CN110272731A - A kind of fluorescence probe DCCO and its preparation method and application - Google Patents
A kind of fluorescence probe DCCO and its preparation method and application Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000010828 elution Methods 0.000 claims abstract description 8
- 239000012043 crude product Substances 0.000 claims abstract description 7
- 235000004237 Crocus Nutrition 0.000 claims abstract description 6
- 241000596148 Crocus Species 0.000 claims abstract description 6
- 238000004440 column chromatography Methods 0.000 claims abstract description 4
- 238000004090 dissolution Methods 0.000 claims abstract description 4
- 238000001914 filtration Methods 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims abstract description 4
- 150000003053 piperidines Chemical class 0.000 claims abstract description 4
- 239000000047 product Substances 0.000 claims abstract description 4
- 238000010992 reflux Methods 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 claims abstract description 4
- 239000002904 solvent Substances 0.000 claims abstract description 4
- QGJXVBICNCIWEL-UHFFFAOYSA-N 9-ethylcarbazole-3-carbaldehyde Chemical compound O=CC1=CC=C2N(CC)C3=CC=CC=C3C2=C1 QGJXVBICNCIWEL-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000012472 biological sample Substances 0.000 claims abstract description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N ethyl acetate Substances CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- 239000003208 petroleum Substances 0.000 claims description 9
- 238000002474 experimental method Methods 0.000 claims description 8
- 230000004044 response Effects 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 239000011550 stock solution Substances 0.000 claims description 7
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000004448 titration Methods 0.000 claims description 4
- 230000001419 dependent effect Effects 0.000 claims description 3
- 238000012417 linear regression Methods 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- 238000002845 discoloration Methods 0.000 claims 1
- 150000002148 esters Chemical class 0.000 claims 1
- 235000019441 ethanol Nutrition 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- QXAMGWKESXGGNV-UHFFFAOYSA-N 7-(diethylamino)-1-benzopyran-2-one Chemical group C1=CC(=O)OC2=CC(N(CC)CC)=CC=C21 QXAMGWKESXGGNV-UHFFFAOYSA-N 0.000 abstract description 2
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 abstract description 2
- PLAZXGNBGZYJSA-UHFFFAOYSA-N 9-ethylcarbazole Chemical compound C1=CC=C2N(CC)C3=CC=CC=C3C2=C1 PLAZXGNBGZYJSA-UHFFFAOYSA-N 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 8
- 150000001450 anions Chemical class 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 7
- 241000252212 Danio rerio Species 0.000 description 6
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 6
- 125000003396 thiol group Chemical class [H]S* 0.000 description 6
- 241000219194 Arabidopsis Species 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 3
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 150000003851 azoles Chemical class 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Natural products O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000283070 Equus zebra Species 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 238000000605 extraction Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
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- 230000028993 immune response Effects 0.000 description 1
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- 238000009331 sowing Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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Abstract
The present invention provides a kind of fluorescence probe DCCO and its preparation method and application.The fluorescence probe is 7- (diethylamino) -3- (E) -3- (9- ethyl -9H- carbazole -3- base) acryloyl group) -2H- pyran-2-one.Preparation method: 3- acetyl group -7- (diethylamino) -2H- pyran-2-one and 9- ethyl -9H- carbazole -3- formaldehyde are dissolved in ethanol/acetonitrile (1/1 by equimolar ratio; V/V) in mixed solution; piperidines is instilled, is heated to reflux, solution becomes red from crocus；Solvent is removed under reduced pressure, linear gradient elution method carries out column chromatography for separation, obtains the crude product of fluorescence probe；With ether dissolution, filtering is dried to get pure products are arrived.The fluorescence probe is to ClO‑Detection be Ratio-type, and show high sensitivity and good selectivity and stability, and have many advantages, such as that detection process is easy, quickly, testing result is accurate.Fluorescence probe DCCO can be in biological sample ClO‑It is applied in detection.
Description
Technical field
The present invention relates to small organic molecule fluorescence probe, a kind of fluorescence probe DCCO and preparation method thereof is particularly belonged to, with
And probe application ClO in biological sample-Detection.
Background technique
Over more than 100 years, ClO-As it is a kind of effectively can anti-multiple-microorganism drug, be used as clinically general disappears
Toxic agent.In animal and human body, ClO-The crucial fungicide being also considered as during immune response, with neutrophil phagocytosis
The digestion process of bacterium is corresponding.However, the excessive HClO of generation may cause or aggravate a variety of diseases under inflammatory conditions, packet
Include Alzheimer disease, cardiovascular disease, atherosclerosis, inflammatory bowel disease, myocardial infarction, organ-graft refection, even
Cancer.Therefore, there is an urgent need to develop one kind for detecting ClO in organism-Quickly and effectively tool.
The method of the detection hypochlorous acid or hypochlorite that currently exist has chromatography, chemoluminescence method, electrical analysis
Deng.However, these method sensitivity are low, and it is complicated for operation, it is often more important that, it may not apply to internal HClO/ClO-Detection.And
Tool one of of the fluorescence probe as most strong detection and analysis object, it is easy to operate not need because they have high sensitivity
The detection to analyte is achieved that by complex instrument, and is also had and be easy to visual cells internal dynamics and high-resolution
The advantages of positioning interested biomolecule, there are two mutually independent fluorescence signals for Ratiometric fluorescent probe tool, can be more preferable
Ground avoids background interference.By laser confocal microscope carry out fluorescence imaging, it can be achieved that in organism hypochlorite detection.
Therefore, this just there is an urgent need to synthesize to have that simple, high sensitivity, selectivity is good, detection limit is low, the fluorescence probe of good light stability
For detecting the ClO of biological sample-。
Summary of the invention
An object of the present invention is to provide a kind of fluorescence probe DCCO.The second purpose is to provide the preparation of probe DCCO
Method, the preparation process is simple, low in cost.The third purpose is to provide the purposes of the probe, that is, is applied in biological sample
ClO-Detection.The probe should have quick response, high sensitivity, selectivity and good light stability, can effectively reduce biology
The advantages that interference of sample autofluorescence.
A kind of fluorescence probe DCCO provided by the invention is 7- (diethylamino) -3- (E) -3- (9- ethyl -9H- click
Azoles -3- base) acryloyl group) -2H- pyran-2-one, structural formula are as follows:
The preparation method of the fluorescence probe DCCO of one kind provided by the invention, includes the following steps:
(1) press 3- acetyl group -7- (diethylamino) -2H- pyran-2-one and 9- ethyl -9H- carbazole -3- formaldehyde etc.
Molar ratio is dissolved in ethanol/acetonitrile (1/1, V/V) mixed solution, is instilled piperidines, is heated to reflux 30 hours, solution is by crocus
Become red, TLC tracking reaction (VEthyl acetate: VPetroleum ether=1:3)；
(2) after reaction, solvent is removed under reduced pressure, linear gradient elution method carries out column chromatography for separation, first uses ethyl acetate/petroleum
Ether (1/3, V/V) elution, then eluted with ethyl acetate/petroleum ether (1/2, V/V), obtain the crude product of fluorescence probe；
(3) crude product ether dissolution, filtering are dry to get the pure products DCCO cotton-shaped to crocus.
Its synthetic route is as follows:
A kind of quantitative fluorescence provided by the invention detects ClO-Method, the steps include:
(1) the fluorescence probe DCCO stock solution of 1mM is prepared with DMSO；
(2) fluorescence cuvette is added in 2.0mL water/DMSO (1/1, v/v) system and 10.0 μ L fluorescence probe stock solutions
In, titration experiments are carried out on Fluorescence spectrophotometer, with ClO-Addition, the fluorescence intensity at 420nm gradually increases,
Fluorescence intensity at 570nm gradually weakens；
(3) with ClO-Concentration is abscissa, with relative intensity of fluorescence I420nm/I570nmFor ordinate drafting figure and carry out
Sigmoidal fitting, equation of linear regression are as follows: I420nm/I570nm=0.033* [NaClO] -1.586, NaClO concentration unit
It is 10-6mol/L；Linearly dependent coefficient is R2=0.994, optimum linear response range is 53.6 μM -375.2 μM.
Stability experiment proves fluorescence probe to ClO-Measurement have good photostability.
Experiments verify that Common Anions and biological thiol not interference system to ClO-Measurement.
Fluorescence probe DCCO of the invention passes through laser confocal microscope imaging technique, it was demonstrated that can be used for detecting biology
ClO in sample-Variation.
Compared with existing fluorescence probe, the present invention synthesizes fluorescence probe DCCO and has the advantage that 1, fluorescence of the invention
Probe synthesis step is simple, low in cost.2, detection means is simple, it is only necessary to can be realized by Fluorescence Spectrometer.3, fluorescence
Probe DCCO is to ClO-Response has the characteristics that short response time, high sensitivity and selectivity are good, and not by Common Anions
With the interference of biological thiol.4, Ratiometric fluorescent probe has effectively eliminated the interference of environmental factor.5, fluorescence probe DCCO can
For ClO in cell, zebra fish, arabidopsis-Detection.
Detailed description of the invention
2 fluorescence probe DCCO of Fig. 1 embodiment is with ClO-The uv absorption spectra of variation
3 fluorescence probe DCCO of Fig. 2 embodiment is with ClO-The fluorescence titration figure of variation
3 fluorescence probe DCCO of Fig. 3 embodiment is to ClO-The working curve of response
Response condition of the 4 fluorescence probe DCCO of Fig. 4 embodiment to Common Anions
Fig. 5 embodiment 5Hella cell imaging figure
6 zebra fish image of Fig. 6 embodiment
7 arabidopsis image of Fig. 7 embodiment
Specific embodiment
The preparation of 1 fluorescence probe DCCO of embodiment
(1) by 3- acetyl group -7- (diethylamino) -2H- pyran-2-one (0.777g, 3mmol), 9- ethyl -9H- click
Azoles -3- formaldehyde (0.669g, 3mmol) is dissolved in ethanol/acetonitrile (1/1, V/V) mixed solution of 15mL, instills piperidines
(0.75mL, 7.5mmol) is heated to reflux 30 hours, and reaction solution becomes red from crocus, TLC tracking reaction (VEthyl acetate: VPetroleum ether
=1:3)；
(2) after reaction, solvent is removed under reduced pressure, linear gradient elution method carries out column chromatography for separation, first uses ethyl acetate/petroleum
Ether (1/3, V/V) elution, then eluted with ethyl acetate/petroleum ether (1/2, V/V), obtain 0.699g crude product；
(3) crude product ether dissolution, filtering are dry to get the pure products for arriving 0.349g crocus, yield 25%.
Fluorescence probe is used1H NMR characterization is as a result as follows:
1H NMR(600MHz,DMSO-d6, δ/ppm): δ 8.61 (s, 1H), 8.55 (s, 1H), 8.24 (d, J=7.6Hz,
1H), 7.99 (d, J=15.6Hz, 1H), 7.91 (d, J=15.6Hz, 1H), 7.87 (d, J=8.4Hz, 1H), 7.71 (s, 1H),
7.69 (s, 1H), 7.66 (d, J=8.1Hz, 1H), 7.50 (t, J=7.6Hz, 1H), 7.26 (t, J=7.3Hz, 1H), 6.82
(d, J=8.9Hz, 1H), 6.63 (s, 1H), 4.48 (d, J=6.8Hz, 2H), 3.51 (d, J=6.6Hz, 4H), 1.34 (t, J=
6.7Hz, 3H), 1.16 (t, J=6.6Hz, 6H)
13C NMR(150MHz,DMSO-d6,δ/ppm):185.89,160.45,158.60,153.30,148.57,
144.41,141.42,140.54,132.70,126.81,126.60,126.31,123.14,122.63,122.47,122.34,
121.19,120.01,116.52,110.61,110.26,110.08,108.38,96.38,44.91,37.68,14.25,
12.85.
2 fluorescence probe DCCO of embodiment is with ClO-The uv absorption spectra of variation
10.0 μ L fluorescence probe stock solutions are added in 2.0mL water/DMSO (1/1, v/v) system, carry out ClO-Ultraviolet drop
Fixed experiment, and record its ultra-violet absorption spectrum (Fig. 1).With ClO-The increase of amount, the purple at 290nm, 321nm and 355nm
Outer absorption rises, the ultraviolet absorption value decline at 480nm.
3 fluorescence probe DCCO of embodiment is with ClO-The fluorescence titration figure of variation
10.0 μ L fluorescence probe stock solutions are added in 2.0mL water/DMSO (1/1, v/v) system and carry out ClO-Fluorescence drop
Fixed experiment, is detected on Fluorescence spectrophotometer, with ClO-Increase, the fluorescence intensity at 570nm gradually weakens,
Occur a new peak at 420nm and gradually increases (Fig. 2).Instrument parameter: the slit width of excitation wavelength and launch wavelength difference
For 5.0nm, 5.0nm, voltage 600V, the maximum excitation wavelength of fluorescence probe solution are as follows: λexFor 335nm and maximum emission wavelength
For λem570nm.With fluorescence ratio I420nm/I570nmIt draws and schemes for ordinate, obtain ClO-The working curve of concentration, linear regression
Equation is I420nm/I570nm=0.033* [NaClO] -1.586, NaClO concentration unit is 10-6mol/L；Linearly dependent coefficient
For R2=0.994, optimum linear response range is 53.6 μM -375.2 μM (Fig. 3).
Response condition of 4 fluorescence probe of embodiment to Common Anions and biological thiol
10.0 μ L fluorescence probe stock solutions are added in 2.0mL water/DMSO (1/1, v/v) system, then are separately added into it
Its anion and biological thiol (F-、Ac-、CNS-、S2-、SO3 2-、CO3 2-、Br-、HCO3 -、SO4 2-、S2O3 2-、Cl-、HSO3 -、GSH、
Cys), make 250 μM of its ultimate density, survey its fluorescence spectrum respectively, draw different anions and biological thiol corresponds to fluorescence
Intensity rate I420nm/I570nmHistogram.Experiment proves that other anion and biological thiol not interference system to ClO-'s
It detects (Fig. 4).
Embodiment 5Hella cell imaging figure
By HeLa cell in DMEM culture medium, 5%CO2, cultivate in 37 DEG C of environment.Before CLSM imaging, cell is connect
Kind is on 14mm glass cover-slip and is incubated for 12 hours, and cell is placed in multiple small culture dishes using preceding.By two small trainings
The culture solution extraction for supporting ware, and is washed with the PBS buffer solution of pH=7.40, be then separately added into two small culture dishes containing
2.0mLPBS (pH 7.40) buffer solutions of 20.0 μ L probes (1mM is dissolved with DMSO) is incubated for 5min or so, rear to extract out, with
The PBS buffer solution washing of pH=7.40 is three times.It is added in a small culture dish thereto and contains 10.0 μ L (1 × 10-2M)ClO-'s
The PBS buffer solution of 2.0mL pH=7.4 is incubated for 10min or so, rear to extract out, then is carried out with the PBS buffer solution of pH=7.40
Washing.It finally will only incubate probe and incubated probe and incubated the cell of hypochlorite again and be respectively placed in small culture dish and be added
The PBS solution of 2.0mL pH=7.40 is observed under laser confocal microscope.Fixed excitation wavelength is 405nm, collects transmitting
Wave band is blue channel (405-490nm) and yellow channels (500-600nm).As can be seen from Figure 5, when probe is only added, carefully
HSO is added in the stronger yellow fluorescence of presentation intracellular and faint blue-fluorescence (A1-D1)3 -Yellow weakens afterwards, and blue-fluorescence enhances
(A2-D2)。
6 zebra fish image of embodiment
Zebra fish is cultivated in E3 culture medium and is maintained at 28 DEG C, and the zebra fish in 5 day age is in the culture solution for containing 10 μM of probes
After middle incubation 15min, partially for being imaged, another part is containing ClO-10min, zebra are further incubated in the culture solution of (μm)
Fish is rinsed three times before imaging with PBS buffer solution.Fixed excitation wavelength is 405nm, and collection emission band is blue channel (405-
490nm) and yellow channels (500-600nm).As shown in fig. 6, zebra fish is very strong in yellow channels fluorescence after incubating probe,
Blue channel less fluorescence (A1-D1).When further using ClO-After incubating, yellow fluorescence weakens, and blue-fluorescence enhances (A2-
D2)。
7 arabidopsis image of embodiment
Carefully manually seedling is moved on to by sowing after arabidopsis seed disinfection on MS/2 solid medium, after 10 days homemade
On buoy, it is put into culture solution；Two days later, (hole is slightly smaller than buoy) is transferred on cystosepiment with holes, and the seedling after taking 10 days is made
It is spare for experimental material.Before imaging experiment, several small section fibres of arabidopsis are taken with tweezers, are put after being rinsed well with PBS buffer solution
Enter in the culture solution containing 10 μM of fluorescence probes, incubate 15min, portion washes are simultaneously imaged, and another part is added containing ClO-(μm)
Culture solution, wash and be imaged after being further incubated for 10min.Fixed excitation wavelength is 405nm, and collecting emission band is that blue is logical
Road (405-490nm) and yellow channels (500-600nm).As shown in fig. 7, arabidopsis is in yellow channels fluorescence after incubating probe
It is very strong, at blue channel less fluorescence (A1-D1).When further using ClO-After incubating, yellow fluorescence weakens, blue-fluorescence enhancing
(A2-D2)。
Claims (5)
1. a kind of fluorescence probe DCCO, is characterized in that, structural formula are as follows:
2. a kind of preparation method of fluorescence probe DCCO as described in claim 1, which is characterized in that synthetic route is as follows:
3. a kind of preparation method of fluorescence probe DCCO as described in claim 1, which comprises the steps of:
(1) 3- acetyl group -7- (diethylamino) -2H- pyran-2-one and 9- ethyl -9H- carbazole -3- formaldehyde are pressed into equimolar
Than being dissolved in the mixed solution of isometric ethyl alcohol and acetonitrile, piperidines is instilled, is heated to reflux 30 hours, solution is by orange discoloration
For red, TLC tracking reaction (VEthyl acetate: VPetroleum ether=1:3)；
(2) after reaction, solvent is removed under reduced pressure, linear gradient elution method carries out column chromatography for separation, first uses the acetic acid second of volume ratio 1:3
The elution of ester and petroleum ether, then with the ethyl acetate of volume ratio 1:2 and the elution of petroleum ether, obtain fluorescence spy
The crude product of needle；
(3) crude product ether dissolution, filtering are dry to get the pure products DCCO for arriving crocus.
4. a kind of quantitative fluorescence detects ClO-Method, which is characterized in that step are as follows:
(1) fluorescence probe DCCO stock solution described in the claim 1 of 1mM is prepared with DMSO；
(2) system and 10.0 μ L DCCO stock solutions of the water of 2.0mL volume ratio 1:1 and DMSO are added in fluorescence cuvette,
Titration experiments are carried out on Fluorescence spectrophotometer, with ClO-Addition, the fluorescence intensity at 420nm gradually increases, 570nm
The fluorescence intensity at place gradually weakens；
(3) with ClO-Concentration is abscissa, with relative intensity of fluorescence I420nm/I570nmFor ordinate drafting figure and carry out
Sigmoidal fitting, equation of linear regression are as follows: I420nm/I570nm=0.033* [NaClO] -1.586, NaClO concentration unit
It is 10-6mol/L；Linearly dependent coefficient is R2=0.994, optimum linear response range is 53.6 μM -375.2 μM.
5. a kind of fluorescence probe DCCO as described in claim 1 is used for biological sample ClO-Detection.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110627756A (en) * | 2019-10-10 | 2019-12-31 | 中国科学院新疆理化技术研究所 | Colorimetric-fluorescent probe for detecting hypochlorite, preparation method and application thereof |
| CN112745287A (en) * | 2020-12-30 | 2021-05-04 | 山西大学 | Fluorescent probe HM and preparation method and application thereof |
| CN113218922A (en) * | 2021-03-12 | 2021-08-06 | 天津理工大学 | Coumarin skeleton-based rapid hypochlorite ratio detection type fluorescent probe and application thereof |
Citations (3)
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| CN105837558A (en) * | 2016-04-22 | 2016-08-10 | 山西大学 | Reagent and method for fluorescence detection of hypochlorous acid |
| CN105968094A (en) * | 2016-05-30 | 2016-09-28 | 山西大学 | Carbazole fluorescent probe for detecting ClO- and preparation method and application thereof |
| CN109988560A (en) * | 2019-05-17 | 2019-07-09 | 济南大学 | A kind of hydrazine fluorescence probe of novel coumarin derivative |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105837558A (en) * | 2016-04-22 | 2016-08-10 | 山西大学 | Reagent and method for fluorescence detection of hypochlorous acid |
| CN105968094A (en) * | 2016-05-30 | 2016-09-28 | 山西大学 | Carbazole fluorescent probe for detecting ClO- and preparation method and application thereof |
| CN109988560A (en) * | 2019-05-17 | 2019-07-09 | 济南大学 | A kind of hydrazine fluorescence probe of novel coumarin derivative |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110627756A (en) * | 2019-10-10 | 2019-12-31 | 中国科学院新疆理化技术研究所 | Colorimetric-fluorescent probe for detecting hypochlorite, preparation method and application thereof |
| CN112745287A (en) * | 2020-12-30 | 2021-05-04 | 山西大学 | Fluorescent probe HM and preparation method and application thereof |
| CN112745287B (en) * | 2020-12-30 | 2022-03-18 | 山西大学 | Fluorescent probe HM and preparation method and application thereof |
| CN113218922A (en) * | 2021-03-12 | 2021-08-06 | 天津理工大学 | Coumarin skeleton-based rapid hypochlorite ratio detection type fluorescent probe and application thereof |
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