH 3 Carrying out dehydration condensation reaction to prepare a compound E;
step S220: reacting the compound E with nitromethane to prepare a compound F;
step S230: reacting the compound F with concentrated sulfuric acid to prepare a compound G;
step S240: reacting compound G with ammonium acetate to prepare a compound A;
wherein, the structural formulas of the compound E, the compound F and the compound G are respectively as follows:、and->。
The synthetic route for compound a is shown below:
。
in some of these embodiments, in step S210, R 2 -C(=O)-CH 3 And R is R 1 The molar ratio of-CHO is (1-2): (1.2 to 3.5).
Optionally, in step S210, a first alkaline reagent, R, is also added 2 -C(=O)-CH 3 The molar ratio of the alkali metal salt to the first alkaline reagent is (1-2): (1.1-2.2). Optionally, in step S210, the first alkaline reagent includes any one or a combination of several of sodium hydroxide, potassium hydroxide and triethylamine.
Alternatively, in step S210, the temperature of the dehydration condensation reaction is room temperature. The room temperature may be, for example, but not limited to, 25 ℃ to 30 ℃.
Optionally, in step S210, the dehydration condensation reaction time is 12h to 14h. For example, the time of the dehydration condensation reaction may be, but not limited to, 12h, 12.5h, 13h, 13.5h, 14h or a range composed of any two of these values.
Alternatively, in step S210, the dehydration condensation reaction is performed in a second solvent. For example, the volume of the second solvent may be, but is not limited to, 70 mL-100 mL. For example, the volume of the second solvent may be, but is not limited to, 70mL, 75mL, 80mL, 85mL, 90mL, 95mL, 100mL, or a range consisting of any two of these values.
In some of these embodiments, compound E is reacted with nitromethane under heating and refluxing in step S220 in the presence of a second basic reagent and a third alcohol solvent to produce compound F.
Optionally, in step S220, the molar ratio of the compound E to nitromethane is (1-2): (5-6).
Optionally, in step S220, the second alkaline reagent includes any one or a combination of several of sodium hydroxide, potassium hydroxide, triethylamine and diethylamine.
Optionally, in step S220, the third alcohol solvent includes any one or a combination of several of methanol, ethanol and isopropanol. The volume of the third alcohol solvent is 100 mL-200 mL. For example, the volume of the third alcohol solvent may be, but is not limited to, 100mL, 110mL, 120mL, 130mL, 140mL, 150mL, 160mL, 170mL, 180mL, 190mL, 200mL, or a range consisting of any two of these values.
Optionally, in step S220, the reaction temperature is 80 ℃ to 120 ℃. For example, the reaction temperature may be, but is not limited to, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃, 105 ℃, 110 ℃, 115 ℃, 120 ℃ or a range composed of any two of these values.
Optionally, in step S220, the reaction time is 12h to 17h. For example, the reaction time may be, but is not limited to, 12h, 13h, 14h, 15h, 16h, 17h or a range consisting of any two of these values.
Optionally, step S220 includes: and (3) reacting the compound E with nitromethane in a third alcohol solvent under the action of a second alkaline reagent at 80-120 ℃ under the condition of heating reflux for 12-17 h to prepare the compound F. Wherein, the mol ratio of the compound E to the nitromethane is (1-2): (5-6). The volume of the third alcohol solvent is 100 mL-200 mL.
In some embodiments, in step S230, compound F is mixed with a fourth alcohol solvent, a third alkaline reagent is added, and then concentrated sulfuric acid is added, and the mixture is heated to react to prepare compound G. Optionally, the volume of the fourth alcohol solvent is 100 ml-150 ml. The molar ratio of the compound 2 to the third alkaline reagent is (1-2): (5-6). The molar ratio of the compound 2 to the concentrated sulfuric acid is (1-2): (0.1 to 0.5). In one specific example, the fourth alcohol solvent may be, but is not limited to, methanol.
It is understood that in the present embodiment, concentrated sulfuric acid refers to sulfuric acid having a mass fraction of 98%.
Optionally, in step S230, the temperature of the heating reaction is 100 ℃ to 120 ℃. For example, the temperature of the heating reaction may be, but not limited to, 100 ℃, 102 ℃, 105 ℃, 108 ℃, 110 ℃, 112 ℃, 115 ℃, 118 ℃, 120 ℃ or a range composed of any two of these values.
Optionally, in step S230, the heating reaction time is 12h to 14h. For example, the time of the heating reaction may be, but is not limited to, 12h, 12.5h, 13h, 13.5h, 14h or a range consisting of any two of these values.
Optionally, in step S230, the third alkaline reagent includes any one or more of sodium hydroxide and potassium hydroxide.
Optionally, step S230 includes: and (3) dissolving the compound F in a fourth alcohol solvent, adding a third alkaline reagent, adding concentrated sulfuric acid, and heating to 100-120 ℃ for reaction for 12-14 h. The volume of the fourth alcohol solvent is 100-150 mL. The molar ratio of the compound 2 to the third alkaline reagent is (1-2): (5-6). The molar ratio of the compound 2 to the concentrated sulfuric acid is (1-2): (0.1 to 0.5).
In some embodiments, in step S240, the molar ratio of compound G to ammonium acetate is (1-2): 10-20.
In some embodiments, acetic acid is also added in step S240. The volume of acetic acid is 100 mL-200 mL.
Optionally, in step S240, the reaction temperature is 120 ℃ to 150 ℃. For example, the reaction temperature may be, but is not limited to, 120 ℃, 125 ℃, 130 ℃, 135 ℃, 140 ℃, 145 ℃, 150 ℃ or a range composed of any two of these values.
Optionally, in step S240, the reaction time is 12h to 16h. For example, the reaction time may be, but is not limited to, 12h, 12.5h, 13h, 13.5h, 14h, 14.5h, 15h, 15.5h, 16h, or a range consisting of any two of these values.
Optionally, step S240 includes: and (3) dissolving the compound G in acetic acid, and adding ammonium acetate to react for 12-16 hours at the temperature of 120-150 ℃. The molar ratio of the compound G to the ammonium acetate is (1-2): 10-20. The volume of acetic acid is 100 mL-200 mL.
In some embodiments, during the preparation of compound a, R 1 Is thienyl, R 2 One selected from thienyl, methylthiophene and bithiophene.
In some embodiments, R 3 Comprising methacrylate groups, the preparation of compound C comprising: reacting a compound H with a reducing agent to prepare a compound J, and then reacting the compound J with methacryloyl chloride to prepare a compound C, wherein the structural formulas of the compound H and the compound J are respectively as follows: And->. Alternatively, the reducing agent may be, but is not limited to, lithium aluminum hydride.
Specifically, the molar ratio of compound H to reducing agent is 1: (0.25 to 0.5). For example, the molar ratio of compound H to reducing agent may be, but is not limited to, 1:0.25, 1:0.28, 1:0.3, 1:0.32, 1:0.35, 1:0.38, 1:0.4, 1:0.42, 1:0.45, 1:0.48, 1:0.5, or a range consisting of any two of these values.
Alternatively, in the step of reacting compound H with a reducing agent, it is carried out in a dry solvent. The drying solvent comprises one or more of tetrahydrofuran, dichloromethane and chloroform.
Alternatively, the temperature at which compound H is reacted with the reducing agent is room temperature, which may be, for example, but not limited to, 25℃to 30 ℃.
Optionally, the reaction time of the compound H and the reducing agent is 12-17H. For example, the reaction time may be, but is not limited to, 12h, 13h, 14h, 15h, 16h, 17h or a range consisting of any two of these values.
In some of these embodiments, compound H is reacted with a reducing agent in a dry solvent at room temperature for 12H to 17H to produce compound J. The molar ratio of the compound H to the reducing agent is 1: (0.25 to 0.5). The synthetic route is as follows:
。
In some of these embodiments, the molar ratio of compound J to methacryloyl chloride is 1: (1-2). For example, the molar ratio of compound J to methacryloyl chloride can be, but is not limited to, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, or a range consisting of any two of these values.
Optionally, in the step of reacting compound J with methacryloyl chloride, a fourth basic agent is also added. The molar ratio of compound J to fourth basic agent is 1: (1-2). For example, the molar ratio of compound J to the fourth alkaline reagent may be, but is not limited to, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, or a range consisting of any two of these values. In some of these embodiments, the fourth basic reagent comprises any one or a combination of several of sodium hydroxide, potassium hydroxide, and triethylamine.
In some of these embodiments, the temperature at which compound J is reacted with methacryloyl chloride is from 25℃to 35 ℃. For example, the reaction temperature may be, but is not limited to, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃ or a range composed of any two of these values.
In some embodiments, compound J is reacted with methacryloyl chloride for a period of 12 to 17 hours. For example, the reaction time may be, but is not limited to, 12h, 13h, 14h, 15h, 16h, 17h or a range consisting of any two of these values.
In some of these embodiments, the step of reacting compound J with methacryloyl chloride comprises: and (3) reacting the compound J with methacryloyl chloride for 12-17 hours under a fourth alkaline reagent, wherein the reaction temperature is 25-35 ℃. The molar ratio of compound J to methacryloyl chloride is 1: (1-2). The synthetic route is as follows:
。
the preparation method of the fluorescent dye has simple process and is easy for industrial production.
The third aspect of the invention provides a magnetic fluorescent coding microsphere, which comprises a magnetic microsphere and a coating layer coated inside the magnetic microsphere, wherein the coating layer comprises a copolymer of a coating monomer and a fluorescent dye, and the coating monomer comprises one or more of styrene, divinylbenzene, methacrylic acid, hydroxyethyl methacrylate and glycidyl methacrylate.
In some embodiments, in the preparation raw materials of the coating layer, the mass ratio of the coating monomer to the fluorescent dye is 100: (1-40). For example, the mass ratio of coating monomer to fluorescent dye may be, but is not limited to, 100:1, 100:5, 100:8, 100:10, 100:15, 100:20, 100:25, 100:30, 100:35, 100:40, or a range of any two of these values. Preferably, the mass ratio of the coating monomer to the fluorescent dye is 100: (1-20).
In some embodiments, the mass ratio of the magnetic microsphere to the coating is (1-3): 1. For example, the mass ratio of magnetic microspheres to coating layer may be, but is not limited to, 1:1, 1.5:1, 2:1, 2.5:1, 3:1 or any two of these values.
In some embodiments, the coating layer includes a first coating layer including a polymer formed from a coating monomer and a second coating layer including a copolymer of the coating monomer and a fluorescent dye. The first coating layer is coated on the surface of the magnetic microsphere, and the second coating layer is coated on the surface of one side, far away from the magnetic microsphere, of the first coating layer. Through the arrangement, the fluorescent dye and the magnetic microspheres can be isolated, and the quenching effect of the magnetic microspheres on the fluorescent dye is reduced.
In some embodiments, the average particle size of the magnetically fluorescent encoded microspheres is 6 μm to 6.5 μm. For example, the average particle size of the magnetically fluorescent encoded microspheres may be, but is not limited to, 6 μm, 6.1 μm, 6.2 μm, 6.3 μm, 6.4 μm, 6.5 μm, or a range consisting of any two of these values.
Alternatively, the magnetic microsphere comprises a polymer core, a brush polymer intermediate layer with magnetic material and a functionalized polymer outer layer, the brush polymer bearing functional groups. Can be prepared by the following steps: (1) Carrying out polymerization reaction on the surface of the polymer core to obtain microspheres with brush-shaped polymer intermediate layers; (2) Carrying out chemical modification on the brush polymer of the microsphere obtained in the step (1), and introducing functional groups to obtain the microsphere with the functional groups; (3) Depositing a magnetic material on the surface of the microsphere with the functional group obtained in the step (2) to obtain the microsphere with the magnetic material; (4) And (3) polymerizing the surfaces of the microspheres with the magnetic materials obtained in the step (3) to form a functional polymer outer layer, so as to obtain the magnetic microspheres.
Specifically, the magnetic microspheres are prepared according to the preparation method in the patent CN108129614a, and are not described herein.
Optionally, in some embodiments, the fluorescent dye comprises a first dye and a second dye, the first dye comprisingThe second dye comprises +>、Is->One or more of them. By adopting the first dye and the second dye to match, the number of codes can be 25 groups, no overlapping phenomenon exists, and the small fluorescence intensity CV indicates uniform dyeing.
Further, the mass ratio of the first dye to the second dye is (1-16): 1. For example, the mass ratio of the first dye to the second dye may be, but is not limited to, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, or a range of any two of these values.
The fourth aspect of the present invention provides a method for preparing magnetic fluorescent coding microspheres, comprising the steps of:
mixing magnetic microsphere, coating monomer and fluorescent dye, and making polymerization reaction under the action of initiator so as to obtain the invented magnetic fluorescent coding microsphere.
In some embodiments, the initiator comprises any one or more of azobisisobutyronitrile, azobisisoheptonitrile, t-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate, potassium persulfate.
In some embodiments, the polymerization reaction is carried out at a temperature of 75-80 ℃ for a time of 24-28 hours. For example, the polymerization reaction temperature may be, but is not limited to, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃ or a range composed of any two of these values. The polymerization time may be, but is not limited to, 24h, 25h, 26h, 27h, 28h or a range of any two of these values.
In some of these embodiments, the step of mixing the magnetic microsphere, the coating monomer, and the fluorescent dye, and performing the polymerization reaction under the action of the initiator comprises: firstly, mixing the magnetic microsphere with part of coating monomers and part of initiator to perform first polymerization to prepare a first polymer;
the first polymer is polymerized a second time with the remainder of the coating monomer, the remainder of the initiator, and the fluorescent dye.
By adopting the mode, the magnetic microsphere is coated with the first coating layer formed by partially coating the monomer, and the rest part of the coating monomer is polymerized with the fluorescent dye to form the second coating layer, so that the fluorescent dye is isolated from the magnetic microsphere, and the quenching effect of the magnetic microsphere on the fluorescent dye is reduced.
It will be appreciated that the meaning of the part and the rest of the coating monomer is as follows: for example, mixing magnetic microspheres, 1g of coating monomer and fluorescent dye may be: the magnetic microspheres were mixed with 0.5g of coating monomer (i.e., the portion of coating monomer) for a first polymerization, and then the first polymer was polymerized with 0.5g of coating monomer (i.e., the remaining portion of coating monomer) and fluorescent dye for a second polymerization. Likewise, some and the rest of the initiator have similar meanings and are not described in detail.
In some embodiments, the solvent used in the polymerization reaction is selected from any one or a combination of several of methanol, ethanol and isopropanol.
The current method for preparing the magnetic fluorescent coding microsphere mainly comprises the following steps: (1) In the copolymerization method, usually, fluorescent dye and magnetic particles are dispersed in an organic phase, then monomer is added, and the fluorescent dye is copolymerized on the microsphere by a free radical polymerization method, but the method has the influence on the microsphere at 300-350 ℃ at high temperature, and the structure of the dye can be possibly damaged. (2) The method is characterized in that fluorescent quantum dots and magnetic particles are jointly swelled in microspheres to prepare magnetic coding microspheres, but the method has limitation, the quenching effect of the magnetic particles on fluorescence is remarkable when the magnetic particles are swelled at the same time, and the fluorescent quantum dots have larger particle volumes and occupy positions, so that the magnetic particles are less, the magnetic response time is longer, the fluorescence intensity can easily reach a saturated state, and coding and magnetic separation are greatly limited; (3) The step method comprises the steps of firstly improving the surface of the magnetic nano particles by using short-chain fatty acid when synthesizing the magnetic particles, and increasing the polarity of the surface of the magnetic nano particles, so that the magnetic nano particles can be uniformly dispersed in a good solvent, and the particle size is more uniform; and then a two-step swelling method is adopted, firstly, magnetic particles enter the swelled microspheres, and then fluorescent dye enters the swelled polymer microspheres, so that the magnetic fluorescent coding microspheres are formed. However, the method has complex process and is not easy for industrial production.
The preparation method of the magnetic fluorescent coding microsphere is characterized in that the magnetic fluorescent coding microsphere is prepared by selecting proper fluorescent dye and polymerizing the fluorescent dye, the coating monomer and the magnetic microsphere, the process is simple, the industrial production is easy, the batch stability is good, the uniformity of microsphere particles is good, and meanwhile, the magnetic and fluorescent properties are good.
In order to make the objects and advantages of the present invention more apparent, the following more particular description of the magnetically and fluorescently encoded microspheres and their effects will be given in connection with specific examples, it being understood that the specific examples described herein are for illustration only and are not intended to be limiting. The following examples, unless otherwise specified, do not include other components than the unavoidable impurities. The drugs and apparatus used in the examples are all routine choices in the art, unless specifically indicated. 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. The magnetic microspheres used in the following examples were prepared according to the preparation method in patent CN108129614 a.
Example 1
This example provides a fluorescent dye, designated compound I, synthesized as follows:
。
The preparation steps of the fluorescent dye of this example are as follows:
(1) Synthesis of Compound C
10mmol of ethyl 4-methyl-2-pyrrolecarboxylate is weighed into a 250mL flask, dissolved in 100mL of tetrahydrofuran to obtain a reaction solution, 3mmol of lithium aluminum hydride is slowly injected into the reaction solution, the reaction is carried out for 15h at 25 ℃, after the reaction is finished, the quenching is carried out by deionized water, the extraction is carried out by 90mL of ethyl acetate, the organic phase is collected, the drying is carried out by anhydrous sodium sulfate, and brown oily matter is obtained by rotary evaporation, wherein the structural formula is that。
In a 250mL flask, 5mmol of the brown oil was dissolved in methylene chloride solution, and 5.2mmol of sodium hydroxide and 6mmol of methacryloyl chloride were further added to react at 25℃for 14 hours. After the reaction is finished, extracting with dichloromethane and deionized water, collecting an organic phase, performing rotary evaporation, and purifying by column chromatography to obtain a colorless oily substance, namely a compound C. Hydrogen nuclear magnetic characterization of compound C 1 H NMR) and High Resolution Mass Spectrometry (HRMS) are characterized as follows:
1 H NMR (500 MHz, CDCl 3 ) δ 8.34 (s, 1H), 6.19 – 6.10 (m, 2H), 5.85 (dq,J= 2.0, 1.0 Hz, 1H), 5.73 (dt,J= 8.4, 0.7 Hz, 1H), 5.19 (s, 2H), 2.21 (d,J= 0.7 Hz, 3H), 1.94 (t,J= 1.0 Hz, 3H)。
HRMS:C 10 H 13 NO 2 ，179.102。
(2) Synthesis of Compound I
10mmol of 2, 4-diphenyl pyrrole is weighed and dissolved in 70mL of ethanol, 0.5mL of hydrochloric acid is added, then 12mmol of aqueous solution of sodium nitrite is slowly added dropwise for 45min, and 1.5mL of hydrochloric acid is added after the dropwise addition is finished for 3h. And after the reaction is finished, adding deionized water to separate out white precipitate, filtering a filter cake, cleaning with deionized water, and drying in a vacuum drying oven at 70 ℃ to obtain a white solid, namely the compound B.
10mmol of compound B and 12mmol of compound C are weighed and dissolved in 7mL of acetic acid solution, 0.8mL of acetic anhydride solution is added dropwise for reaction at 120 ℃ for 2h, ethanol is added after the reaction is finished, a dark blue solid is separated out, the solution is filtered by suction and washed 3 times by ethanol, and the solution is dried in a vacuum drying oven at 120 ℃ for 12h to obtain a dark blue solid, namely the compound D.
10mmol of compound D is weighed and dissolved in 100mL of dichloromethane, 60mmol of triethylamine and 60mmol of boron trifluoride diethyl etherate solution are added for reaction for 14h at 25 ℃, deionized water and dichloromethane are used for extraction after the reaction is finished, and ethyl acetate and normal hexane are used for column chromatography purification, so that a solid with metallic luster is obtained, namely the compound I. The characterization of compound I is as follows:
1 H NMR (500 MHz, CDCl 3 ) δ 7.88 (q, J = 1.4 Hz, 1H), 7.81 – 7.75 (m, 2H), 7.56 – 7.50 (m, 3H), 7.48 –7.40 (m, 2H), 7.39 – 7.26 (m, 4H), 6.21 (dq, J = 1.8, 1.0 Hz, 1H), 5.87 (dq, J = 2.0, 1.0 Hz, 1H), 5.12 (s, 2H), 2.23 (d, J = 1.3 Hz, 3H), 1.94 (t, J = 1.0 Hz,3H)。
HRMS：C 26 H 22 BF 2 N 3 O 2 ，457.18。
example 2
This example provides a fluorescent dye, designated compound II, synthesized as follows:
。
the preparation steps of the fluorescent dye of this example are as follows:
12mmol of 2-acetylthiophene and 24mmol of thiophenal are weighed and dissolved in 120mL of ethanol, 30mL of aqueous solution of 10% by mass sodium hydroxide is slowly added to the mixture, the mixture is reacted for 13 hours at 25 ℃, after the reaction is finished, suction filtration is carried out, a filter cake is washed by deionized water, and the mixture is dried in vacuum at 120 ℃ to obtain a yellow solid which is marked as a compound E.
Weighing 5mmol of compound E, dissolving in 150mL of methanol solution, adding 25mmol of nitromethane and 25mmol of triethylamine, reacting for 14h at 100 ℃, cooling to room temperature after the reaction is finished, distilling under reduced pressure, adding deionized water, precipitating yellow solid, filtering, flushing a filter cake with deionized water, and drying to obtain yellow solid, which is marked as compound F.
Weighing 10mmol of compound F, dissolving in 120mL of methanol, adding 50mmol of sodium hydroxide, dissolving 2mmol of concentrated sulfuric acid in 20mL of methanol, slowly dropwise adding the solution into the solution by using a constant pressure dropping funnel, heating to 100 ℃ after the dropwise adding is finished, reacting for 12 hours, returning to room temperature after the reaction is finished, extracting the solution by using dichloromethane and deionized water, distilling the solution under reduced pressure, and using the volume ratio of 1:3 and n-hexane to give a white solid, designated as compound G.
6mmol of compound G was weighed and dissolved in 150mL of acetic acid solution, 80mmol of ammonium acetate was added, and the mixture was reacted at 120℃for 14 hours, after the reaction was completed, the mixture was extracted with methylene chloride and deionized water, and the mixture was purified by column chromatography under the condition of methylene chloride and n-hexane=3:1 (volume ratio), to obtain a white solid powder, which was designated as compound A.
Weighing 5mmol of compound A, dissolving in 60mL of ethanol, adding 0.5mL of hydrochloric acid, slowly dropwise adding 12mmol of aqueous solution of sodium nitrite for 0.5h, adding 1.5mL of hydrochloric acid after dropwise adding, heating to 80 ℃ for reaction for 3h, filtering to obtain red solid after the reaction is finished, flushing with diethyl ether, and vacuum drying at 40 ℃ for 12h to obtain red solid powder which is marked as compound B.
0.5mmol of compound B and 0.6mmol of compound C prepared in accordance with example 1 were weighed and dissolved in 5mL of acetic acid and 0.5mL of acetic anhydride solution, the temperature was raised to 100℃for 2 hours, after the reaction was completed, the mixture was cooled to room temperature, 20mL of ice water and 20mL of 2mol/L aqueous sodium hydroxide solution were added, stirred for 30 minutes, extracted with dichloromethane and deionized water, the amount of dichloromethane was 100mL each time, and the mixture was accumulated twice, dried with anhydrous sodium sulfate and distilled under reduced pressure to obtain a dark blue solid, which was designated as compound D.
1mmol of compound D is weighed and dissolved in 100mL of dichloromethane solution, 8mmol of triethylamine and 8mmol of boron trifluoride diethyl etherate solution are added into the reaction solution to react for 16h at 25 ℃, dichloromethane and deionized water are adopted for extraction after the reaction is finished, 50mL of dichloromethane is used for three times each time, the three times of dichloromethane consumption are accumulated, anhydrous sodium sulfate is adopted for drying, and reduced pressure distillation is carried out to obtain solid powder containing metallic luster, namely the compound II. The characterization of compound II is as follows:
1 H NMR (500 MHz, CDCl 3 ) δ 7.88 (q, J = 1.4 Hz, 1H), 7.50 – 7.41 (m, 2H), 7.39 (s, 1H), 7.35 – 7.27 (m,2H), 7.30 – 7.19 (m, 2H), 6.23 (dq, J = 2.0, 1.0 Hz, 1H), 5.89 (dq, J = 2.0, 1.0 Hz, 1H), 5.86 (s, 2H), 2.28 (d, J = 1.5 Hz, 3H), 1.94 (t, J = 1.0 Hz, 3H)。
HRMS：C 22 H 18 BF 2 N 3 O 2 S 2 ，469.09。
Example 3
The present example provides a fluorescent dye, designated compound III, having the following structural formula:the procedure was similar to example 2, except that 2-acetylthiophene was changed to 2-acetyl-5-methylthiophene in the synthesis of Compound II (Shanghai Bi's medicine, cat# BD 5026), and the remaining procedures were the same as in example 2. Chemical treatmentThe characterization of compound III is as follows:
1 H NMR (500 MHz, CDCl 3 ) δ 7.88 (q, J = 1.4 Hz, 1H), 7.49 – 7.41 (m, 2H), 7.37 (s, 1H), 7.35 – 7.26 (m,2H), 6.95 (dq, J = 8.6, 0.9 Hz, 1H), 6.23 (dq, J = 1.8, 1.0 Hz, 1H), 5.91 – 5.84 (m, 3H), 2.41 (d, J = 0.7 Hz, 3H), 2.28 (d, J = 1.2 Hz, 3H), 1.94 (t, J =1.0 Hz, 3H)。
HRMS：C 23 H 20 BF 2 N 3 O 2 S 2 ，483.12。
example 4
The present example provides a fluorescent dye, designated compound IV, of the formula:the procedure is similar to example 2, except that 2-acetylthiophene is replaced by 5-acetyl-2, 2' -bithiophene in the synthesis of compound II (Beijing carbofuran technologies Co., ltd.; cat.: 35770). The characterization of compound IV is as follows:
1 H NMR (500 MHz, CDCl 3 ) δ 7.69 – 7.61 (m, 2H), 7.51 – 7.43 (m, 2H), 7.41 (s, 1H), 7.36 – 7.27 (m, 2H),7.21 (dd, J = 4.2, 3.1 Hz, 1H), 7.13 – 7.05 (m, 2H), 6.22 (dq, J = 2.0, 1.0 Hz, 1H), 5.88 (dq, J = 2.0, 1.0 Hz, 1H), 5.75 (s, 2H), 2.27 (d, J = 1.2 Hz, 3H),1.94 (t, J = 1.0 Hz, 3H)。
HRMS：C 26 H 20 BF 2 N 3 O 2 S 3 ，551.09。
example 5
The embodiment provides a magnetic fluorescent coding microsphere, which is prepared by the following steps:
dispersing 10.0g of the microspheres subjected to magnetic precipitation in 100mL of ethanol, uniformly mixing by ultrasonic, adding 1.1g of polyvinylpyrrolidone (K30), mechanically stirring, charging nitrogen for 30min, weighing 1g of styrene monomer and 0.01g of azobisisobutyronitrile, dissolving into a 10mL ethanol system, slowly dripping the mixture into a reaction bottle through a dropping funnel, heating to 75 ℃ for reaction for 12h, weighing 0.8g of styrene monomer and 0.01g of the compound I fluorescent dye prepared in example 1, dissolving 0.01g of azobisisobutyronitrile into 10mL of ethanol, slowly adding into the reaction bottle, heating to 75 ℃ for reaction for 12h, cooling after the reaction is finished, centrifugally cleaning three times by deionized water, centrifugally cleaning 100mL each time, and dispersing the mixture into deionized water to obtain the magnetic fluorescent coded microspheres with average particle size of 6.2 mu m, namely the magnetic fluorescent coded microspheres 5-1.
The mass of the fluorescent dye of the compound I prepared in the example 1 in the preparation process is adjusted to be 0.02g, 0.04g, 0.08g and 0.16g respectively, and other conditions are unchanged, so that magnetic fluorescent coding microspheres which are respectively marked as magnetic fluorescent coding microspheres 5-2, 5-3, 5-4 and 5-5 are obtained.
Example 6
The embodiment provides a magnetic fluorescent coding microsphere, which is prepared by the following steps:
dispersing 10g of the microspheres subjected to magnetic precipitation in 100mL of ethanol, uniformly mixing by ultrasonic, adding 1.34g of polyvinylpyrrolidone (K30), mechanically stirring, charging nitrogen for 30min, weighing 1.3g of styrene monomer and 0.01g of azobisisobutyronitrile into a 10mL ethanol system, slowly dripping the mixture into a reaction bottle through a dropping funnel, heating to 75 ℃ for reaction for 12h, weighing 1g of styrene monomer and 0.01g of the compound III fluorescent dye prepared in example 3, and 0.01g of azobisisobutyronitrile into 10mL of ethanol, slowly adding the mixture into the reaction bottle, heating to 75 ℃ for reaction for 12h, cooling after the reaction is finished, centrifugally cleaning three times by deionized water, using 100mL of deionized water each time, and dispersing the mixture into deionized water to obtain the magnetic fluorescent coded microspheres with the average particle size of 6.3 mu m, and recording the magnetic fluorescent coded microspheres as 6-1.
The mass of the fluorescent dye of the compound III prepared in the example 3 in the preparation process is adjusted to be 0.02g, 0.04g, 0.08g and 0.16g respectively, and other conditions are unchanged, so that magnetic fluorescent coding microspheres are obtained and are respectively marked as magnetic fluorescent coding microspheres 6-2, 6-3, 6-4 and 6-5.
Example 7
The embodiment provides a magnetic fluorescent coding microsphere, which is prepared by the following steps:
dispersing 10g of the microspheres subjected to magnetic precipitation in 100mL of ethanol, uniformly mixing by ultrasonic, adding 1.3g of polyvinylpyrrolidone (K30), mechanically stirring, charging nitrogen for 30min, weighing 1.2g of styrene monomer and 0.01g of azodiisobutyronitrile, dissolving into a 10mL ethanol system, slowly dripping the mixture into a reaction bottle through a dropping funnel, heating to 75 ℃ for reaction for 12h, weighing 1g of styrene monomer and 0.01g of the compound I fluorescent dye prepared in example 1, dissolving 0.01g of the compound III fluorescent dye prepared in example 3 and 0.01g of azodiisobutyronitrile into 10mL of ethanol, slowly adding into the reaction bottle, heating to 75 ℃ for reaction for 12h, cooling after the reaction, centrifugally cleaning three times by deionized water, using 100mL of deionized water each time, and dispersing in deionized water to obtain the magnetic fluorescent coded microspheres with average particle size of 6.2 mu m, namely the magnetic fluorescent coded microspheres 7-1.
The mass of the compound I fluorescent dye prepared in example 1 was adjusted to be 0.02g, 0.04g, 0.08g and 0.16g, respectively, and the mass of the compound III fluorescent dye prepared in example 3 was adjusted to be 0.02g, 0.04g, 0.08g and 0.16g, respectively, under the other conditions, to obtain magnetic fluorescent coding microspheres, which were designated as magnetic fluorescent coding microspheres 7-2, 7-3, 7-4 and 7-5, respectively. Namely: the mass of the compound I fluorescent dye added in the preparation process of the magnetic fluorescent coding microsphere 7-2 is 0.02g, and the mass of the compound III fluorescent dye is 0.02g; the mass of the compound I fluorescent dye added in the preparation process of the magnetic fluorescent coding microsphere 7-3 is 0.04g, and the mass of the compound III fluorescent dye is 0.04g; the mass of the compound I fluorescent dye added in the preparation process of the magnetic fluorescent coding microsphere 7-4 is 0.08g, and the mass of the compound III fluorescent dye is 0.08g; the mass of the compound I fluorescent dye added in the preparation process of the magnetic fluorescent coding microsphere 7-5 is 0.16g, and the mass of the compound III fluorescent dye is 0.16g.
Example 8
The embodiment provides a magnetic fluorescent coding microsphere, which is prepared by the following steps:
dispersing 10g of the microspheres subjected to magnetic precipitation in 100mL of ethanol, uniformly mixing by ultrasonic, adding 1.3g of polyvinylpyrrolidone (K30), mechanically stirring, charging nitrogen for 30min, weighing 1.2g of styrene monomer and 0.01g of azobisisobutyronitrile into a 10mL ethanol system, slowly dripping the mixture into a reaction bottle through a dropping funnel, heating to 75 ℃ for reaction for 12h, weighing 1g of styrene monomer and 0.16g of the compound II fluorescent dye prepared in example 2 and 0.01g of azobisisobutyronitrile into 10mL of ethanol, slowly adding the mixture into the reaction bottle, heating to 75 ℃ for reaction for 12h, cooling after the reaction is finished, centrifugally cleaning three times by deionized water, centrifugally cleaning 100mL of deionized water each time, and dispersing the mixture into deionized water to obtain the magnetic fluorescent coded microspheres with average particle size of 6.4 mu m, namely the magnetic fluorescent coded microspheres 8-1.
The mass of the fluorescent dye of the compound II prepared in the example 2 in the preparation process is adjusted to be 0.32g, 0.64g and 1.28g respectively, and other conditions are unchanged, so that magnetic fluorescent coding microspheres which are respectively marked as magnetic fluorescent coding microspheres 8-2, 8-3 and 8-4 are obtained.
Example 9
The embodiment provides a magnetic fluorescent coding microsphere, which is prepared by the following steps:
dispersing 10g of the microspheres subjected to magnetic precipitation in 100mL of ethanol, uniformly mixing by ultrasonic, adding 1.3g of polyvinylpyrrolidone (K30), mechanically stirring, charging nitrogen for 30min, weighing 1.2g of styrene monomer and 0.01g of azobisisobutyronitrile into a 10mL ethanol system, slowly dripping the mixture into a reaction bottle through a dropping funnel, heating to 75 ℃ for reaction for 12h, weighing 1g of styrene monomer and 0.16g of the compound IV fluorescent dye prepared in example 4 and 0.01g of azobisisobutyronitrile into 10mL of ethanol, slowly adding the mixture into the reaction bottle, heating to 75 ℃ for reaction for 12h, cooling after the reaction is finished, centrifugally cleaning three times by deionized water, centrifugally cleaning 100mL of deionized water each time, and dispersing the mixture into deionized water to obtain the magnetic fluorescent coded microspheres with average particle size of 6.4 mu m, namely the magnetic fluorescent coded microspheres 9-1.
The mass of the fluorescent dye of the compound IV prepared in the example 4 in the preparation process is adjusted to be 0.32g, 0.64g and 1.28g respectively, and other conditions are unchanged, so that magnetic fluorescent coding microspheres which are respectively marked as magnetic fluorescent coding microspheres 9-2, 9-3 and 9-4 are obtained.
Comparative example 1
Comparative example 1 provides a fluorescent dye having the structural formula:the procedure was similar to example 1 except that 2, 4-diphenylpyrrole was replaced with pyrrole (aladine, cat. No. P104879) in the synthesis of compound I, and the rest of the procedure was the same as in example 1. />
Comparative example 2
Comparative example 2 provides a fluorescent dye of the formulaThe procedure was similar to example 1 except that 2, 4-diphenylpyrrole was replaced with 2, 4-dimethylpyrrole (Allatin, cat. D123132) in the synthesis of compound I, and the rest of the procedure was the same as in example 1.
The following are the test parts:
FIG. 1 is a scanning electron microscope image of a magnetic microsphere used in the example, and it can be seen from the image that the magnetic microsphere has a uniform particle size, a large number of magnetic particles are provided on the surface, and the upper magnetic flux is high.
Fig. 2 is an emission wavelength spectrum of the compound I prepared in example 1, and fig. 3 is an emission wavelength spectrum of the compounds II, III and IV prepared in examples 2 to 4. In both FIGS. 2 and 3, the compound was dissolved in methylene chloride at a concentration of 10 -6 Emission wavelength spectrum tested at mol/L. As can be seen from fig. 2 and 3, the maximum emission wavelength of compound I is 640nm, the maximum emission wavelength of compound II is 720nm, the maximum emission wavelength of compound III is 760nm, and the maximum emission wavelength of compound IV is 780nm.
Fig. 4 to 6 are graphs showing the results of detection by a flow cytometer, fig. 4 shows graphs showing the results of the detection by a flow cytometer, fig. 5 shows graphs showing the results of the detection by a flow cytometer, and fig. 6 shows graphs showing the results of the detection by a flow cytometer. In the figure, SSC-A represents the side scatter of the flow instrument, APC-A is the channel on the flow instrument that receives with 670+ -7 nm fluorescence, and APC-Cy7-A is the channel on the flow instrument that receives with 780+ -30 nm fluorescence. In FIG. 4, the magnetic fluorescent encoding microspheres 5-1, 5-2, 5-3, 5-4 and 5-5 are respectively corresponding from left to right, in FIG. 5, the magnetic fluorescent encoding microspheres 6-1, 6-2, 6-3, 6-4 and 6-5 are respectively corresponding from left to right, and in FIG. 6, the magnetic fluorescent encoding microspheres 7-1, 7-2, 7-3, 7-4 and 7-5 are respectively corresponding from left to right. As can be seen from the figure, as the fluorescent dye increases, the fluorescent intensity of the microspheres increases. As can be seen from FIGS. 4 and 5, the magnetic fluorescent coding microspheres prepared by using the fluorescent dyes of the compound I and the compound III can be obviously separated in the APC channel without overlapping phenomenon, and the fluorescent intensity CV is small and uniform in dyeing. As can be seen from FIG. 6, the magnetic encoding microsphere is prepared by using the fluorescent dyes of the compound I and the compound III, the encoding number can be 25 groups, no overlapping phenomenon exists, and the small fluorescence intensity CV indicates uniform dyeing.
FIGS. 7 and 8 are graphs showing the test results of the magnetically fluorescent encoded microspheres prepared in example 8 and example 9, respectively. As can be seen from the figure, the four sets of magnetically fluorescent encoded microspheres 8-1, 8-2, 8-3, 8-4 of example 8 are present at the same location, and the four sets of magnetically fluorescent encoded microspheres 9-1, 9-2, 9-3, 9-4 of example 9 are present at the same location, indicating that the results of the test are the same for the microspheres of compound II or compound IV at different concentrations, and that the fluorescence values of the magnetically fluorescent encoded microspheres prepared from four different concentrations of fluorescent dye are substantially the same for the channels of APC and APC-Cy-7.
FIG. 9 is se:Sub>A graph showing fluorescence spectrse:Sub>A of the fluorescent dyes of comparative examples 1 and 2, and it can be seen from the graph that the maximum emission wavelengths of the compound V and the compound VI are about 520nm and 590nm, which do not meet the requirements of the channels APC-A (670+ -7 nm) and APC-Cy-7-A (780+ -30 nm), and fluorescence overflows on the two channels of FITC (525+ -15 nm) and PE (575+ -13 nm), which has se:Sub>A great influence on the practical application in the later period. So in the examples by varying R 1 And R is 2 The benzene ring and thiophene can increase the conjugation of the whole structure,so that the fluorescence spectrum can be red shifted to match the encoding channel.
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 protection scope 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 (15)
1. A fluorescent dye comprising one or both of the following structures: 、/>The fluorescent dye can be used for preparing magnetic fluorescent coding microspheres.
2. The method for preparing the fluorescent dye according to claim 1, comprising the steps of:
reacting the compound A with sodium nitrite to prepare a compound B;
reacting the compound B with a compound C to prepare a compound D;
reacting the compound D with triethylamine and boron trifluoride to prepare the fluorescent dye;
the structural formula of the compound A isThe structural formula of the compound B isThe structural formula of the compound C is +.>The structural formula of the compound D is +.>The method comprises the steps of carrying out a first treatment on the surface of the Alternatively, the compound A has the structural formulaThe structural formula of the compound B is +.>The structural formula of the compound C is +.>The structural formula of the compound D is。
3. The method for preparing a fluorescent dye according to claim 2, wherein the step of reacting the compound a with sodium nitrite comprises: dissolving the compound A in a first alcohol solvent, adding a part of acidic reagent, then dropwise adding an aqueous solution containing sodium nitrite, adding the rest of acidic reagent after the dropwise adding is finished, and reacting for 2-5 hours, wherein the molar ratio of the compound A to the sodium nitrite is 1: (1.15-3).
4. The method for preparing a fluorescent dye according to claim 2, wherein the step of reacting the compound B with the compound C satisfies at least one of the following conditions:
(1) The molar ratio of the compound B to the compound C is 1: (1-1.5);
(2) The reaction temperature is 100-150 ℃ and the reaction time is 1-3 hours;
(3) Acetic acid and acetic anhydride are also added during the reaction.
5. The method for preparing a fluorescent dye according to claim 2, wherein the step of reacting the compound D with triethylamine, boron trifluoride satisfies at least one of the following conditions:
(1) The molar ratio of the compound D to the triethylamine to the boron trifluoride is 1 (5-10): 5-10;
(2) The reaction temperature is 25-30 ℃ and the reaction time is 12-17 h.
6. The method for preparing a fluorescent dye according to any one of claims 2-5, wherein the structural formula of the compound A isThe preparation steps of the compound A comprise:
r is R 1 -CHO and R 2 -C(=O)-CH 3 Carrying out dehydration condensation reaction to prepare a compound E;
reacting the compound E with nitromethane to prepare a compound F;
reacting the compound F with concentrated sulfuric acid to prepare a compound G;
Reacting the compound G with ammonium acetate to prepare the compound A;
wherein the structural formulas of the compound E, the compound F and the compound G are respectively、/>And->Wherein R is 1 Is thienyl, R 2 Is methylthio.
7. The method for preparing a fluorescent dye according to any one of claims 2-5, wherein the step of preparing the compound C comprises: reacting a compound H with a reducing agent to prepare a compound J, and reacting the compound J with methacryloyl chloride to prepare a compound C, wherein the structural formulas of the compound H and the compound J are respectivelyAnd->The R is 4 Is methyl.
8. A magnetically fluorescent encoded microsphere, comprising:
magnetic microspheres; a kind of electronic device with high-pressure air-conditioning system
A coating layer coated inside the magnetic microsphere, wherein the coating layer comprises a copolymer of a coating monomer and a fluorescent dye, and the fluorescent dye is the fluorescent dye of claim 1;
the coating monomer is one or more of styrene, divinylbenzene, methacrylic acid, hydroxyethyl methacrylate and glycidyl methacrylate.
9. The magnetically fluorescent encoded microsphere of claim 8, wherein the magnetically fluorescent encoded microsphere satisfies at least one of the following conditions:
(1) In the preparation raw materials of the coating layer, the mass ratio of the coating monomer to the fluorescent dye is 100: (1-40);
(2) The mass ratio of the magnetic microspheres to the coating layer is (1-3) 1;
(3) The average particle diameter of the magnetic fluorescent coding microsphere is 6-6.5 mu m.
10. The magnetically fluorescent encoded microsphere of claim 8 or 9, wherein the fluorescent dye comprises a first dye and a second dye, the first dye beingThe second dye is。
11. The magnetically encoded fluorescent microsphere of claim 10, wherein the mass ratio of the first dye to the second dye is (1-16): 1.
12. The preparation method of the magnetic fluorescent coding microsphere is characterized by comprising the following steps:
the magnetic microsphere, the coating monomer and the fluorescent dye are mixed and subjected to polymerization reaction under the action of an initiator to prepare the magnetic fluorescent coding microsphere, wherein the fluorescent dye is the fluorescent dye of claim 1, and the coating monomer is one or more of styrene, divinylbenzene, methacrylic acid, hydroxyethyl methacrylate and glycidyl methacrylate.
13. The method of preparing magnetically fluorescent encoded microspheres according to claim 12, wherein the preparation method satisfies at least one of the following conditions:
(1) The initiator comprises any one or more of azodiisobutyronitrile, azodiisoheptonitrile, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate and potassium persulfate;
(2) The temperature of the polymerization reaction is 75-80 ℃ and the time is 24-28 h;
(3) The magnetic microsphere, the coating monomer and the fluorescent dye are mixed, and the steps of polymerization reaction under the action of an initiator comprise:
mixing the magnetic microsphere with part of coating monomer and part of initiator for first polymerization to prepare a first polymer;
and polymerizing the first polymer with the rest of the coating monomers, the rest of the initiator and the fluorescent dye for the second time.
14. Use of the magnetically fluorescent encoded microsphere according to any one of claims 8 to 11 in the preparation of a diagnostic kit.
15. A diagnostic kit comprising the magnetically fluorescently encoded microsphere of any one of claims 8 to 11 or prepared by the method of any one of claims 12 to 13.
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