CN112342018B - Compound APS5-CQD, preparation method thereof and chemiluminescent substrate solution - Google Patents
Compound APS5-CQD, preparation method thereof and chemiluminescent substrate solution Download PDFInfo
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- 239000000758 substrate Substances 0.000 title claims abstract description 55
- 150000001875 compounds Chemical class 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title abstract description 25
- 239000000243 solution Substances 0.000 claims abstract description 48
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 21
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 14
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 10
- 239000007983 Tris buffer Substances 0.000 claims abstract description 4
- 230000009471 action Effects 0.000 claims abstract description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 34
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 26
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 18
- 239000003054 catalyst Substances 0.000 claims description 18
- 239000003960 organic solvent Substances 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 229910052786 argon Inorganic materials 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims description 10
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 claims description 9
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 8
- CQPGDDAKTTWVDD-UHFFFAOYSA-N 4-bromobutanenitrile Chemical compound BrCCCC#N CQPGDDAKTTWVDD-UHFFFAOYSA-N 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 229910021617 Indium monochloride Inorganic materials 0.000 claims description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 4
- APHGZSBLRQFRCA-UHFFFAOYSA-M indium(1+);chloride Chemical compound [In]Cl APHGZSBLRQFRCA-UHFFFAOYSA-M 0.000 claims description 4
- RLOWWWKZYUNIDI-UHFFFAOYSA-N phosphinic chloride Chemical compound ClP=O RLOWWWKZYUNIDI-UHFFFAOYSA-N 0.000 claims description 4
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 claims description 4
- GFAUNYMRSKVDJL-UHFFFAOYSA-N formyl chloride Chemical compound ClC=O GFAUNYMRSKVDJL-UHFFFAOYSA-N 0.000 claims description 3
- UZXDEYWUHSZPLQ-UHFFFAOYSA-N ClC1=CC=CC=C1.[S] Chemical compound ClC1=CC=CC=C1.[S] UZXDEYWUHSZPLQ-UHFFFAOYSA-N 0.000 claims description 2
- 238000004020 luminiscence type Methods 0.000 abstract description 9
- 230000008569 process Effects 0.000 abstract description 5
- 230000007774 longterm Effects 0.000 abstract description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 abstract description 3
- 238000001378 electrochemiluminescence detection Methods 0.000 abstract 1
- 230000002708 enhancing effect Effects 0.000 abstract 1
- 229920000136 polysorbate Polymers 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 16
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 13
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 13
- ZCSHNCUQKCANBX-UHFFFAOYSA-N lithium diisopropylamide Chemical group [Li+].CC(C)[N-]C(C)C ZCSHNCUQKCANBX-UHFFFAOYSA-N 0.000 description 11
- 239000012224 working solution Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- POZJERCVPSQRFG-UHFFFAOYSA-N acridine-9-carbonyl chloride Chemical compound C1=CC=C2C(C(=O)Cl)=C(C=CC=C3)C3=NC2=C1 POZJERCVPSQRFG-UHFFFAOYSA-N 0.000 description 3
- FZEYVTFCMJSGMP-UHFFFAOYSA-N acridone Chemical class C1=CC=C2C(=O)C3=CC=CC=C3NC2=C1 FZEYVTFCMJSGMP-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- VZXOZSQDJJNBRC-UHFFFAOYSA-N 4-chlorobenzenethiol Chemical compound SC1=CC=C(Cl)C=C1 VZXOZSQDJJNBRC-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000012300 argon atmosphere Substances 0.000 description 2
- 235000011089 carbon dioxide Nutrition 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 238000002165 resonance energy transfer Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000007086 side reaction Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 150000000077 1,2-dioxanes Chemical class 0.000 description 1
- -1 10-acridylmethylene Chemical group 0.000 description 1
- CQZIEDXCLQOOEH-UHFFFAOYSA-N 3-bromopropanenitrile Chemical compound BrCCC#N CQZIEDXCLQOOEH-UHFFFAOYSA-N 0.000 description 1
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 description 1
- NWWWGAKVHCSAEU-UHFFFAOYSA-N 5-bromopentanenitrile Chemical compound BrCCCCC#N NWWWGAKVHCSAEU-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical class C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- KNJDBYZZKAZQNG-UHFFFAOYSA-N lucigenin Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.C12=CC=CC=C2[N+](C)=C(C=CC=C2)C2=C1C1=C(C=CC=C2)C2=[N+](C)C2=CC=CC=C12 KNJDBYZZKAZQNG-UHFFFAOYSA-N 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- 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
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
- C07F9/576—Six-membered rings
- C07F9/64—Acridine or hydrogenated acridine ring systems
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
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- C09K2211/1007—Non-condensed systems
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- 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
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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Abstract
The invention discloses a compound APS5-CQD, a preparation method thereof and chemiluminescent substrate solution, wherein the structural general formula of the compound APS5-CQD is as follows: wherein n is 3 to 5 and the R group is a carbon quantum dot. The preparation method comprises the following steps: obtaining a compound IV shown as a formula II; carrying out grafting reaction on the compound IV and carboxyl fluorescent carbon quantum dots under the action of a condensing agent to obtain a compound APS 5-CQD; the formula of the chemiluminescent substrate solution is as follows: 10-300 mM Tris, 0.01-1.0% SDS, 20-1000 mg/L APS5-CQD, 200.01-0.1% Tween and 8.5-10 pH; the invention grafts APS-5 to the carbon quantum dot to form an enhanced chemiluminescence substrate APS5-CQD, thereby enhancing the luminescence property and long-term stability of the substrate and simplifying the configuration process.
Description
Technical Field
The invention relates to the technical field of chemiluminescence immunity, in particular to a compound APS5-CQD, a preparation method thereof and chemiluminescence substrate liquid.
Background
Chemiluminescence immunoassay technology, a nonradioactive immunoassay technology, has rapidly developed worldwide after Enzyme Immunoassay (EIA), radioimmunoassay, and fluoroimmunoassay.
The luminescent substrate is one of important basic reagents of a chemiluminescence immune system, and the performance of the substrate directly influences the indexes of the detection system, such as the analysis sensitivity, the precision, the stability and the like, and is an important factor for determining the quality of the analysis system. Currently, luminol, isoluminol, acridinium esters, 1, 2-dioxanes and acridones are used as luminescent substances in chemiluminescent immunoassays. Among them, acridones are substrates for alkaline phosphatase (ALP) with high sensitivity. They are hydrolyzed and provide strong optical signals when they are contacted with ALP in an appropriate buffer. A representative compound of the acridones at present is APS-5 ((4-chlorobenzenethiol) (10-methyl-9, 10-acridylmethylene) phosphate disodium salt). APS-5 is a chemical substrate with low background. However, since the organic chemical substance itself has low luminous efficiency and poor stability in an aqueous solution, various components such as an antioxidant, a stabilizer, a luminescence enhancer and the like are required in the process of preparing the chemical substrate working solution, and the preparation process is complicated.
Therefore, how to develop a compound APS5-CQD which has low luminous efficiency, good stability in aqueous solution and simple preparation of a luminous substrate working solution and a preparation method thereof becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a compound APS5-CQD, a preparation method thereof and a chemiluminescent substrate solution, wherein APS-5 is grafted to a carbon quantum dot to form an enhanced chemiluminescent substrate APS5-CQD, so that the luminescent property and long-term stability of the substrate are enhanced, the chemiluminescent substrate working solution is optimized, and the preparation process is simplified.
In a first aspect of the invention, there is provided a compound APS5-CQD, the compound APS5-CQD having the general structural formula:
wherein n is 3 to 5 and the R group is a carbon quantum dot.
In a second aspect of the invention, there is provided a method of preparing the compound APS5-CQD, the method comprising:
obtaining a compound IV;
carrying out grafting reaction on the compound IV and carboxyl fluorescent carbon quantum dots under the action of a condensing agent to obtain a compound APS 5-CQD;
wherein the structural formula of the compound IV is shown as follows:
further, the condensing agent is DCC.
Further, before the grafting reaction, the compound IV and the carboxyl fluorescent carbon quantum dots are dissolved in a THF solution, and then a condensing agent is added for the grafting reaction.
Further, the mass ratio of the compound IV to the carboxyl fluorescent carbon quantum dots is (0.5-1): (3-4).
Further, when n is 4, the compound IV is obtained, which specifically includes:
obtaining a compound I;
carrying out a first reaction on the compound I and 4-bromobutyronitrile in an organic solvent A and a catalyst A to obtain a compound II;
reacting the compound II with 3-hydroxypropionitrile and POCl 3 Carrying out a second reaction in an organic solvent B and a catalyst B, and then stirring under an alkaline and anaerobic condition to obtain a compound III;
reacting said compound III with NaBH 4 Carrying out a third reaction under an organic solvent C and a catalyst C to obtain a compound IV; the structural formula and the reaction formula of the compound I, the compound II and the compound III are as follows:
further, the obtaining compound I comprises:
dissolving 4-chlorobenzene sulfurIn CH 2 Cl 2 Dissolving 9-acridine formyl chloride in a pyridine solution, stirring at room temperature under the protection of argon, and then carrying out solid-liquid separation and washing to obtain a brownish yellow solid;
dissolving the brownish yellow solid in CH 2 Cl 2 And adding zinc powder and acetic acid into the solution under the protection of argon at room temperature to react, and then carrying out solid-liquid separation and washing to obtain the compound I.
Further, the organic solvent A is a sodium hydroxide solution, and the catalyst A is tetrabutylammonium bromide; the organic solvent B is a tetrahydrofuran solution, and the catalyst B is LDA and pyridine; the organic solvent C is tetrahydrofuran solution, and the catalyst C is InCl 3 And (3) solution.
Further, the first reaction, the second reaction and the third reaction are all carried out under the protection of argon at room temperature.
In a third aspect of the present invention, there is provided a chemiluminescent substrate solution, wherein the chemiluminescent substrate solution has the following formula: 10-300 mM Tris, 0.01-1.0% SDS, 20-1000 mg/L APS5-CQD, 200.01-0.1% Twen, and 8.5-10 pH.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
according to the compound APS5-CQD and the preparation method thereof, APS-5 is grafted to a carbon quantum dot to form an enhanced chemiluminescent substrate APS5-CQD, so that the luminescent property and the long-term stability of the substrate are enhanced, the chemiluminescent substrate working solution is optimized, and the preparation process is simplified.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a flow chart of a preparation method of a compound APS5-CQD provided by the embodiment of the invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.
According to an exemplary embodiment of the present invention, there is provided a compound APS5-CQD, the compound APS5-CQD having the general structural formula:
wherein n is 3 to 5, and the R group is a carbon quantum dot.
As a preferred embodiment, n is 4, specifically, the structural formula of the compound APS5-CQD is as follows:
wherein the R group is an alkyl group.
The carbon quantum dots are a novel luminescent substance, have high fluorescence quantum yield, can form resonance energy transfer with a chemiluminescent substrate to enhance the luminous efficiency of the chemiluminescent substrate, and have rich functional groups, so that the carbon quantum dots can protect the chemiluminescent substrate and enhance the stability of the chemiluminescent substrate. According to the compound APS5-CQD and the preparation method thereof, APS-5 is grafted to a carbon quantum dot to form an enhanced chemiluminescent substrate APS5-CQD, so that the luminescent property and the long-term stability of the substrate are enhanced, the chemiluminescent substrate working solution is optimized, and the preparation process is simplified.
The structural formula of APS-5 is shown below:
n-linked in APS-5 is CH 3 Comparison of structural formulas shows that N is connected with C in the compound IV 4 H 8 NH 2
The compound APS5-CQD of the invention is prepared from amino-NH in compound IV 2 Is obtained by condensation reaction with carboxyl-COOH on carboxyl fluorescent carbon quantum dots, and adopts a compound IV (N in the compound IV is connected with C) 4 H 8 NH 2 ) Without using the carbon chain CH of APS-5 3 Directly connected with carboxyl fluorescent carbon quantum dots because:
the applicant of the present invention found that: the N-connected compound IV with 3-5 carbon atoms is connected with the carboxyl fluorescent carbon quantum dots, so that side reactions are less; therefore, in the formula, n is 3-5; the best connecting effect is achieved when n is 4. If the carbon chain is too long, namely n is more than 5, side reactions are increased; if the carbon chain is too short, namely n is less than 3, the carbon chain is influenced by steric hindrance in the process of connecting with the carbon quantum dot, so that the connecting efficiency is greatly reduced.
According to another exemplary embodiment of the present invention, there is provided a method for preparing compound APS5-CQD, as shown in fig. 1, the method comprising:
s1, obtaining compound IV;
s2, carrying out a grafting reaction on the compound IV and carboxyl fluorescent carbon quantum dots under the action of a condensing agent to obtain a compound APS 5-CQD;
wherein the structural formula of the compound IV is shown as follows:
as a preferred embodiment of the method of the present invention,
in step S1, when n is 4, the obtaining of compound IV specifically includes:
s101, obtaining a compound I;
s102, carrying out a first reaction on the compound I and 4-bromobutyronitrile in an organic solvent A and a catalyst A to obtain a compound II;
s103, reacting the compound II with 3-hydroxypropionitrile and POCl 3 Carrying out a second reaction in an organic solvent B and a catalyst B, and then stirring under an alkaline and anaerobic condition to obtain a compound III;
s104, mixing the compound III with NaBH 4 Carrying out a third reaction under an organic solvent C and a catalyst C to obtain a compound IV;
the structural formula and the reaction formula of the compound I, the compound II and the compound III are as follows:
in step S101, the obtaining of compound I includes:
dissolving 4-chlorobenzene sulfur in CH 2 Cl 2 Dissolving 9-acridine formyl chloride in a pyridine solution in the solution, stirring at room temperature under the protection of argon, and then carrying out solid-liquid separation and washing to obtain a brownish yellow solid;
dissolving the brownish yellow solid in CH 2 Cl 2 And adding zinc powder and acetic acid into the solution under the protection of argon at room temperature to react, and then carrying out solid-liquid separation and washing to obtain the compound I.
In other embodiments, in step S101, the intermediate reagent for reacting compound I to compound II can be 3-bromopropionitrile, 4-bromobutyronitrile, 5-bromovaleronitrile;
in the steps S102-S104, the organic solvent A is sodium hydroxide solution, and the catalyst isA is tetrabutylammonium bromide; the organic solvent B is a tetrahydrofuran solution, and the catalyst B is LDA and pyridine; the organic solvent C is tetrahydrofuran solution, and the catalyst C is InCl 3 And (3) solution.
The first reaction, the second reaction and the third reaction are all carried out under the protection of argon at room temperature.
In the step S1, when n is 3 or 5, the compound IV is obtained by the method described above with reference to n being 4, but specific reaction conditions are slightly different and the compound IV can be searched as needed.
In a preferred embodiment, in step S2,
the condensing agent is DCC.
Before the grafting reaction, the compound IV and the carboxyl fluorescent carbon quantum dots are dissolved in a THF solution, and then a condensing agent is added for the grafting reaction.
The mass ratio of the compound IV to the carboxyl fluorescent carbon quantum dots is (0.5-1): (3-4). The ratio carbon quantum dot-grafted compound IV tends to saturate, resulting in waste of compound IV. Too small a content results in a low chemiluminescence efficiency because the luminescent substance per unit mass contains few groups which can be catalyzed by alkaline phosphatase.
According to another exemplary embodiment of the present invention, there is provided a chemiluminescent substrate fluid having a formulation comprising: 10-300 mM Tris, 0.01-1.0% SDS, 20-1000 mg/L APS5-CQD, 200.01-0.1% Twen, and 8.5-10 pH.
TABLE 1 table of preparation of chemical luminous substrate solution APS5-CQD working solution
The chemiluminescent substrate working solution is simple to prepare and easy to operate.
A compound APS5-CQD of the present application will be described in detail below with reference to examples and experimental data.
The experimental materials used in the examples of the present invention and the comparative examples include:
key chemical reagents: 4-Chlorobenzothiophenol (CAS: 106-54-7); 9-acridinecarbonyl chloride (CAS: 66074-67-7); 4-bromobutyronitrile (CAS: 5332-06-9); lithium diisopropylamide (LDA, CAS: 4111-54-0); 3-hydroxypropionitrile (CAS: 109-78-4).
Example 1
1. The preparation method of the compound APS5-CQD provided by the embodiment of the invention comprises the following steps:
(1) 4-Chlorobenzothiaol (7.43g) and 9-acridinecarbonyl chloride (10.85g) were dissolved in 100mL of CH, respectively 2 Cl 2 And 12.1mL pyridine. Stirring overnight at room temperature under argon, after evaporation of the solvent, the solid was washed with 100mL of hexane, filtered, repeated 2 times, then washed with 50mL of ultrapure water and filtered, repeated 3 times, and air dried. 13.41g of a brownish yellow solid are obtained.
(2) The product (5.0g) from (1) was dissolved in 100mL of CH 2 Cl 2 In (1). Under the protection of argon at room temperature, 9.3g of zinc powder and 0.9mL of acetic acid are added. The reaction was stirred for 20 minutes, filtered, the solid was washed with 100mL of hexane, filtered, repeated 2 times, then washed with 50mL of ultrapure water and filtered, repeated 3 times, and air-dried.
(3) The product (4.0g) obtained in (2) is dissolved in 60mL 50% sodium hydroxide solution, and 5.14g of 4-bromobutyronitrile and 0.8g of tetrabutylammonium bromide are added under the protection of argon at room temperature. 20mL of toluene were added, the mixture was refluxed for 12 hours, extracted three times with 60mL of ethyl acetate, and the organic phase was washed with saturated sodium chloride and then with anhydrous Na 2 SO 4 And (5) drying. Purification by silica gel chromatography with dichloromethane as eluent gave the pure product (2.4 g).
(4) The product (2.0g) from (3) was dissolved in 30mL of anhydrous tetrahydrofuran, added dropwise slowly to the solution of 1.8mL of LDA under argon at room temperature, and stirred in a dry ice bath for 60 min. To this was added slowly dropwise a solution of 1.55g of POCl diluted with 12mL of THF 3 And 4.56mL of a pre-dilution of pyridine. Stirred in a dry ice bath for 30 min. Next, 3-hydroxypropionitrile (2.67g) and 3.0mL of pyridine were added thereto. The mixture was stirred at room temperature overnight under an argon atmosphere and the precipitate was washed with tetrahydrofuran. Will obtainThe filtrate of (2) was evaporated in vacuo and the resulting material was dissolved in ethyl acetate and washed 4 times with 25mL of water. The ethyl acetate solution was then dried and concentrated. Purifying by column chromatography.
(5) The product of (4) (1.2g) was added to 10mL of acetone. 1mL of 2M NaOH was added. The mixture was stirred overnight at room temperature under an argon atmosphere. The precipitate formed was filtered off with suction and air-dried.
(6) The product of (5) (1.0g) was added to 20mL of THF, diluted slowly with 10mL of THF under argon at room temperature, containing 0.3g of NaBH4 and 0.65g of InCl 3 The pre-dilution of (1). After 30 min. Purifying by column chromatography.
(7) The product (0.7g) obtained in (6) and (3.5g) of the carboxyl group-containing carbon quantum dots was added to 25mL of THF, 5mL of 0.2g DCC (THF solution) was added thereto, the reaction was stirred at room temperature for 1 hour, and the precipitate was washed with 10mL of THF and air-dried. 4.0g of APS5-CQD compound was obtained.
The specific synthetic route in this example is as follows:
2. preparation of chemiluminescent substrate solution
1L of chemiluminescent substrate solution was prepared according to Table 2.
TABLE 2 table of preparation of chemical luminous substrate solution APS5-CQD working solution
Comparative example 1
1. The luminescent substrate of the comparative example of the present invention was compound APS-5;
2. preparation of chemiluminescent substrate solution: 1L of chemiluminescent substrate solution was prepared according to Table 2.
Comparative example 2
1. The luminescent substrate of the comparative example of the present invention was compound APS-5;
2. preparation of chemiluminescent substrate solution: 1L of chemiluminescent substrate solution was prepared according to Table 3.
TABLE 3 table of working solution preparation for chemiluminescent substrate solution APS5
Experimental example 1
The chemiluminescent substrate solutions obtained in the examples and comparative examples were evaluated:
1. detection of the Linear relationship of luminous intensity to ALP
(1) An aliquot of 50000pg/mL ALP solution was diluted 5-fold with an enzyme diluent (formula for the enzyme diluent is shown in Table 4). Diluted 6 times to 8 concentration points including 0 concentration. Respectively adding 10uL of ALP solution with different concentrations into the bottom of a test tube, then adding 300uL of substrate, vortex uniformly mixing, immediately loading to a detection position of a luminescence detector of a MODULUS luminescence detector of TURER BIOSYSTEMS company, and detecting to obtain the luminescence intensity. The substrates of different examples 1, comparative examples 1 and comparative examples 2 were analyzed for their emission intensity as a function of ALP concentration.
Table 4: enzyme diluent preparation table
(2) The results of the linear relationship of the luminous intensity of the different substrates with ALP are shown in Table 5.
Table 5: linear relationship of different substrate luminescence intensity to ALP
As is clear from the data in Table 5, in comparison with example 1, in comparative example 1, the luminescence value was much lower than that of example 1 at the same concentration of ALP, because QDS was known as a fluorescence enhancer of APS-5 structure in APS5-CQD particles. Meanwhile, in comparison with example 1, in case of the same concentration of ALP, the luminous value of example 1 is still 1.6 times higher than that of comparative example 2, because QDS is combined with the modified APS-5 structure through chemical bonds, the resonance energy transfer effect is stronger. And the QDS has higher fluorescence quantum yield than the lucigenin, and is more suitable for being used as a fluorescence enhancer.
2. Substrate stability assay
(1) The substrates of example 1, comparative example 1 and comparative example 2 were dispensed in 100mL portions per bottle, placed at 25 ℃ and tested on days 1, 3, 7, 14 and 30, respectively. Luminescence values of different substrates were measured with ALP of 2ng/mL for 3 replicates each and the average was calculated.
(2) The results of the luminescent substrate stability measurements are shown in table 6:
table 6: substrate stability
As can be seen from the above data, example 1 compares comparative example 1 and comparative example 2, the droop was-5.90% in example 1 at 30 days; comparative example 1 and comparative example 2 accelerated by-11.90% and-12.30%, respectively, over 30 days.
In conclusion, the compound APS5-CQD and the preparation method thereof provided by the invention enhance the luminescence property and stability of the substrate, optimize the working solution of the chemiluminescent substrate and simplify the preparation process.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A compound APS5-CQD, wherein the structural formula of the compound APS5-CQD is as follows:
wherein n = 3-5, and the R group is a carbon quantum dot.
2. The compound APS5-CQD according to claim 1, wherein n = 4.
3. A method for preparing the compound APS5-CQD of any one of claims 1-2, comprising:
obtaining a compound IV;
carrying out grafting reaction on the compound IV and carboxyl fluorescent carbon quantum dots under the action of a condensing agent to obtain a compound APS 5-CQD;
wherein the structural formula of the compound IV is shown as follows:
wherein n =3 to 5.
4. The method of claim 3, wherein the condensing agent is DCC, for preparing the compound APS 5-CQD.
5. The method of claim 3, wherein the compound of APS5-CQD is prepared by dissolving the compound IV and the carboxyfluorescing carbon quantum dot in THF solution before the grafting reaction, and then adding a condensing agent to perform the grafting reaction.
6. The method for preparing the compound APS5-CQD according to claim 3, wherein the mass ratio of the compound IV to the carboxyl fluorescent carbon quantum dots is (0.5-1): (3-4).
7. The method for preparing the compound APS5-CQD according to claim 3, wherein the step of obtaining the compound IV, when n =4, specifically comprises:
obtaining a compound I;
carrying out a first reaction on the compound I and 4-bromobutyronitrile in an organic solvent A and a catalyst A to obtain a compound II;
reacting the compound II with 3-hydroxypropionitrile and POCl 3 Carrying out a second reaction in an organic solvent B and a catalyst B, and then stirring under an alkaline and anaerobic condition to obtain a compound III;
reacting said compound III with NaBH 4 Carrying out a third reaction under an organic solvent C and a catalyst C to obtain a compound IV;
the structural formula and the reaction formula of the compound I, the compound II and the compound III are as follows:
8. the method of claim 7, wherein obtaining compound I comprises:
dissolving 4-chlorobenzene sulfur in CH 2 Cl 2 Dissolving 9-acridine formyl chloride in pyridine solution, stirring at room temperature under argon protection, and solidifyingLiquid separation and washing to obtain a slightly brownish yellow solid;
dissolving the brownish yellow solid in CH 2 Cl 2 And adding zinc powder and acetic acid into the solution under the protection of argon at room temperature to react, and then carrying out solid-liquid separation and washing to obtain the compound I.
9. The method for preparing the compound APS5-CQD according to claim 7, wherein the organic solvent A is sodium hydroxide solution and the catalyst A is tetrabutylammonium bromide; the organic solvent B is a tetrahydrofuran solution, and the catalyst B is LDA and pyridine; the organic solvent C is tetrahydrofuran solution, and the catalyst C is InCl 3 And (3) solution.
10. A chemiluminescent substrate solution is characterized by comprising the following components in percentage by weight: tris 10-300 mM, SDS 0.01-1.0%, APS5-CQD 20-1000 mg/L, Twen 200.01% -0.1%, pH8.5-10 as described in claim 1 or 2.
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Denomination of invention: A compound APS5 CQD and its preparation method, chemiluminescent substrate liquid Granted publication date: 20220819 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: WUHAN LIFE ORIGIN BIOTECH JOINT STOCK Co.,Ltd. Registration number: Y2024980009814 |