CN106749359B - A kind of synthesis and application for detecting hydrogen peroxide novel fluorescence probe - Google Patents
A kind of synthesis and application for detecting hydrogen peroxide novel fluorescence probe Download PDFInfo
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 102
- 239000000523 sample Substances 0.000 title claims abstract description 57
- 230000015572 biosynthetic process Effects 0.000 title abstract description 4
- 238000003786 synthesis reaction Methods 0.000 title abstract description 4
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 238000002360 preparation method Methods 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 6
- 238000004458 analytical method Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 229960002163 hydrogen peroxide Drugs 0.000 description 43
- 238000006243 chemical reaction Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 10
- -1 peroxide Free radical Chemical class 0.000 description 9
- 239000000975 dye Substances 0.000 description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 8
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 229920006391 phthalonitrile polymer Polymers 0.000 description 6
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 5
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 5
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 5
- 229910001424 calcium ion Inorganic materials 0.000 description 5
- 238000004821 distillation Methods 0.000 description 5
- 229960003180 glutathione Drugs 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 229910001425 magnesium ion Inorganic materials 0.000 description 5
- 238000010992 reflux Methods 0.000 description 5
- 229910001415 sodium ion Inorganic materials 0.000 description 5
- 238000010792 warming Methods 0.000 description 5
- ICSNLGPSRYBMBD-UHFFFAOYSA-N 2-aminopyridine Chemical compound NC1=CC=CC=N1 ICSNLGPSRYBMBD-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 239000012065 filter cake Substances 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000006837 decompression Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 150000002431 hydrogen Chemical class 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- XQZYPMVTSDWCCE-UHFFFAOYSA-N phthalonitrile Chemical compound N#CC1=CC=CC=C1C#N XQZYPMVTSDWCCE-UHFFFAOYSA-N 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 3
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 3
- BMIBJCFFZPYJHF-UHFFFAOYSA-N 2-methoxy-5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine Chemical compound COC1=NC=C(C)C=C1B1OC(C)(C)C(C)(C)O1 BMIBJCFFZPYJHF-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- SVENKGKXRLHCTC-UHFFFAOYSA-N B(O)(O)O.BrCC=1C=CC=CC1 Chemical class B(O)(O)O.BrCC=1C=CC=CC1 SVENKGKXRLHCTC-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 235000004237 Crocus Nutrition 0.000 description 2
- 241000596148 Crocus Species 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000003818 flash chromatography Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- OUUQCZGPVNCOIJ-UHFFFAOYSA-N hydroperoxyl Chemical compound O[O] OUUQCZGPVNCOIJ-UHFFFAOYSA-N 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 229910001414 potassium ion Inorganic materials 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 235000010288 sodium nitrite Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- LXNFWIOKXNVNHY-UHFFFAOYSA-N 4-ethyl-4,5,5-trimethyl-2-phenyl-1,3,2-dioxaborolane Chemical group CCC1(OB(OC1(C)C)C1=CC=CC=C1)C LXNFWIOKXNVNHY-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- KWGACYZYFZTYRN-UHFFFAOYSA-N OC(C)(C)C(C)(C)O.B(O)(O)O.BrCC1=CC=CC=C1 Chemical class OC(C)(C)C(C)(C)O.B(O)(O)O.BrCC1=CC=CC=C1 KWGACYZYFZTYRN-UHFFFAOYSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 125000005211 alkyl trimethyl ammonium group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 230000031709 bromination Effects 0.000 description 1
- 238000005893 bromination reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
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- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/025—Boronic and borinic acid compounds
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
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- C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms
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Abstract
The invention discloses a kind of fluorescence probes for detecting hydrogen peroxide and its preparation method and application.Chemical structural formula is：The probe raw material of the present invention is cheap and easy to get, and synthesis is simple and convenient.Probe does not emit fluorescence in itself, and fluorescence intensity gradually enhances (highest increases to original 77.8 times) after being acted on hydrogen peroxide, and maximum emission wavelength has the Stokes shift (Stokes shifts) of up to 217nm in near-infrared.Detection limits low (6.6nM), and high sensitivity, detection range is wide, has good selectivity and anti-interference to the identification of hydrogen peroxide, can have broad application prospects in vitro with detection hydrogen peroxide inside living cells in chemical analysis detection field.
Description
Technical field
The present invention relates to chemical analysis detection technique fields, and in particular to the new feux rouges of a kind of identification hydrogen peroxide-near red
The preparation method of outer fluorescence probe and its in vitro in living cells detect hydrogen peroxide in terms of application.
Background technology
Active oxygen includes superoxide radical (O2), hydroperoxy radical (HO2), hydroxy radical (HO), peroxide
Free radical (RO2), singlet oxygen (1O2) and hypochlorous acid (HOCl) etc..Hydrogen peroxide is as indispensable in active oxygen family
A member plays an important role in the physiology of organism, metabolism and pathology.Content of hydrogen peroxide liter in cell
Height is likely to result in DNA damage, variation and Genomic instability.Its still with aging and the instruction of the relevant oxidative stress of disease
Agent, the second messenger that cell signal transfers, immunocyte and bacterium are struggled the deoxidier generated in the process.In life system
In, the generation of hydrogen peroxide, accumulation and elimination can all generate far different physiology and pathology results, Just because of this mistake
For the detection technique of hydrogen oxide in chemistry, biology and medical domain are all particularly important.But since the instantaneous of hydrogen peroxide is deposited
In property, the technology development directly detected is had difficulty in taking a step, and the detection technique of hydrogen peroxide is to utilize itself and substrate reactions mostly at present,
Then with a series of means detection substrates be changed into product process or product in itself, with realize hydrogen peroxide detection mesh
Ground.These detection techniques include electrochemical process, colorimetric method, gas detection method, fluorescence method etc..Wherein since fluorescence probe has
Easy to operate, radiationless, highly sensitive and high-resolution advantage becomes the effective means for detecting hydrogen peroxide.In recent years, mistake
The fluorescence probe of hydrogen oxide response excitation has been reported very much, however most probes have short launch wavelength and small stoke
The shortcomings that this displacement (Stokes shifts).It is well known that long launch wavelength and big Stokes shift are imaged cell fluorescence
It is very favorable, because the photon with long wavelength can reduce the light scattering of environmental induction, so as to reduce in detection process
The influence of interior raw chromophore, and the light injury of living cells can be reduced, big Stokes shift can improve the sensitive of detection
Degree.Feux rouges-the near infrared fluorescent probe for being used to detect hydrogen peroxide with big Stokes shift is also rarely reported now.
The content of the invention
For the present situation of current hydrogen peroxide detection, it is cheap and easy to get that the purpose of the present invention one is to provide a kind of raw material, synthesis
The fluorescence probe synthetic method that step is simple, reaction condition is mild, yield is higher；Two be to provide one kind can to hydrogen peroxide into
The fluorescence probe of row specific recognition, and it has good selectivity, highly sensitive, strong anti-interference ability, big Stokes shift
The advantage of (Stokes shifts) and long launch wavelength.
The technical solution of the present invention that solves the problems, such as to take is as follows, and a kind of novel fluorescence of specific recognition hydrogen peroxide is visited
Pin has following molecular structural formula：The synthetic route of the fluorescence probe is as follows：Details as Follows for preparation method：(1) 3- nitros are taken
Phthalonitrile (1.0g, 5.8mmol), potassium carbonate (0.9g, 6.35mmol) and sodium nitrite (0.4g, 5.8mmol) are in 25mL
In round-bottomed flask, 15mL dmso solutions are added in, oil bath is warming up to 130 DEG C and is stirred at reflux, the detection of TLC thin-layer chromatographic analysis
Extent of reaction is cooled to room temperature after the completion of reaction in 30 minutes, is slowly added to distilled water (45mL) dilute reaction solution, dilute hydrochloric acid (2M)
PH to 3, Precipitation are adjusted, then decompression filters, and filter cake three times, is recrystallized to give brown needles with distillation washing in glacial acetic acid
Crystal filters to obtain all crystal, is placed in drying over night in vacuum desiccator, obtains product 3- hydroxyl phthalonitriles.Yield：
0.51g.Yield 60%.(2) take 3- hydroxyls phthalonitrile (288mg, 2mmol), 2-aminopyridine (385mg, 4.1mmol) and
Calcium chloride (46mg, 0.41mmol) adds in 6mL n-Butanol solubles in 25mL round-bottomed flasks.Under protection of argon gas, oil bath heats up
It is stirred at reflux to 110 DEG C, TLC thin-layer chromatographic analysis detection extent of reaction is reacted after 5 days and completed, reaction solution is cooled to room temperature, subtracts
Solvent is distilled off in pressure, and with distillation washing three times, filtering, filter cake is placed in vacuum desiccator overnight solid, and column chromatography is further
Purifying, gained crocus powder under vacuum are dried overnight.Yield：150.8mg.Yield 26.6%.(3) step products therefrom is taken
(48mg, 0.15mmol), potassium carbonate (400mg, 3mmol), potassium iodide (80mg, 0.5mmol) add in 100mL round-bottomed flasks
Enter the dissolving of 30mL acetonitriles, stirring at normal temperature adds in 4- bromomethyl benzene boric acids pinacol ester (47.6mg, 1.6mmol) after 30 minutes, so
Oil bath is warming up to 61 DEG C and is stirred at reflux afterwards, and TLC thin-layer chromatographic analysis detection extent of reaction is reacted after 30 minutes and completed, reaction solution
It is cooled to room temperature, vacuum rotary steam removes solvent acetonitrile, and flash column chromatography obtains yellow powder.Yield：59mg.Yield：
73.8%.
That Details as Follows is described for detection mechanism of the fluorescence probe to hydrogen peroxide of the present invention, and the phenolic hydroxyl group of dye molecule is by 4-
After the substitution of bromomethyl benzene boric acid pinacol ester, excited state intramolecular proton transfer (ESIPT) is blocked, thus gained probe point
Son does not have fluorescence.And under the action of hydrogen peroxide, methylphenylboronic acid pinacol ester part is left away, and dye molecule is recovered,
The very strong red fluorescence of transmitting.Response process is as follows：
High performance liquid chromatography demonstrates the mechanism of action of the fluorescence probe of the present invention to hydrogen peroxide, liquid chromatogram well
In, probe molecule occurred unimodal at 23.32 minutes, and dye molecule is unimodal appears in 15.55 minutes, when adding in 5 times of amounts
Hydrogen peroxide, when 37 DEG C of reactions 1 are small after, probe molecule 23.32 minutes while dye molecule 15.55 minutes unimodal occurs
Unimodal remitted its fury.When adding in 80 times of amount hydrogen peroxide, when 37 DEG C of reactions 1 are small after, the only dye molecule list of 15.55 minutes
Peak, the probe molecule peak of 23.32 minutes disappear.This result is consistent with the Response Mechanism predicted.
The fluorescence probe launch wavelength of the present invention is in near-infrared, itself does not emit fluorescence, after being acted on hydrogen peroxide
There are very strong fluorescence, maximum emission wavelength 585nm.
The Stokes shift of the fluorescence probe of the present invention is up to 217nm, a length of 368nm of maximum absorption wave, with peroxide
Maximum emission wavelength is 585nm after changing hydrogen reaction.
The fluorescence probe of the present invention has hydrogen peroxide good selectivity, in PBS buffer solutions of the PH equal to 7.4,
Probe solution does not emit fluorescence, adds in the hydrogen peroxide of 80 times of amounts, and fluorescence intensity gradually enhances, increases to after sixty minutes 0 minute
77.8 times, other chemical substances that may interfere with are separately added under similarity condition, including TBHP, OH, OtBu, ClO-,
NO, ROO-, ONOO-, O2 -,1O2, Na+, K+, Mg2+, Ca2+, HS-, SO3 2-, S2O3 2-, GSH, Hcy, Cys, the fluorescence intensity of probe is simultaneously
There is no significant change.
The fluorescence probe of the present invention has a very strong antijamming capability, other chemical substances that may interfere with (TBHP,
OH, OtBu, ClO-, NO, ROO-, ONOO-, O2 -,1O2, Na+, K+, Mg2+, Ca2+, HS-, SO3 2-, S2O3 2-, GSH, Hcy, Cys)
Presence do not influence response of the probe molecule to hydrogen peroxide.
The fluorescence probe of the present invention goes out the detected representation of hydrogen peroxide very high sensitivity, the fluorescence intensity of probe solution
It is incremented by with concentration of hydrogen peroxide, about reaches peak value when adding in 80 times of amount hydrogen peroxide.Hydrogen peroxide is measured at 0 to 20 times
In section, probe solution fluorescence intensity has good linear relationship with concentration of hydrogen peroxide.The detection limit of fluorescence probe of the present invention
For 6.6nM.
The PH scope of applications of the fluorescence probe of the present invention are wider, and probe molecule is not affected in the range of 7 to 13 to mistake
The detection of hydrogen oxide.
Advantages of the present invention：Probe molecule raw material is cheap and easy to get, and building-up process is easy to operate, and yield is higher, detects peroxide
Long with launch wavelength during change hydrogen, Stokes shift is big, and the good, strong antijamming capability of selectivity, high sensitivity, detection limit is low,
The advantages of detection range is wide.Therefore, fluorescence probe of the invention is that one kind is simple, sensitive, can be examined in vitro with inside living cells
The reagent of hydrogen peroxide is surveyed, is had broad application prospects in chemical analysis detection field.
Description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram of the fluorescence probe of the present invention.(1) liquid chromatogram of fluorescent probe molecule (5 μM).
(2) fluorescent probe molecule (5 μM) and the liquid chromatogram after 5 times of amount hydrogen peroxide effects.(3) fluorescent probe molecule (5 μM) and 80
Liquid chromatogram after the effect of amount hydrogen peroxide again.(4) liquid chromatogram of dye molecule (5 μM).
Fig. 2 is block diagram of the fluorescence probe of the present invention to different ions and molecule selectivity.Point table represents wherein from 1 to 16
H2O2, TBHP, OH, OtBu, ClO-, NO, ROO-, ONOO-, O2 -,1O2, Na+, K+, Mg2+, Ca2+, HS-, SO3 2-, S2O3 2-,
GSH, Hcy, Cys.
Fig. 3 is block diagram of the fluorescence probe of the present invention to different ions and molecule antijamming capability.With other ions and point
When son coexists, the fluorescence intensity ratio (I/I after (5 μM) effects of hydrogen peroxide and fluorescence probe0) block diagram.I represent other from
Fluorescence intensity when son and molecule coexist with hydrogen peroxide, I0Fluorescence intensity when representing there was only hydrogen peroxide.
Fig. 4 is the fluorescence probe (5 μM) of the present invention in PBS buffer solutions (10mM, pH=7.4,1.0Mm cetyl
Base trimethylammonium) in, add in progressive concentration hydrogen peroxide (0-400 μM), 37 DEG C heat preservation 1 it is small when after fluorescence spectra.
Fig. 5 is the fluorescence probe (5 μM) of the present invention in PBS buffer solutions (10mM, pH=7.4,1.0Mm cetyl
Base trimethylammonium) in, the linear relationship chart with concentration of hydrogen peroxide (0-100 μM).
Fig. 6 is the fluorescence probe (5 μM) of the present invention in PBS buffer solutions (10mM, pH=7.4,1.0Mm cetyl
Base trimethylammonium) in, with (400 μM) collection of illustrative plates for acting on fluorescence and changing with time of hydrogen peroxide.
Fig. 7 be the fluorescence probe (5 μM) of the present invention in different pH (2-13) buffer solution, add in hydrogen peroxide (400 μ
M the fluorescence spectra before and after).
Fig. 8 is that cell (Hela cells) imaging (a) probe of the fluorescence probe (10 μM) of the present invention at different conditions exists
Light field is imaged in the cell of hydrogenperoxide steam generator processing, fluorescence imaging in the cell that (b) probe is handled in hydrogenperoxide steam generator,
(c) probe light field in cell is imaged (d) probe fluorescence imaging in cell.
Example is embodied
Embodiment 1：The preparation of 3- hydroxyl phthalonitriles
Take 3- nitrophthalonitriles (1.0g, 5.8mmol), potassium carbonate (0.9g, 6.35mmol) and sodium nitrite
(0.4g, 5.8mmol) is added in 15mL dimethyl sulfoxide (DMSO)s and dissolved, oil bath is warming up to 130 DEG C and stirs back in 25ml round-bottomed flasks
Stream, TLC thin-layer chromatographic analysis detection extent of reaction, is cooled to room temperature after the completion of reaction in 30 minutes, is slowly added to distilled water
(45mL) dilute reaction solution, dilute hydrochloric acid (2M) adjust PH to 3, Precipitation, and then decompression filters, and filter cake uses distillation washing three times,
Brown needle-like crystals are recrystallized to give in glacial acetic acid, filter to obtain all crystal, are placed in drying over night in vacuum desiccator, are produced
Object 3- hydroxyl phthalonitriles.Yield：0.51g.Yield 60%.
Embodiment 2：The preparation of dyestuff
Take 3- hydroxyls phthalonitrile (288mg, 2mmol), 2-aminopyridine (385mg, 4.1mmol) and calcium chloride
(46mg, 0.41mmol) adds in 6mL n-Butanol solubles in 25mL round-bottomed flasks.Under protection of argon gas, oil bath is warming up to 110
It DEG C is stirred at reflux, TLC thin-layer chromatographic analysis detection extent of reaction is reacted after 5 days and completed, and reaction solution is cooled to room temperature, and decompression is steamed
Solvent is removed in distillation, and with distillation washing three times, filtering, filter cake is placed in vacuum desiccator overnight solid, and column chromatography is further purified,
Gained crocus powder under vacuum is dried overnight.Yield：150.8mg.Yield 26.6%.1H NMR (400MHz, CDCl3)δH
13.70 (s, 1H), 8.62 (m, 2H), 7.78 (t, J=7.9Hz, 2H), 7.60 (s, 1H), 7.52 (d, J=7.3Hz, 2H),
7.38 (d, J=8.0Hz, 1H), 7.19-7.09 (m, 3H).13C NMR (101MHz, CDCl3)δc160.10 159.34,
155.81,155.59,153.70,147.98,147.76,138.12,138.08,135.66,133.58,123.43,122.31,
120.50,120.27,118.98,118.29,114.52.
Embodiment 3：The preparation of probe molecule
Take dyestuff (48mg, 0.15mmol) obtained by step, potassium carbonate (400mg, 3mmol), potassium iodide (80mg,
0.5mmol) in 100mL round-bottomed flasks, the dissolving of 30mL acetonitriles is added in, stirring at normal temperature adds in 4- bromomethyl benzene boric acids after 30 minutes
Pinacol ester (47.6mg, 1.6mmol), then oil bath are warming up to 61 DEG C and are stirred at reflux, TLC thin-layer chromatographic analysis detection react into
Degree is reacted after 30 minutes and completed, and reaction solution is cooled to room temperature, and vacuum rotary steam removes solvent acetonitrile, and flash column chromatography obtains
Yellow powder.Yield：59mg.Yield：73.8%.
Embodiment 4：The application of fluorescence probe of the present invention
Take probe molecule mother liquor (1.0 × 10-3Mol/L) in PBS buffer solutions (10mM, pH=7.4,1.0Mm bromination 16
Alkyltrimethylammonium) in be made into solution to be measured, then be separately added into various particles and molecule (tert-butyl hydroperoxide, hydroxy radical, uncle
Butoxy free radical, hypochlorous acid, nitric oxide, sodium ion, potassium ion, magnesium ion, calcium ion, sulfydryl, inferior sulfate radical, thiosulfuric acid
Root, glutathione, homocysteine, cysteine) 37 DEG C of heat preservations 1 it is small when after measure, solution fluorescence becomes almost without apparent
Change, and the probe solution fluorescence of human hydrogen peroxidase is only added to be greatly enhanced, it is seen that the fluorescence probe can realize hydrogen peroxide
Selective recognition.When other ions and molecule (tert-butyl hydroperoxide, hydroxy radical, tertiary butoxy free radical, hypochlorous acid, an oxygen
Change nitrogen, sodium ion, potassium ion, magnesium ion, calcium ion, sulfydryl, inferior sulfate radical, thiosulfate anion, glutathione, half Guang ammonia of homotype
Acid, cysteine) each added in simultaneously with hydrogen peroxide, 37 DEG C heat preservation 1 it is small when after measure, probe solution fluorescence still significantly increases
By force, fluorescence probe of the invention comes out very strong antijamming capability to the detected representation of hydrogen peroxide.Probe molecule detection limit
It is low, 6.6nM can be reached, be suitble to trace detection.PH does not affect detection of the probe molecule to hydrogen peroxide 7 to 13, it is seen that
The fluorescence probe of the present invention has good biological adaptation ability.
Claims (1)
1. a kind of detection hydrogen peroxide novel fluorescence probe, structure are：
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| CN110243790B (en) * | 2019-04-02 | 2021-06-22 | 华东理工大学 | Gene coding type fluorescent biological probe specifically responding to hydrogen peroxide and construction method and application thereof |
| CN110172070B (en) * | 2019-06-05 | 2021-11-02 | 商丘师范学院 | Fluorescent probe for detecting viscosity and hydrogen peroxide as well as synthesis method and application thereof |
| CN110540837B (en) * | 2019-09-23 | 2022-12-13 | 湘潭大学 | Preparation and application of hydrogen peroxide near-infrared fluorescent probe |
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| "Development of a Highly Sensitive Fluorescence Probe for Hydrogen Peroxide";Masahiro A. et al;《J. Am. Chem. Soc》;20110621;第133卷;第10629-10637页 * |
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