CN118005625A - Compound for reticulocyte staining, preparation method of compound, fluorescent dye solution, detection reagent and detection method - Google Patents
Compound for reticulocyte staining, preparation method of compound, fluorescent dye solution, detection reagent and detection method Download PDFInfo
- Publication number
- CN118005625A CN118005625A CN202211389918.0A CN202211389918A CN118005625A CN 118005625 A CN118005625 A CN 118005625A CN 202211389918 A CN202211389918 A CN 202211389918A CN 118005625 A CN118005625 A CN 118005625A
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- China
- Prior art keywords
- compound
- alkyl
- reticulocyte
- fluorescent dye
- detection reagent
- Prior art date
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 114
- 210000001995 reticulocyte Anatomy 0.000 title claims abstract description 112
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 102
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 91
- 238000001514 detection method Methods 0.000 title claims abstract description 67
- 238000010186 staining Methods 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title abstract description 18
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 111
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 36
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 36
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 21
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 21
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 21
- 229910052736 halogen Chemical group 0.000 claims abstract description 17
- 150000002367 halogens Chemical class 0.000 claims abstract description 14
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 14
- 125000004185 ester group Chemical group 0.000 claims abstract description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 12
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims abstract description 12
- 125000001033 ether group Chemical group 0.000 claims abstract description 12
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 12
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 60
- 239000003085 diluting agent Substances 0.000 claims description 39
- -1 quinoline aromatic compound Chemical class 0.000 claims description 35
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 24
- 239000000523 sample Substances 0.000 claims description 21
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 239000003093 cationic surfactant Substances 0.000 claims description 15
- 239000002736 nonionic surfactant Substances 0.000 claims description 14
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 11
- 239000003755 preservative agent Substances 0.000 claims description 11
- 230000002335 preservative effect Effects 0.000 claims description 10
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 8
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 8
- 239000012472 biological sample Substances 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 7
- 239000012895 dilution Substances 0.000 claims description 7
- 150000004820 halides Chemical class 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- 229910052711 selenium Inorganic materials 0.000 claims description 7
- 229910052717 sulfur Inorganic materials 0.000 claims description 7
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 6
- 239000002280 amphoteric surfactant Substances 0.000 claims description 6
- 239000002563 ionic surfactant Substances 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 claims description 6
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 6
- 159000000000 sodium salts Chemical class 0.000 claims description 6
- 239000004094 surface-active agent Substances 0.000 claims description 6
- 239000005711 Benzoic acid Substances 0.000 claims description 5
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 5
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 claims description 5
- 235000010233 benzoic acid Nutrition 0.000 claims description 5
- 125000006702 (C1-C18) alkyl group Chemical group 0.000 claims description 4
- OBTZDIRUQWFRFZ-UHFFFAOYSA-N 2-(5-methylfuran-2-yl)-n-(4-methylphenyl)quinoline-4-carboxamide Chemical compound O1C(C)=CC=C1C1=CC(C(=O)NC=2C=CC(C)=CC=2)=C(C=CC=C2)C2=N1 OBTZDIRUQWFRFZ-UHFFFAOYSA-N 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- SMWDFEZZVXVKRB-UHFFFAOYSA-N anhydrous quinoline Natural products N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- CEYULKASIQJZGP-UHFFFAOYSA-L disodium;2-(carboxymethyl)-2-hydroxybutanedioate Chemical compound [Na+].[Na+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O CEYULKASIQJZGP-UHFFFAOYSA-L 0.000 claims description 4
- MGIYRDNGCNKGJU-UHFFFAOYSA-N isothiazolinone Chemical compound O=C1C=CSN1 MGIYRDNGCNKGJU-UHFFFAOYSA-N 0.000 claims description 4
- XXTISPYPIAPDGY-UHFFFAOYSA-N n,n-diphenylmethanimidamide Chemical compound C=1C=CC=CC=1N(C=N)C1=CC=CC=C1 XXTISPYPIAPDGY-UHFFFAOYSA-N 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
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- 125000005843 halogen group Chemical group 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 2
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical group C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims 1
- 238000004043 dyeing Methods 0.000 abstract description 11
- 239000000243 solution Substances 0.000 description 70
- 239000000975 dye Substances 0.000 description 44
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 42
- 239000000203 mixture Substances 0.000 description 38
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 35
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 27
- 238000006243 chemical reaction Methods 0.000 description 22
- 210000003743 erythrocyte Anatomy 0.000 description 22
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 21
- 210000000601 blood cell Anatomy 0.000 description 19
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 18
- 210000004185 liver Anatomy 0.000 description 18
- 238000005259 measurement Methods 0.000 description 18
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 16
- 238000001816 cooling Methods 0.000 description 14
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- 150000001450 anions Chemical class 0.000 description 11
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- 125000006545 (C1-C9) alkyl group Chemical group 0.000 description 10
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- 238000005160 1H NMR spectroscopy Methods 0.000 description 9
- 229940126062 Compound A Drugs 0.000 description 9
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 description 9
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- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 9
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- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 description 6
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- FDCJDKXCCYFOCV-UHFFFAOYSA-N 1-hexadecoxyhexadecane Chemical compound CCCCCCCCCCCCCCCCOCCCCCCCCCCCCCCCC FDCJDKXCCYFOCV-UHFFFAOYSA-N 0.000 description 4
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- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 150000002891 organic anions Chemical class 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 1
- 229910001948 sodium oxide Inorganic materials 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 229940117986 sulfobetaine Drugs 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- ACOJCCLIDPZYJC-UHFFFAOYSA-M thiazole orange Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1.C1=CC=C2C(C=C3N(C4=CC=CC=C4S3)C)=CC=[N+](C)C2=C1 ACOJCCLIDPZYJC-UHFFFAOYSA-M 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a compound for reticulocyte staining, a preparation method of the compound, fluorescent dye, a detection reagent and a detection method, wherein the compound has the following general formula: Wherein: m is 1,2 or 3; n is an integer of 1 to 18; y is C (CH 3)2, O, S OR Se; R 1 and R 2 are respectively and independently selected from hydrogen atom, alkyl chain with hydroxyl, alkyl chain with ether group, alkyl chain with ester group and halogen substituted alkyl chain, and R 1 and R 2 are alkyl groups with different carbon numbers when being alkyl chains, R 3 is C 1‑18 alkyl, benzyl OR (CH 2)nNR1R2;R4 and R 5 are respectively and independently selected from H, C 1‑18 alkyl, OR 6 OR halogen; R 6 is H OR C 1‑18 alkyl; Z ‑ is negative ion), the compound, fluorescent dye solution and detection reagent thereof have lower fluorescence background when no nucleic acid exists, have higher fluorescence quantum efficiency after being combined with nucleic acid in reticulocyte, and are used for RNA specific dyeing of the reticulocyte and improve resolution of the reticulocyte.
Description
Technical Field
The invention relates to the technical field of biological sample dyeing, in particular to a compound for reticulocyte dyeing, a preparation method of the compound, fluorescent dye liquor, a detection reagent and a detection method.
Background
Most automatic counters now measure reticulocyte numbers by nucleic acid staining. Such as flow cytometry, to detect thiazole orange stained cells. Currently, basic pigments are generally used to color RNA in reticulocytes for identification and enumeration. There are generally two methods, non-fluorescent labeling and fluorescent labeling.
Non-fluorescent labeling and fluorescent labeling generally employ a combination of two reagents, one of which is a spheroidizing reagent for spheroidizing red blood cells, reticulocytes, and platelets, and the other of which is a non-fluorescent dye or fluorescent dye for selectively staining reticulocytes. Fluorescent dyes currently in use are susceptible to fluorescence quenching and photobleaching. Fluorescence quenching refers to the phenomenon that fluorescent dyes interfere with each other due to molecular aggregation or too close a distance between them, and the emitted fluorescent signal becomes weak. Fluorescence quenching is a reversible process, and if the distance between molecules is increased, the fluorescence intensity is recovered. Even though the fluorescence quenching complex may dissociate in the excited state, the dissociation of the complex may be slower. Fluorescence quenching occurs mainly in such cases as dye molecules being insoluble, or dye molecules being too concentrated, and dye molecule labeling being too dense to the surface of the macromolecule.
Fluorescence bleaching is manifested as chemical structural destruction of the dye fluorescent parent nucleus. Under the high energy of the excited state, the chemical bond part of the dye parent nucleus is broken, so that the structure of the parent nucleus which emits fluorescence is destroyed, and the fluorescence can not be emitted any more. Fluorescence bleaching is a common phenomenon, and almost all dyes have bleaching phenomena, but the difficulty of fluorescence bleaching is greatly different due to the difference of chemical structures and luminous principles. In addition to photobleaching, fluorescent dyes tend to break down in chemical structure at higher temperatures. Therefore, in the production, the raw materials of the fluorescent dye are protected from light and kept at a low temperature. Avoiding that the fluorescent dye can react with surrounding particles more easily after obtaining energy from the environment, thereby destroying the structure.
RNA fluorescent dye used for detecting biological samples of reticulocytes at present has high dye concentration and large dosage. The fluorescent dye is easy to generate fluorescence quenching phenomenon under high concentration to reduce the fluorescence intensity, so that the utilization rate of the fluorescent dye is reduced. The fluorescent dye with high concentration has great adhesive force to the plastic tube in the flow cytometer, which results in increased fluorescent background and pollution to the pipeline and raised cleaning difficulty. In blood detection, when the nonspecific fluorescence of erythrocytes becomes large and the specific fluorescence of reticulocytes becomes small, the distinction between erythrocytes and reticulocytes becomes difficult, and the sorting accuracy is reduced. The ideal reticulocyte detection reagent needs to meet the conditions of specific staining of reticulocytes, simultaneous inhibition of nonspecific staining of erythrocytes, low staining concentration, stable dye molecules and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a compound for reticulocyte staining, a preparation method of the compound, fluorescent dye solution, a detection reagent and a detection method, wherein the compound for reticulocyte has the following advantages: the fluorescent dye has lower fluorescent background in the absence of nucleic acid, higher fluorescent quantum yield after being combined with the nucleic acid in reticulocyte detection, and no affinity to biomolecules other than the nucleic acid; has higher photo-thermal stability and is not easy to generate photo-bleaching phenomenon. Meanwhile, fluorescent dye molecules do not aggregate under the high-concentration condition; the spectral range is greatly different from that of biological samples.
The technical scheme adopted for solving the technical problems is as follows: a compound for reticulocyte staining having the following structural formula I:
Wherein:
m is 1,2 or 3;
n is an integer of 1 to 18;
Y is C (CH 3)2, O, S or Se;
R 1 and R 2 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, an alkyl chain having a hydroxyl group, an alkyl chain having an ether group, an alkyl chain having an ester group, a halogen-substituted alkyl chain, and R 1 and R 2 are not alkyl groups of the same carbon number when both are alkyl chains;
R 3 is C 1-18 alkyl, benzyl or (CH 2)nNR1R2;
R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 OR halogen;
R 6 is H or C 1-18 alkyl;
Z - is halide or OTs -.
Further, it is preferable that R 1 and R 2 are the same or different and are each independently selected from a hydrogen atom, a C 1-9 alkyl group, a C 1-9 alkyl chain having a hydroxyl group, a C 1-9 alkyl chain having an ether group, a C 1-9 alkyl chain having an ester group, a halogen-substituted C 1-9 alkyl chain, and R 1 and R 2 are not alkyl groups of the same carbon number when they are both alkyl chains.
Further, it is preferable that Z - is a halogen anion generated by the reaction or OTs -.
Further, n is preferably an integer of 2 to 9.
Further, preferably R 4 and R 5 are each independently selected from H, C 2-9 alkyl, OR 6 OR halogen; r 6 is H or C 2-9 alkyl.
Further, the preferred compounds are:
Further, the preferred compounds are:
Further, the preferred compounds are:
The invention also provides a fluorescent dye for dyeing reticulocytes, which comprises the compound and alcohol, wherein the alcohol is at least one of isopropyl alcohol and ethylene glycol, and the compound is a polymethine dye and has the following general formula I:
Wherein:
m is 1,2 or 3;
n is an integer of 1 to 18;
Y is C (CH 3)2, O, S or Se;
R 1 and R 2 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, an alkyl chain having a hydroxyl group, an alkyl chain having an ether group, an alkyl chain having an ester group, a halogen-substituted alkyl chain, and R 1 and R 2 are not alkyl groups of the same carbon number when both are alkyl chains;
R 3 is C 1-18 alkyl, benzyl or (CH 2)nNR1R2;
R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 OR halogen;
R 6 is H or C 1-18 alkyl;
Z - is halide or OTs -.
Further, the concentration of the compound in the fluorescent dye is preferably 0.005 to 1g/L.
The invention also provides a preparation method of the compound for reticulocyte staining, which comprises the following steps: s1, reacting a quinoline aromatic compound with R 5 Z to prepare a first quaternary ammonium salt intermediate III;
S2, preparing a first quaternary ammonium salt intermediate III and N, N-diphenyl formamidine to obtain a compound IV;
S3, preparing a second quaternary ammonium salt intermediate VI from the 2-methylbenzo five-membered ring and R 1R2N(CH2)n Br;
s4, reacting the IV compound with a second quaternary ammonium salt intermediate VI to obtain a compound of a formula VII:
S5, carrying out anion replacement on the compound VII and sodium salt or potassium salt containing Y - to obtain a compound of the formula I:
Wherein m is 1, 2 or 3;
n is an integer of 1 to 18;
Y is C (CH 3)2, O, S or Se;
R 1 and R 2 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, an alkyl chain with a hydroxyl group, an alkyl chain with an ether group, an alkyl chain with an ester group, a halogen-substituted alkyl chain, and R 1 and R 2 are not C 1-18 alkyl groups of the same carbon number when both are alkyl chains;
r 3 is C 1-18 alkyl, benzyl or (CH 2)nNR1R2;
R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 OR halogen;
R 6 is H or C 1-18 alkyl;
Z - is halide or OTs -.
The invention also discloses a reticulocyte detection reagent, which comprises a diluent and a fluorescent dye solution for reticulocyte staining, wherein the pH of the diluent is 8-9, and the diluent comprises a buffer system, inorganic salt, multivalent anion salt, a surfactant and a preservative.
Further, it is preferable that the concentration of the buffer system in the diluent is 0.1g to 10g/L, and the buffer system is one or more of a phosphate buffer system, a tris buffer system and an N-tris (hydroxymethyl) methylglycine buffer system.
Further, it is preferable that the concentration of the inorganic salt in the diluent is 1 to 30g/L, and the inorganic salt is one or more of sodium hydrogen citrate, sodium chloride and potassium chloride. In order to prevent hemolysis of erythrocytes under low osmotic pressure, the reagent of the present invention is usually prepared by adjusting the osmotic pressure to 150mOsm/kg to 600mOsm/kg with an inorganic salt. Preferably, the osmotic pressure is adjusted to between 200 and 250 mOsm/kg.
Further, it is preferable that the concentration of the polyvalent anion salt in the dilution is 0.1 to 10g/L, and the polyvalent anion salt is at least one of sulfate SO 4 2-, phosphate PO 4 3-, sodium salt or potassium salt of phosphite PO 3 2-.
Further, it is preferable that the surfactant is at least one of a nonionic surfactant, an amphoteric surfactant, a cationic surfactant, and a negative surfactant.
Further, it is preferable that the nonionic surfactant is a polyoxyethylene nonionic surfactant, and the concentration of the nonionic surfactant in the diluent is 0.01 to 5g/L; the cationic surfactant is quaternary ammonium salt cationic surfactant, and the concentration of the cationic surfactant in the diluent is 0.1-10g/L; the ampholytic surfactant is betaine type ampholytic surfactant, and the concentration of the ampholytic surfactant in the diluent is 0.1-5g/L.
Further, the preservative is preferably one or more of sodium azide, benzoic acid and salts thereof, bisimidazolidinyl urea and isothiazolinone, the common use amount of the preservative is 0.05-5g/L, different concentrations of different preservatives can be selected, and the normal use of the reagent in the effective period is ensured.
The invention also discloses a reticulocyte detection method, which comprises the following steps: the reticulocyte detection reagent is adopted to treat a sample to be detected, and the detection reagent is used to spheroidize reticulocytes in the sample to be detected, so that nucleic acid of the reticulocytes is specifically combined with fluorescent dye liquor, and the reticulocyte count is realized.
Further, the detection method further comprises: and (3) preparing the fluorescent dye solution and the diluent into preset volume according to a certain proportion, uniformly stirring at normal temperature, filtering by using a 0.22um filter membrane, and detecting a sample to be detected by using the filtered filtrate.
The invention also provides an application of the fluorescent dye solution or the reticulocyte detection reagent for dyeing the reticulocyte in dyeing the biological sample.
The invention has the beneficial effects that: the compound provided by the invention has good dyeing effect on reticulocytes in biological products, and the fluorescent dye prepared by the compound provided by the invention has lower fluorescent background when no nucleic acid exists, and has higher fluorescent quantum efficiency after being combined with the nucleic acid. The nucleic acid of the reticulocyte is still specifically stained when the concentration of the fluorescent dye (compound) is as low as 0.005g/L, and the reduction of the reagent concentration can reduce the pollution of the dye to the pipeline in the blood cell analyzer. The dye has good thermal stability, and the improvement of the stability is beneficial to reducing the failure risk in the use process of the dye for customers. When the anions in the diluent of the detection reagent are multivalent anions (sulfate SO 4 2-, phosphate PO 4 3- and phosphite PO 3 2-), the nonspecific staining of erythrocytes is inhibited, and the specific staining of reticulocytes by combining with fluorescent dyes is easy to distinguish erythrocytes from reticulocytes. The fluorescent dye prepared by the compound of the invention increases the fat solubility on thiazole rings by introducing long-chain alkyl groups so as to improve the cell membrane permeability of the dye, meanwhile, the fluorescent dye is combined with nucleic acid in reticulocytes in a high-efficiency and specific manner, the erythrocyte groups are obviously distinguished from the reticulocytes, and in addition, multivalent anions of the diluent in the reagent can inhibit nonspecific fluorescence of the erythrocytes. The fluorescent dye and the diluent act together to improve the accuracy of reticulocyte count.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a graph showing fluorescence emission spectra before and after binding of Compound A of example 10 of the present invention with Amyda sinensis liver RNA;
FIG. 2 is a graph showing fluorescence emission spectra before and after binding of Compound B of example 11 to Amyda sinensis liver RNA according to the present invention;
FIG. 3 is a graph showing fluorescence emission spectra before and after binding of Compound C of example 12 with Amyda sinensis liver RNA according to the present invention;
FIG. 4 is a graph of RET channel measured on a complete set of blood cell analyzers for sample 1 when the XN-1000RET channel dye solution of Hissen-Meikang is diluted to the same concentration as the dye solution of the present invention;
FIG. 5 is a graph of RET channel measured on a complete set of blood cell analyzers for sample 2 when the XN-1000RET channel dye solution of Hissen-Meikang is diluted to the same concentration as the dye solution of the present invention;
FIG. 6 is a graphical representation of RET channels of sample 1 measured on a companion blood cell analyzer using the first reagent of example 16 in accordance with the present invention;
FIG. 7 is a graphical representation of RET channels of sample 2 measured on a companion blood cell analyzer using the first reagent of example 16 in accordance with the present invention;
FIG. 8 is a graphical representation of RET channels of sample 1 measured on a companion blood cell analyzer using the second reagent of example 17 in accordance with the present invention;
FIG. 9 is a graphical representation of RET channels of sample 2 measured on a companion blood cell analyzer using the second reagent of example 17 in accordance with the present invention;
FIG. 10 is a graphical representation of RET channels of sample 1 measured on a companion blood cell analyzer using the third reagent of example 18 in accordance with the present invention;
FIG. 11 is a graphical representation of RET channels of sample 2 measured on a companion blood cell analyzer using the third reagent of example 18 in accordance with the present invention;
FIG. 12 is a graphical representation of RET channels of sample 1 measured on a companion blood cell analyzer using the fourth reagent of example 19 in accordance with the present invention;
FIG. 13 is a graph showing RET pathway of sample 2 measured on a complete blood cell analyzer using the fourth reagent of example 19 according to the present invention.
Detailed Description
Specific embodiments of the present invention will now be described in detail for a clearer understanding of the technical features, objects and effects of the present invention.
A compound for reticulocyte staining having the following structural formula I:
Wherein:
m is 1,2 or 3;
n is an integer of 1 to 18;
Y is C (CH 3)2, O, S or Se;
R 1 and R 2 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, an alkyl chain with a hydroxyl group, an alkyl chain with an ether group, an alkyl chain with an ester group, a halogen-substituted alkyl chain, and R 1 and R 2 are not alkyl groups of the same carbon number when both are alkyl chains;
The term "alkyl" as used herein includes both straight chain alkyl and branched alkyl groups. If a single alkyl group such as "propyl" is mentioned, only straight chain alkyl groups are specified, if a single branched alkyl group such as "isopropyl" is mentioned, only branched alkyl groups are specified. For example, "C 1-6 alkyl" includes C 1-4 alkyl, C 1-3 alkyl, methyl, ethyl, n-propyl, isopropyl, and t-butyl. Similar rules apply to other groups used in this specification;
R 3 is C 1-18 alkyl, benzyl or (CH 2)nNR1R2; the term "benzyl" as used herein refers to the-CH 2 -Ph group when the benzyl group is modified with "optional substitution," it is intended that the benzyl group may exist in unsubstituted form or may be substituted at any suitable position with suitable substituents including, but not limited to, halogen, hydroxy, mercapto, cyano, nitro, alkyl, aryl, alkoxy, heterocyclyl, haloalkyl, amino, alkylamino, amido, carboxyl, etc., provided that the resulting compound has the desired properties of the invention.
R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 OR halogen;
R 6 is H or C 1-18 alkyl;
Z - is halide or OTs -.
The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.
Z is used herein to represent an anion, which may be any suitable anion, including but not limited to inorganic anions or organic anions
Further, it is preferable that R 1 and R 2 are the same or different and are each independently selected from a hydrogen atom, a C 1-9 alkyl group, a C 1-9 alkyl chain having a hydroxyl group, a C 1-9 alkyl chain having an ether group, a C 1-9 alkyl chain having an ester group, a halogen-substituted C 1-9 alkyl chain, and R 1 and R 2 are not alkyl groups of the same carbon number when they are both alkyl chains.
Further, preferably R 4 and R 5 are each independently selected from H, C 2-9 alkyl, OR 6 OR halogen; r 6 is H or C 2-9 alkyl.
Further, preferred compounds are:
further, preferred compounds are:
further, preferred compounds are:
The compounds of the invention can be used directly as salt forms herein for staining biological samples. Alternatively, the compounds of the present invention may be used as derivatives of the compounds of formula I, including but not limited to conjugates. As used herein, "conjugate" refers to a compound of the general formula of the present invention that is formed by a covalent bond to other molecules.
The invention also provides a composition containing the compound of the formula I, the composition is formed by combining at least two compounds, and the composition is used for dyeing reticulocytes.
The invention also provides a fluorescent dye for dyeing reticulocytes, which comprises a compound and alcohol, wherein the alcohol is at least one of isopropyl alcohol and ethylene glycol, and the compound is a polymethine dye and has the following general formula I:
Wherein:
m is 1,2 or 3;
n is an integer of 1 to 18;
Y is C (CH 3)2, O, S or Se;
R 1 and R 2 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, an alkyl chain having a hydroxyl group, an alkyl chain having an ether group, an alkyl chain having an ester group, a halogen-substituted alkyl chain, and R 1 and R 2 are not alkyl groups of the same carbon number when both are alkyl chains;
R 3 is C 1-18 alkyl, benzyl or (CH 2)nNR1R2;
R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 OR halogen;
R 6 is H or C 1-18 alkyl;
Z - is halide or OTs -.
Further, the concentration of the compound in the fluorescent dye is preferably 0.005 to 1g/L. The amount of the fluorescent dye (compound) in the reagent of the present invention can be appropriately adjusted by the kind of the dye and the combination of the reagents. The compound and the composition have good dyeing effect on the reticulocytes of biological products, and the fluorescent dye liquor prepared by the compound and the composition has good dyeing effect on the reticulocytes in biological products. The nucleic acid of the reticulocyte is still specifically stained when the concentration of the fluorescent dye (compound) is as low as 0.005g/L, and the reduction of the reagent concentration can reduce the pollution of the dye to the pipeline in the blood cell analyzer. The dye has good thermal stability, and the improvement of the stability is beneficial to reducing the failure risk in the use process of the dye for customers. When the anions in the diluent in the detection reagent are multivalent anions (sulfate SO 4 2-, phosphate PO 4 3- and phosphite PO 3 2-), the nonspecific staining of erythrocytes is inhibited, and the specific staining of reticulocytes by combining with fluorescent dyes is easy to distinguish erythrocytes from reticulocytes. The fluorescent dye prepared by the compound of the invention increases the fat solubility on thiazole rings by introducing long-chain alkyl groups so as to improve the cell membrane permeability of the dye, meanwhile, the fluorescent dye is combined with nucleic acid in reticulocytes in a high-efficiency and specific manner, the erythrocyte groups are obviously distinguished from the reticulocytes, and in addition, multivalent anions of the diluent in the reagent can inhibit nonspecific fluorescence of the erythrocytes. The fluorescent dye and the diluent act together to improve the accuracy of reticulocyte count.
The invention also provides a reticulocyte detection reagent, which comprises a diluent and the fluorescent dye for reticulocyte staining, wherein the pH value of the diluent is controlled to be 8-10. When the pH is too low in the above range, the erythrocytes tend to be fragile and hemolyzed, and the amount of broken erythrocytes may be increased. And when the pH value is too high, the acidic functional groups on the erythrocyte membrane are easy to dissociate and combine with the cationic pigment, so that the non-specific staining opportunity of broken erythrocytes is increased.
In order to ensure that the pH of the diluent is stable in the range of 8 to 10, a buffer system may be used for adjustment. Thus, the diluent comprises a buffer system for maintaining the pH, the buffer system being selected from one or more of a phosphate buffer system, a tris buffer system, an N-tris (hydroxymethyl) methylglycine buffer system, and the like, the buffer system being present in a concentration of 0.1g to 10g/L, preferably 1g to 5g/L.
In order to adjust the osmolarity of the detection reagent of the present invention to a range suitable for hemolysis of erythrocytes, the osmolarity of the dilution is adjusted to physiological osmolarity (e.g., 150-600 mOsm/kg). The diluent thus also comprises an inorganic salt, which may be selected from one or more of sodium hydrogen citrate, sodium oxide and potassium chloride, in a concentration of 1-30g/L, preferably 10-20g/L.
For inhibiting nonspecific staining of erythrocytes, the diluent further comprises a multivalent anion salt, preferably at least one of sulfate SO 4 2-, phosphate PO 4 3-, sodium salt or potassium salt of phosphite PO 3 2-, and the concentration of the multivalent anion salt is 0.1-10g/L, preferably 0.5-2g/L; the addition of the multivalent anion salt inhibits the nonspecific staining of the erythrocytes, and the specific staining of the fluorescent dye to the reticulocytes is improved.
In order to significantly reduce the surface tension of the detection reagent, the diluent further comprises a surfactant, which may be at least one of a nonionic surfactant, an amphoteric surfactant, and a cationic surfactant. Cationic surfactants quaternary ammonium cationic surfactants are commonly used. The quaternary ammonium salt cationic surfactant includes at least one of dodecylmonoammonium bromide (DTAB), dodecyltrimethylammonium bromide (CTAB) and octyltrimethylammonium bromide (OTAB), and the cationic surfactant is used at a concentration of 0.1g to 10g/L, wherein the preferred concentration of OTAB is 1g to 10g/L, the concentration of CTAB is 0.5g to 5g/L, and the concentration of DTAB is 0.1g to 3g/L, and the amount of the cationic surfactant used decreases as the total carbon number of the cationic surfactant increases. The amphoteric surfactant can be betaine type amphoteric surfactant, and is usually at least one of laurylbetaine, tetradecylbetaine and lauramidopropyl betaine, and the concentration of the amphoteric surfactant is 0.1g-5g/L. The nonionic surfactant can be polyoxyethylene nonionic surfactant, the concentration of the nonionic surfactant is 0.1-5g/L, preferably 0.1-2g/L, and the common polyoxyethylene nonionic surfactant comprises polyethylene glycol cetyl ether (n=20), polyethylene glycol cetyl ether (n=30), polyethylene glycol monostearate (n=25) and polyoxyethylene oil ether (n=20), and the nonionic surfactant can play roles in dispersion and solubilization.
The diluent also comprises a preservative, the concentration of the preservative in the diluent is 0.5-4g/L, the preservative can effectively control the growth of microorganisms in the reagent, and the preservative is one or more of sodium azide, benzoic acid and salts thereof, bisimidazolidinyl urea, isothiazolinone and the like.
The invention also provides a reticulocyte detection method, which comprises the following steps: by adopting the reticulocyte detection reagent, the sample to be detected is treated by the detection reagent, and the detection reagent spheroidizes the erythrocytes in the sample to be detected, so that the nucleic acid of the reticulocyte is specifically combined with the compound in the fluorescent dye solution, and the reticulocyte count is realized.
Further, the detection method further comprises: after the fluorescent dye solution and the diluent are prepared into preset volume according to a certain proportion, stirring uniformly at normal temperature, filtering by using a 0.22um filter membrane, and detecting a sample to be detected by the filtered filtrate.
The invention also provides an application of the fluorescent dye solution or the reticulocyte detection reagent for reticulocyte staining in the biological sample.
The invention also discloses a preparation method of the compound for reticulocyte staining, which comprises the following steps:
M in the following is 1,2 or 3; n is an integer of 1 to 18; y is C (CH 3)2, O, S OR Se; R 1 and R 2 are identical OR different and are each independently selected from the group consisting of hydrogen atom, alkyl group, alkyl chain with hydroxyl group, alkyl chain with ether group, alkyl chain with ester group, halogen-substituted alkyl chain, and R 1 and R 2 are not alkyl groups of the same carbon number when they are both alkyl chains; R 3 is C 1-18 alkyl, benzyl OR (CH 2)nNR1R2;R4 and R 5 are each independently selected from the group consisting of H, C 1-18 alkyl, OR 6 OR halogen; R 6 is H OR C 1-18 alkyl; Z - is a halide anion OR OTs-.
S1, preparing a first quaternary ammonium salt intermediate III by adopting a quinoline aromatic compound II to react with R 5 Z; for example, 4-methylquinoline and ethyl iodide were added in a molar ratio of 1:1.2 to a round bottom flask containing 20mL toluene under argon. The reaction was heated to reflux for 8h and stopped. The mixture was cooled and the precipitate was filtered and the filter cake was washed thoroughly with diethyl ether. After drying in vacuo, a pale yellow solid powder was obtained.
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S2, preparing the first quaternary ammonium salt intermediate III obtained in the step S1 and N, N-diphenyl formamidine to obtain a compound IV, specifically, heating and stirring the first quaternary ammonium salt intermediate III and the N, N-diphenyl formamidine on an oil bath at 160 ℃ according to a molar ratio of 1:1, and reacting in a molten state for 0.5h. The reddish brown solid obtained by the reaction was repeatedly washed with diethyl ether and recrystallized from ethanol (100 mL) to obtain compound IV.
S3, obtaining a second quaternary ammonium salt intermediate VI through the 2-methylbenzene five-membered ring and R 1R2N(CH2)n Br; specifically, the molar ratio of the 2-methylbenzo five-membered ring to R 1R2N(CH2)n Br is 1:1.2 into a round bottom flask containing 20mL of toluene under argon. The reaction was heated to reflux for 14h and stopped. The mixture was cooled and the precipitate was filtered and the filter cake was washed thoroughly with diethyl ether. And (5) obtaining a second quaternary ammonium salt intermediate VI after vacuum drying.
S4, reacting the IV compound obtained in the S2 step with the second quaternary ammonium salt intermediate VI obtained in the S2 step to obtain a compound of a formula VII;
Carrying out anion replacement on the compound VII and Y - -containing or potassium salt to obtain a compound of the formula I:
the compound product of the formula I prepared by the method can be confirmed by nuclear magnetic resonance hydrogen spectrum or mass spectrum. The structure can be confirmed by assisting nuclear magnetic resonance carbon spectrum and nuclear magnetic resonance secondary spectrum.
The compound provided by the invention has the following beneficial effects when used as reticulocyte fluorescent dye:
1. the fluorescent dye compound is introduced with nitrogen substituent, and polar functional groups such as hydroxyl, halohydrocarbon, ester group, acyl group and the like are introduced into the nitrogen substituent, so that the molecular polarity is properly increased, the binding force to internal hydrophobic areas such as membrane lipid, protein and the like is reduced, and the specific binding to nucleic acid is improved.
2. The fluorescent dye compound molecules introduce hydroxyl or halogen atoms and other substituents which are easy to form hydrogen bonds on nitrogen atoms of alkyl chains, so that the action sites of the intermolecular hydrogen bonds between the dye and the nucleic acid are increased, the binding efficiency of the dye and the nucleic acid is improved, and the use concentration of the fluorescent dye is reduced.
3. The dye compound leads the fluorescence emission wavelength of the dye to be more than 650nm by introducing quinoline heterocycle or increasing the number of methine chains, thereby avoiding the self fluorescence background interference of organisms.
4. The dye compound is stable under the high temperature condition, and is not easy to decompose.
5. The dye compound can adopt a red semiconductor laser with low price and good stability as a light source, so that the use cost is reduced.
Example 1
A compound A for reticulocyte staining, which has the specific chemical structural formula:
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the preparation method of the fluorescent dye compound A comprises the following specific synthetic route:
20mmol of 1-ethylphenyl-4-methylquinoline quaternary ammonium salt and 30mmol of N, N-dimethylformamide were combined in 30ml of dichloromethane/methanol=1: 1 in a solvent at 160 ℃ in an oil bath for 0.5 hour. After cooling, the reddish brown solid obtained by the reaction is washed out by diethyl ether and recrystallized in ethanol to obtain mauve crystals, which are filtered and dried in a yield of 52%. To this was added 10mmol of 1-N, -diethanolamino-butyl-2-methylbenzothiazole quaternary ammonium salt and 1.5mL of triethylamine, and the mixture was stirred under reflux in ethylene glycol monomethyl ether for 1.5 hours. After cooling, the reaction solution is poured into diethyl ether, dark purple small particles are separated out, the dye is separated through a neutral alumina column, the eluent dichloromethane and methanol are used for gradient elution, and the blue component is collected, so that the compound A is obtained, and the yield is 44%. Confirmation of the above Compounds Using Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,MeOH-d4)δ＝8.43(d,J＝8.5Hz,1H),8.14(d,J＝7.0Hz,1H),8.08(t,J＝12.8Hz,1H),7.88(d,J＝5.4Hz,2H),7.65-7.57(m,3H),7.43–7.27(m,7H),7.20(t,J＝7.3Hz,1H),7.06(d,J＝13.4Hz,1H),6.49(d,J＝12.2Hz,1H),5.10(s,2H),4.49(q,J＝6.8Hz,2H),4.23(q,J＝7.1Hz,2H),3.42(t,J＝6.8Hz,2H),3.28–3.11(m,6H),2.10–1.92(m,2H),1.91–1.80(m,2H).Q-TOF-MS m/zFound:552.2671;Calculated:C34H38N3O2S+:552.27.
Example 2
A compound B for reticulocyte staining, which has the specific chemical structural formula:
The preparation method of the fluorescent dye compound B comprises the following specific synthetic route:
20mmol of 1-ethyl-4-methylquinoline quaternary ammonium salt and 30mmol of N, N-dimethylformamide were stirred in 30ml of acetic anhydride at 120℃for 1.5 hours with heating in an oil bath. After cooling, the orange-yellow solid precipitated in the reaction solution was filtered, dried, separated by a silica gel column, eluted with a gradient of eluent dichloromethane and methanol, and the yellow component was collected in a yield of 52%. To this was added 10mmol of 1-N, N-dibromoethylamino-butyl-6-methoxybenzoxazole quaternary ammonium salt, 1.5 ml of triethylamine, and the mixture was stirred under reflux in ethylene glycol monomethyl ether for 1.5 hours. After cooling, pouring the reaction solution into diethyl ether, separating out dark purple small particles, separating the dye by a neutral alumina column, gradient eluting with eluent dichloromethane and methanol, and collecting purple component to obtain the compound B with the yield of 47%. Confirmation of Compound B by Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,DMSO-d6)δ＝8.39(d,J＝8.3Hz,1H),8.36(d,J＝7.1Hz,1H),8.07(t,J＝12.8Hz,1H),8.02(d,J＝8.7Hz,1H),7.91(t,J＝7.8Hz,1H),7.80(d,J＝7.8Hz,1H),7.76(d,J＝7.2Hz,1H),7.66(t,J＝7.6Hz,1H),7.50(d,J＝8.2Hz,1H),7.40(t,J＝7.8Hz,1H),7.04(d,J＝13.3Hz,1H),6.48(d,J＝12.2Hz,1H),4.55(q,J＝7.1Hz,2H),4.23(t,J＝7.2Hz,2H),3.85(s,3H),3.58(t,J＝7.2Hz,4H),3.30-3.14(m,6H),1.83(m,J＝7.2Hz,2H),1.71(m,J＝6.8Hz,2H),1.44(t,J＝7.2Hz,3H).Q-TOF-MS m/z Found:630.1115;Calculated:C30H36N3Br2O2 +:630.11.
Example 3
A compound C for reticulocyte staining, which has the specific chemical structural formula:
the preparation method of the fluorescent dye compound C comprises the following steps of:
To a 100mL round bottom flask was added 5mmol of N-ethyl-2, 3-dimethyl-4, 5-benzindole methanol solution, 5mmol of the quaternary ammonium salt intermediate and 2 drops of triethylamine, nitrogen blanket. The reaction was carried out at room temperature for 8 hours in the dark. After the reaction is finished, the solvent is distilled off under reduced pressure, and the deep purple dye compound C is separated by column chromatography. The yield was 64%. Confirmation of the above Compounds Using Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,DMSO-d6)δ＝8.08(d,J＝8.9Hz,1H),8.04(m,2H),7.97(m,2H),7.75(d,J＝7.8Hz,1H),7.65(d,J＝7.5Hz,1H),7.62(t,J＝7.9Hz,2H),7.49(d,J＝7.2Hz,1H),7.32(t,J＝7.7Hz,2H),6.16(d,J＝7.3Hz,1H),5.80(d,J＝12.3Hz,1H),4.96(s,J＝12.5Hz,1H),4.14(q,J＝7.1Hz,2H),3.35(quart,J＝7.4Hz,4H),2.46(t,J＝7.4Hz,2H),1.39(s,6H),1.41(t,J＝7.4Hz,3H),1.36(q,J＝7.1Hz,2H),1.30(m,4H),0.99(t,J＝8.0Hz,6H).Q-TOF-MS m/z Found:553.3684.Calculated:C36H47N3O2 +:553.37.
Example 4
A compound D for reticulocyte staining, which has the specific chemical structural formula:
the preparation method of the fluorescent dye compound D comprises the following specific synthetic route:
To a 100mL round bottom flask was added 5mmol of N-ethylamino-2, 3-dimethyl-4, 5-benzindole methanol solution, 5mmol of the quaternary ammonium salt intermediate and 2 drops of triethylamine, nitrogen blanket. The reaction was carried out at room temperature for 8 hours in the dark. After the reaction is finished, the solvent is distilled off under reduced pressure, and the deep purple dye D pure product is obtained through column chromatography separation. The final product was anion exchanged with OTs - containing sodium salt. Confirmation of the above Compounds Using Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,DMSO-d6)δ＝8.12(d,J＝8.6Hz,1H),7.80(d,J＝7.5Hz,2H),7.65(d,J＝7.5Hz,1H),7.62(d,J＝7.9Hz,2H),7.49(d,J＝7.2Hz,1H),7.32(d,J＝7.4Hz,1H),6.15(s,1H),5.84(d,J＝12.3Hz,1H),5.66(t,J＝12.3Hz,3H),4.96(s,J＝12.5Hz,1H),4.14(s,J＝7.1Hz,3H),2.46(t,J＝7.4Hz,2H),1.67(s,3H),1.48(s,6H),1.38(s,3H).Q-TOF-MS m/z Found:410.2589;Calculated:C36H47N3O2 +:410.26.
Example 5
A compound E for reticulocyte staining, which has the specific chemical structural formula:
The preparation method of the fluorescent dye compound E comprises the following specific synthetic route:
20mmol of 1-benzyl-4-methoxy-6-methyl-quinoline quaternary ammonium salt and 30mmol of N, N-dimethylformamide were stirred in 30ml of acetic anhydride at 120℃for 1.5 hours with heating in an oil bath. After cooling, the orange-yellow solid precipitated in the reaction solution was filtered, dried, separated by a silica gel column, eluted with a gradient of eluent dichloromethane and methanol, and the yellow component was collected in 63% yield. To this was added 10mmol of 1-N, N-ethoxymethyl ammonium acetate-octyl-6-methoxybenzoselenazole quaternary ammonium salt, 1.5 ml of triethylamine, and the mixture was stirred under reflux in ethylene glycol monomethyl ether for 1.5 hours. After cooling, the reaction solution was poured into diethyl ether to precipitate dark purple small particles, the dye was separated by a neutral alumina column, and the dark purple fraction was collected by gradient elution with eluent dichloromethane and methanol to give compound E in 42% yield. Confirmation of the above Compounds Using Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,DMSO-d6)δ＝8.10(d,J＝8.7Hz,1H),8.05(m,2H),7.90(m,2H),7.81(d,J＝7.8Hz,1H),7.67(d,J＝7.5Hz,1H),7.62(t,J＝7.9Hz,2H),7.49(d,J＝7.2Hz,1H),7.32(t,J＝7.7Hz,3H),6.58(d,J＝12.5Hz,1H),6.51(t,J＝12.3Hz,4H),6.47(d,J＝12.5Hz,1H),5.33(d,J＝12.3Hz,1H),4.04(d,J＝7.1Hz,2H),3.80(d,J＝7.1Hz,2H),3.35(quart,J＝7.4Hz,4H),3.10(quart,J＝7.2Hz,4H),2.46(t,J＝7.4Hz,2H),2.39(s,3H),1.41(q,J＝7.4Hz,26H),1.30(t,J＝7.2,6H),0.99(t,J＝8.0Hz,3H).Q-TOF-MS m/z Found:894.4289.Calculated:C52H68N3O5Se+:894.43.
Example 6
A compound F for reticulocyte staining, which has the specific chemical structural formula:
The preparation method of the fluorescent dye compound F comprises the following specific synthetic route: (note: tolene was toluene, reflux was reflux, the same applies below)
20Mmol of 1-N-octadecyl-4-methyl-6-nonenoxyquinoline quaternary ammonium salt and 30mmol of N, N-dimethylformamide were stirred in 30ml of acetic anhydride at 120℃for 1.5 hours with heating on an oil bath. After cooling, the orange-yellow solid precipitated in the reaction solution was filtered, dried, separated by a silica gel column, eluted with a gradient of eluent dichloromethane and methanol, and the yellow component was collected in a yield of 74%. To this was added 10mmol of 1-N, N-ethylvinylacetate-dodecyl6-methoxybenzoselenazole quaternary ammonium salt, 1.5 ml of triethylamine, and the mixture was stirred under reflux in ethylene glycol monomethyl ether for 1.5 hours. After cooling, the reaction solution was poured into diethyl ether to precipitate dark purple small particles, the dye was separated by a neutral alumina column, the purple fraction was collected by gradient elution with eluent dichloromethane and methanol to give compound F in 61% yield. Confirmation of the above Compounds Using Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,DMSO-d6)δ＝8.36(d,J＝7.3Hz,2H),7.90(d,J＝7.2Hz,2H),7.72(d,J＝7.2Hz,2H),7.60(s,1H),7.50(s,1H),6.82(d,J＝13.3Hz,1H),6.48(d,J＝12.2Hz,1H),6.20(t,J＝12.2Hz,1H),4.55(t,J＝7.1Hz,4H),4.23(t,J＝7.2Hz,2H),3.85(quart,J＝7.2Hz,2H),3.58(t,J＝7.2Hz,4H),3.30-3.14(m,6H),2.69(t,J＝7.2Hz,4H),1.83(m,98H),1.71(t,J＝7.2,6H),1.44(t,J＝7.2Hz,9H).Q-TOF-MS m/z Found:1296.9765;Calculated:C81H138N3O4Se+:1296.98.
Example 7
A compound G for reticulocyte staining, which has the specific chemical structural formula:
The preparation method of the fluorescent dye compound G comprises the following steps of:
20mmol of 1-N-iodobutylpropanaminyl-4-methyl-6-methylquinoline quaternary ammonium salt and 30mmol of N, N-dimethylformamide were stirred in 30ml of acetic anhydride at 120℃for 1.5 hours with heating in an oil bath. After cooling, the orange-yellow solid precipitated in the reaction solution was filtered, dried, separated by a silica gel column, eluted with a gradient of eluent dichloromethane and methanol, and the yellow component was collected in a yield of 47%. To this was added 10mmol of 1-N-iodobutylammonium-dodecyl-6-methoxybenzoxazole quaternary ammonium salt, 1.5 ml of triethylamine, and the mixture was stirred under reflux in ethylene glycol monomethyl ether for 1.5 hours. After cooling, the reaction solution was poured into diethyl ether to precipitate dark purple small particles, the dye was separated by a neutral alumina column, and the dark purple fraction was collected by gradient elution with eluent dichloromethane and methanol to give compound G in 58% yield. Confirmation of the above Compounds Using Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,DMSO-d6)δ＝10.02(s,2),7.81(d,J＝7.6Hz,2H),7.70(s,1H),7.65(d,J＝7.5Hz,2H),7.49(s,1H),7.32(d,J＝7.3Hz,2H),6.48(d,J＝12.5Hz,1H),6.34(t,J＝12.3Hz,4H),6.20(d,J＝12.5Hz,1H),5.39(d,J＝12.3Hz,1H),4.14(t,J＝7.1Hz,8H),2.45(t,J＝7.4Hz,2H),1.41(q,J＝7.4Hz,62H),1.30(t,J＝7.2,4H),0.97(t,J＝8.0Hz,3H).Q-TOF-MS m/z Found:1246.5659.Calculated:C64H101ClI2N4O2 +:1246.57.
Example 8
A compound H for reticulocyte staining, which has the specific chemical structural formula:
the preparation method of the fluorescent dye compound H comprises the following steps of:
20mmol of 1-N, N-ethylpropylammonium-4-methyl-6-nonylquinoline quaternary ammonium salt and 30mmol of N, N-dimethylformamide were stirred in 30ml of acetic anhydride at 120℃for 1.5 hours with heating in an oil bath. After cooling, the orange-yellow solid precipitated in the reaction solution was filtered, dried, separated by a silica gel column, eluted with a gradient of eluent dichloromethane and methanol, and the yellow component was collected in 43% yield. To this was added 10mmol of 1-N, N-propylbutylammonio-dodecyl-6-nonylbenzoxazole quaternary ammonium salt, 1.5 ml of triethylamine, and the mixture was stirred under reflux in ethylene glycol monomethyl ether for 1.5 hours. After cooling, the reaction solution was poured into diethyl ether to precipitate dark purple small particles, the dye was separated by a neutral alumina column, the purple fraction was collected by gradient elution with eluent dichloromethane and methanol to give compound H in 59% yield. Confirmation of the above Compounds Using Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,DMSO-d6)δ＝7.92(d,J＝7.6Hz,2H),7.80(s,1H),7.66(d,J＝7.5Hz,2H),7.52(s,1H),7.30(d,J＝7.7Hz,2H),6.28(d,J＝12.3Hz,1H),6.01(t,J＝12.2Hz,1H),5.80(d,J＝12.3Hz,1H),4.14(s,2H),3.35(t,J＝7.4Hz,10H),3.30(quart,J＝7.4Hz,2H),2.46(t,J＝7.4Hz,2H),1.41(quart,J＝7.2Hz,56H),1.36(quart,J＝7.2Hz,2H),1.30(t,J＝7.2Hz,4H),1.12(t,J＝7.3Hz,12H),0.99(t,J＝7.2Hz,6H).Q-TOF-MS m/z Found:919.8065.Calculated:C62H103N4O+:919.81.
Example 9
A compound I for reticulocyte staining, which has the specific chemical structural formula:
the preparation method of the fluorescent dye compound I is as follows, and the specific synthetic route is as follows:
20mmol of 1-N-octadecyl-4-methyl-6-nonenoxyquinoline quaternary ammonium salt and 30mmol of N, N-dimethylformamide were stirred in 30ml of acetic anhydride at 120℃for 1.5 hours with heating on an oil bath. After cooling, the orange-yellow solid precipitated in the reaction solution was filtered, dried, separated by a silica gel column, eluted with a gradient of eluent dichloromethane and methanol, and the yellow component was collected in 67% yield. To this was added 10mmol of 1-N, N-alkoxymethyl ammonium-dodecyl-6-nonylbenzoxazole quaternary ammonium salt, 1.5 ml of triethylamine, and the mixture was stirred under reflux in ethylene glycol monomethyl ether for 1.5 hours. After cooling, the reaction solution was poured into diethyl ether to precipitate dark purple small particles, the dye was separated by a neutral alumina column, and the dark purple fraction was collected by gradient elution with eluent dichloromethane and methanol to give compound I in 54% yield. Confirmation of the above Compounds Using Nuclear magnetic resonance Hydrogen Spectrometry ,1HNMR(400MHz,DMSO-d6)δ＝7.95(d,J＝7.6Hz,2H),7.82(s,1H),7.70(d,J＝7.5Hz,2H),7.56(s,1H),7.34(d,J＝7.7Hz,2H),6.18(d,J＝12.3Hz,1H),5.82(t,J＝12.3Hz,3H),5.35(d,J＝12.5Hz,1H),4.14(s,2H),3.35(t,J＝7.4Hz,8H),3.0(s,3H),2.39(s,6H),1.30(t,J＝7.4Hz,4H),1.36(quart,J＝7.1Hz,56H),0.99(t,J＝7.2Hz,6H).Q-TOF-MS m/z Found:981.7880.Calculated:C63H102FN4O3 +:981.79.
Example 10
The fluorescence emission spectrum and the relative fluorescence intensity of the compound A before and after being combined with the turtle liver RNA are measured, and the specific operation is as follows:
EG (ethylene glycol) solution of Compound A was prepared at a concentration of 0.008g/L, 25. Mu.L was taken, and then phosphate buffer (PBS solution) having a pH of 7.4 and 10mM was added thereto to dilute it to 1mL, and the mixture was placed in a cuvette and the fluorescence intensity thereof was measured. PBS solution of soft-shelled turtle liver RNA with a certain concentration is prepared. In addition, 25 mu L of EG (ethylene glycol) solution of the compound A with the concentration of 0.008g/L is taken in a cuvette, PBS solution of the turtle liver RNA is added into the solution, and finally tris (hydroxymethyl) aminomethane hydrochloride buffer solution with the pH of 7.4 and 10mM is added into the solution to dilute the solution to 1mL, and the fluorescence intensity of the PBS solution of the turtle liver RNA with the final concentrations of 5ug/mL, 10ug/mL and 15ug/mL is respectively tested. As shown in the specific detection results of FIG. 1, as can be seen from FIG. 1, the relative fluorescence intensity of the fluorescence spectrum emission peak at 659nm can be increased by 21.26 times (I/I 0 = 245.75/11.56=13.75) after the compound A is combined with 15ug/mL of turtle liver RNA. Fluorescence spectrophotometer, model: HITACHI F-7100.
Example 11
The fluorescence emission spectrum and the relative fluorescence intensity of the compound B before and after being combined with the turtle liver RNA are measured, and the specific operation is as follows:
An EG (ethylene glycol) solution of Compound B was prepared at a concentration of 0.008g/L, 25. Mu.L was taken, and then a phosphate buffer (PBS solution) having a pH of 7.4 and 10mM was added thereto to dilute it to 1mL, and the mixture was placed in a cuvette and the fluorescence intensity thereof was measured. PBS solution of soft-shelled turtle liver RNA with a certain concentration is prepared. In addition, 25 mu L of EG (ethylene glycol) solution of the compound B with the concentration of 0.008g/L is taken in a cuvette, PBS solution of the turtle liver RNA is added into the solution, and finally tris (hydroxymethyl) aminomethane hydrochloride buffer solution with the pH of 7.4 and 10mM is added into the solution to dilute the solution to 1mL, and the fluorescence intensity of the PBS solution of the turtle liver RNA with the final concentrations of 5ug/mL, 10ug/mL and 15ug/mL is respectively tested. As shown in fig. 2, the relative fluorescence intensity of the fluorescence spectrum emission peak at 657nm increases by 23.79 times (I/I 0 = 250.73/10.54=13.75) after the compound B is combined with 15ug/mL of turtle liver RNA as shown in fig. 2. Fluorescence spectrophotometer, model: HITACHI F-7100.
Example 12
Determination of fluorescence emission spectra and relative fluorescence intensities before and after binding of compound C with turtle liver RNA:
EG (ethylene glycol) solution of compound C was prepared at a concentration of 0.008g/L, 25. Mu.L was taken, and then phosphate buffer (PBS solution) having a pH of 7.4 and 10mM was added thereto to dilute it to 1mL, and the mixture was placed in a cuvette and the fluorescence intensity thereof was measured. PBS solution of soft-shelled turtle liver RNA with a certain concentration is prepared. In addition, 25 mu L of EG (ethylene glycol) solution of compound C with the concentration of 0.008g/L is taken in a cuvette, PBS solution of turtle liver RNA is added into the solution, and finally tris (hydroxymethyl) aminomethane hydrochloride buffer solution with the pH of 7.4 and 10mM is added into the solution to dilute the solution to 1mL, and the fluorescence intensity of PBS solutions of the turtle liver RNA with the final concentrations of 5ug/mL, 10ug/mL and 15ug/mL are respectively tested. As shown in the specific detection results of FIG. 3, as can be seen from FIG. 3, the fluorescence spectrum emission peak of the compound C combined with 15ug/mL of turtle liver RNA is increased by 13.75 times (I/I 0 = 158.66/11.56=13.75) at 661 nm. Fluorescence spectrophotometer, model: HITACHI F-7100.
Example 13
A fluorescent dye a for reticulocyte staining comprising compound a of example 1 and an alcohol, wherein the alcohol is a mixture of isopropyl alcohol and ethylene glycol, the formulation of which is given in the following table:
TABLE 1 formulation of fluorescent dye solution a of example 13
| Fluorescent dye liquor a | Concentration of |
| Compound A in example 1 | 0.005g |
| Isopropyl alcohol | 70.0g |
| Ethylene glycol | Quantifying to 1000mL |
The above fluorescent dye solution a was placed at 56℃and then the ultraviolet absorption intensity of the fluorescent dye solution a at a maximum absorption wavelength of 625nm was measured for 29 days, and the stability of the reagent under high temperature conditions was examined, and the measurement results are shown in Table 2. Ultraviolet spectrophotometer, model: shimadzu UV-2600i.
TABLE 2 absorbance values of fluorescent dye solution a at 56℃for different times
| / | Day 0 | For 5 days | For 8 days | 11 Days | 14 Days | For 17 days | For 20 days | 26 Days | 29 Days |
| Absorbance value | 0.551 | 0.549 | 0.55 | 0.548 | 0.548 | 0.546 | 0.544 | 0.544 | 0.543 |
| Rate of decrease | 0.00％ | -0.36％ | -0.18％ | -0.54％ | -0.54％ | -0.91％ | -1.27％ | -1.27％ | -1.45％ |
As is clear from Table 2, the absorbance value of the maximum absorption peak of the fluorescent dye solution a after accelerating for 29 days at 56℃was reduced by only 1.45%, and the stability at high temperature was good.
Example 14
A fluorescent dye B for reticulocyte staining comprising compound B of example 2 and an alcohol, wherein the alcohol is a mixture of isopropyl alcohol and ethylene glycol, the formulation of which is given in the following table:
TABLE 3 formulation of fluorescent dye b
| Fluorescent dye liquor b | Concentration of |
| Compound B in example 2 | 0.5g |
| Isopropyl alcohol | 70.0g |
| Ethylene glycol | Quantifying to 1000mL |
The fluorescent dye b is placed under the condition of 56 ℃, then the ultraviolet absorption intensity of the fluorescent dye b at the maximum absorption wavelength of 621nm lasts for 29 days, the stability of the reagent of the dye under the high-temperature condition is inspected, and the detection result is shown in the table below. Ultraviolet spectrophotometer, model: shimadzu UV-2600i.
TABLE 4 absorbance values of fluorescent dye b at 56℃for different times
| / | Day 0 | For 5 days | For 8 days | 11 Days | 14 Days | For 17 days | For 20 days | 26 Days | 29 Days |
| Absorbance value | 2.641 | 2.64 | 2.657 | 2.649 | 2.634 | 2.661 | 2.653 | 2.643 | 2.631 |
| Rate of decrease | 0.00％ | -0.04％ | 0.61％ | 0.30％ | -0.27％ | 0.76％ | 0.45％ | 0.08％ | -0.38％ |
As is clear from the above table, the absorbance value of the maximum absorption peak of the fluorescent dye b after accelerating for 29 days at 56 ℃ is reduced by only 0.38%, and the high-temperature stability of the fluorescent dye b is good.
Example 15
A fluorescent dye C for reticulocyte staining comprising compound C of example 3 and an alcohol, wherein the alcohol is a mixture of isopropyl alcohol and ethylene glycol, the formulation of which is given in the following table:
TABLE 5 formula of fluorescent dye solution c
The above fluorescent dye solution c was placed at 56℃and then the ultraviolet absorption intensity of the fluorescent dye solution c at 624nm of maximum absorption wavelength was measured for 29 days, and the stability of the reagent under high temperature conditions was examined, and the measurement results are shown in the following table. Ultraviolet spectrophotometer, model: shimadzu UV-2600i.
TABLE 6 absorbance change of fluorescent dye solution c at 56℃
| / | Day 0 | For 5 days | For 8 days | 11 Days | 14 Days | For 17 days | For 20 days | 26 Days | 29 Days |
| Absorbance value | 5.492 | 5.492 | 5.491 | 5.491 | 5.48 | 5.481 | 5.482 | 5.482 | 5.482 |
| Rate of decrease | 0.00％ | 0.00％ | -0.02％ | -0.02％ | -0.22％ | -0.20％ | -0.18％ | -0.18％ | -0.18％ |
As is clear from the above table, the absorbance value of the maximum absorption peak of the fluorescent dye c after accelerating for 29 days at 56 ℃ is reduced by only 0.18%, and the high-temperature stability of the fluorescent dye c is good.
Example 16
A reticulocyte detection reagent (first reagent) comprising a diluent and fluorescent dye a of example 13, the specific formulation is as follows:
TABLE 7 formulation of first reagent
Wherein the N-tris (hydroxymethyl) methylglycine buffer system may be replaced with at least one of a phosphate buffer system, a tris buffer system or a mixture with the N-tris (hydroxymethyl) methylglycine buffer system; naOH may be replaced with at least one of sodium hydrogen citrate and potassium chloride or a mixture with sodium hydroxide; at least one of tetradecyl sulfobetaine, dodecyl trimethyl ammonium bromide, octyl Trimethyl Ammonium Bromide (OTAB), dodecyl Trimethyl Ammonium Bromide (DTAB), lauryl betaine, lauramidopropyl betaine, polyethylene glycol cetyl ether (n=20), polyethylene glycol cetyl ether (n=30), polyethylene glycol monostearate (n=25), polyoxyethylene oleyl ether (n=20) or a mixture thereof; benzoic acid, formaldehyde may be replaced with at least one of sodium azide, benzoate, bisimidazolidinyl urea and isothiazolinone or a mixture with benzoic acid, formaldehyde; the sodium sulfate may be replaced with at least one of sulfate SO 4 2-, phosphate PO 4 3-, sodium salt or potassium salt of phosphite PO 3 2-.
The diluted solution and the fluorescent dye solution a are prepared according to the table, the volume is fixed to a specified amount, and the mixture is stirred uniformly at normal temperature and then filtered by using a 0.22um filter membrane. The matched reagent is compared with the model of the self-made reagent by using a Hissen Meikang XN1000 instrument. Details of the results are shown in Table 8 and FIGS. 4-7.
Table 8: comparison of detection reagent of example 16 with Hissen Meikang XN1000 Instrument
Table 8 and the results in FIGS. 4 to 7 show that the measurement results of RET (percentage of reticulocytes) of the detection reagent in the embodiment and the measurement results of XN1000 instrument (RET channel dye is undiluted; RET dye concentration: 0.28 g/L) deviate within the allowable range, the measurement results are accurate, wherein FIGS. 4 to 5 are Xisenthan XN-1000RET channel dye dilution (original RET matched dye dilution 35 times; RET dye concentration: 0.008 g/L) are the same as the concentration of the compound of the fluorescent dye in the invention, RET channel patterns measured on the matched blood cell analyzer of sample 1 and sample 2 show that RET expansion degree is not good after RET channel dye dilution 35 times of XN-1000, and the number of low, medium and high fluorescent reticulocytes cannot be counted correctly; FIGS. 6-7 show the RET (percent reticulocytes) measurement results of the detection reagents of the present example on a matched blood cell analyzer, and at the same concentration, RET channel patterns of the detection reagents of the present invention on samples can be well developed, and RET maturity differentiation limit is obvious, so that the prepared RET counting results and LFR, MFR, HFR ratios are obtained.
Example 17
A reticulocyte detection reagent (second reagent) comprising a diluent and fluorescent dye b of example 14, the specific formulation is as follows:
TABLE 9 formulation of the second agent
The diluted solution and the fluorescent dye b were prepared according to the above table to a predetermined volume, and then stirred uniformly at room temperature, and filtered with a 0.22um filter membrane. The matched reagent is compared with the model of the self-made reagent by using a Hissen Meikang XN1000 instrument. The details of the results are shown in Table 10 and FIGS. 8-9.
TABLE 10 comparative detection results of the detection reagents of example 17 and the kit of Hiziram XN1000
The above table and the results in fig. 8 to 9 show that the measurement result of RET (percentage of reticulocytes) of the detection reagent in this embodiment deviates from the measurement result of XN1000 in the allowable range, and the measurement result is accurate, and fig. 8 to 9 show that the measurement result of RET (percentage of reticulocytes) of the detection reagent in this embodiment on the matching blood cell analyzer, and at the same concentration, the RET channel diagram of the detection reagent of the present invention on the sample can be well developed, and the RET maturity differentiation limit is obvious, so as to obtain the prepared RET count result and LFR, MFR, HFR ratio.
Example 18
A reticulocyte detection reagent (third reagent) comprising a diluent and fluorescent dye c of example 15, the specific formulation is as follows:
TABLE 11 formulation of third reagent
The diluted solution and the fluorescent dye solution c were prepared according to the above table to a predetermined volume, and then stirred uniformly at room temperature, and filtered with a 0.22um filter membrane. The matched reagent is compared with the model of the self-made reagent by using a Hissen Meikang XN1000 instrument. The details of the results are shown in Table 12 and FIGS. 10-11.
TABLE 12 comparative detection results of the detection reagents of example 18 with the Hissen Meikang XN1000 instrument
As shown by the above table and the results in FIGS. 10 to 11, the measurement results of RET (percentage of reticulocytes) on the matched blood cell analyzer of the detection reagent in this embodiment deviate from the measurement results of the XN1000 instrument within the allowable range, the measurement results are accurate, and FIGS. 10 to 11 are the measurement results of RET (percentage of reticulocytes) on the matched blood cell analyzer of the detection reagent in this embodiment, and at the same concentration, RET channel patterns of the detection reagent of the present invention on samples can be well developed, and RET maturity differentiation limit is obvious, so that the prepared RET count result and LFR, MFR and HFR proportion are obtained.
Comparative example 1
A reticulocyte detection reagent (fourth reagent) comprising a diluent and the fluorescent dye d of example 16, the specific formulation is as follows:
TABLE 13 formulation of fourth reagent
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Preparing a diluent and a fluorescent dye d according to the table, fixing the volume to a specified amount, uniformly stirring at normal temperature, filtering by using a 0.22um filter membrane, and comparing the matched reagent with the model of the self-made reagent by using a Hissen Meikang XN1000 instrument. Details of the results are shown in Table 14 and FIGS. 12-13.
TABLE 14 comparative detection results of the detection reagents of comparative example 1 and the matched reagents of the Hissen Meikang XN1000 instrument
As shown in the above table and the results in FIGS. 12 to 13, the measurement results of RET (percentage of reticulocytes) on the matched blood cell analyzer by the detection reagent in this comparative example deviate from the measurement results of XN1000 instrument within the allowable range, the measurement results are accurate, and FIGS. 12 to 13 are the measurement results of RET (percentage of reticulocytes) on the matched blood cell analyzer by the detection reagent in comparative example 1, and when sodium sulfate, which is a polyvalent anion salt, is not added, the fluorescence of RBC clusters moves (high fluorescence direction), resulting in a decrease in the spread of RET fluorescence direction, and the low, middle and high fluorescence RET ratios, that is, LFR, MFR and HFR ratios, cannot be accurately calculated.
In summary, the compound for reticulocyte staining, the preparation method of the compound, the fluorescent dye solution and the detection reagent can be used for a blood analyzer, the accurate and rapid detection requirement on reticulocyte counting is met, the reticulocyte counting is processed by the reagent, the specific erythrocyte nucleic acid dye and the erythrocyte membrane processing reagent are increased, and the reticulocyte can be accurately counted in a single channel, namely a RET channel by adopting nucleic acid fluorescent staining and semiconductor laser flow cytometry. The method is characterized in that the form of the reticulocyte is treated by the diluent, and the reticulocyte is spheroidized, and meanwhile, the form of various cell membranes is changed, so that the nucleic acid of the reticulocyte is conveniently and specifically combined with the dye, and the count of the reticulocyte is realized.
The data and the drawings show that the compound, the preparation method of the compound, the fluorescent dye solution and the detection reagent are used for performing relevant performance tests, and the obtained results all meet the acceptance standard. The feasibility and rationality of the invention is verified by the specific embodiments described above, which are only illustrative of alternative individual embodiments of the invention, but not limiting. All changes, modifications and substitutions that come within the meaning and range of equivalency of the claims are intended to be embraced by the scope of the invention.
Claims (19)
1. A compound for reticulocyte staining characterized by having the following structural formula I:
Wherein:
m is 1,2 or 3;
n is an integer of 1 to 18;
Y is C (CH 3)2, O, S or Se;
R 1 and R 2 are each independently selected from the group consisting of a hydrogen atom, an alkyl group, an alkyl chain having a hydroxyl group, an alkyl chain having an ether group, an alkyl chain having an ester group, a halogen-substituted alkyl chain, and R 1 and R 2 are not C 1-18 alkyl groups of the same number of carbon atoms when both are alkyl chains;
r 3 is C 1-18 alkyl, benzyl or (CH 2)nNR1R2;
R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 OR halogen;
R 6 is H or C 1-18 alkyl;
Z - is halide or OTs -.
2. The compound of claim 1, wherein R 1 and R 2 are the same or different and are each independently selected from the group consisting of a hydrogen atom, a C 1-18 alkyl group, a C 1-18 alkyl chain with a hydroxyl group, a C 1-18 alkyl chain with an ether group, a C 1-18 alkyl chain with an ester group, a halogen substituted C 1-18 alkyl chain, and R 1 and R 2 are not alkyl groups of the same carbon number when both are alkyl chains.
3. The compound of claim 1, wherein R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6, OR halogen; r 6 is H or C 1-18 alkyl.
4. The compound of claim 1, wherein n is an integer from 2 to 9.
5. The compound of claim 1, wherein the compound is:
6. the compound of claim 1, wherein the compound is:
7. The compound of claim 1, wherein the compound is:
8. A fluorescent dye for reticulocyte staining comprising a compound according to any one of claims 1-7 and an alcohol, wherein the alcohol is at least one of isopropyl alcohol and ethylene glycol.
9. The fluorescent dye for reticulocyte staining according to claim 8, wherein the concentration of the compound in the fluorescent dye is 0.005-1 g/L.
10. A method for preparing a compound for reticulocyte staining according to any one of claims 1 to 7, comprising the steps of:
S1, reacting a quinoline aromatic compound with R 5 Z to prepare a first quaternary ammonium salt intermediate III;
S2, preparing a first quaternary ammonium salt intermediate III and N, N-diphenyl formamidine to obtain a compound IV;
S3, preparing a second quaternary ammonium salt intermediate VI by using the 2-methylbenzene five-membered ring and a compound R 1R2N(CH2)n Br;
s4, reacting the IV compound with a second quaternary ammonium salt intermediate VI to obtain a compound of the formula I:
Wherein m is 1, 2 or 3;
n is an integer of 1 to 18;
Y is C (CH 3)2, O, S or Se;
R 1 and R 2 are each independently selected from the group consisting of a hydrogen atom, an alkyl group, an alkyl chain having a hydroxyl group, an alkyl chain having an ether group, an alkyl chain having an ester group, a halogen-substituted alkyl chain, and R 1 and R 2 are not C 1-18 alkyl groups of the same number of carbon atoms when both are alkyl chains;
r 3 is C 1-18 alkyl, benzyl or (CH 2)nNR1R2;
R 4 and R 5 are each independently selected from H, C 1-18 alkyl, OR 6 OR halogen;
R 6 is H or C 1-18 alkyl;
Z - is halide or OTs -.
11. A reticulocyte detection reagent, characterized by comprising a diluent and the fluorescent dye for reticulocyte staining according to any one of claims 8-9, wherein the pH of the diluent is 8-10, and the diluent comprises a buffer system, inorganic salt, multivalent anion salt, surfactant and preservative.
12. The reticulocyte detection reagent according to claim 11, wherein a concentration of said buffer system in said diluent is from 0.1 g/L to 10g/L, said buffer system being one or more of a phosphate buffer system, a tris buffer system, and an N-tris (hydroxymethyl) methylglycine buffer system.
13. The reticulocyte detection reagent according to claim 11, wherein a concentration of said inorganic salt in said dilution is 1-30g/L, and said inorganic salt is one or more of sodium hydrogen citrate, sodium chloride, and potassium chloride.
14. The reticulocyte detection reagent according to claim 11, wherein a concentration of said multivalent anion salt in said dilution is 0.1-10g/L, said multivalent anion salt being at least one of sulfate SO 4 2-, phosphate PO 4 3-, sodium salt or potassium salt of phosphite PO 3 2-.
15. The reticulocyte detection reagent according to claim 14, wherein said surfactant is at least one of a nonionic surfactant, an amphoteric surfactant, and a cationic surfactant.
16. The reticulocyte detection reagent according to claim 15, wherein said nonionic surfactant is a polyoxyethylene type nonionic surfactant, and wherein a concentration of said nonionic surfactant in said diluent is 0.01-5g/L; the cationic surfactant is quaternary ammonium salt cationic surfactant, and the concentration of the cationic surfactant in the diluent is 0.1-10g/L; the ampholytic surfactant is betaine type ampholytic surfactant, and the concentration of the ampholytic surfactant in the diluent is 0.1-5g/L.
17. The reticulocyte detection reagent according to claim 11, wherein said preservative is at a concentration of 0.5-4g/L in said diluent, said preservative being one or more of sodium azide, formaldehyde, benzoic acid and salts thereof, bisimidazolidinyl urea, and isothiazolinone.
18. A method for detecting reticulocytes, said method comprising the steps of: the reticulocyte detection reagent according to any one of claims 11-17 is used to treat a sample to be detected, and the detection reagent spheroidizes reticulocytes in the sample to be detected, so that nucleic acid of the reticulocytes is specifically combined with fluorescent dye to realize the counting of the reticulocytes.
19. Use of a fluorescent dye for reticulocyte staining according to any of claims 8-9 or a reticulocyte detection reagent according to any of claims 11-17 for staining a biological sample.
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