CN108717055A - The purposes of high selection hypersensitive peroxynitrite ratio fluorescent probe - Google Patents
The purposes of high selection hypersensitive peroxynitrite ratio fluorescent probe Download PDFInfo
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- CN108717055A CN108717055A CN201810543675.9A CN201810543675A CN108717055A CN 108717055 A CN108717055 A CN 108717055A CN 201810543675 A CN201810543675 A CN 201810543675A CN 108717055 A CN108717055 A CN 108717055A
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- peroxynitrite
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- fluorescent probe
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- 0 CCCC(c1c(CO)c(*2*C2)c(*C)cc1)(c(cc(-c1nc(C(*)=C(*)C*)c(C*)[s]1)c(O)c1*)c1OC1=C2)C1=CC(*)C2N(CC)CC Chemical compound CCCC(c1c(CO)c(*2*C2)c(*C)cc1)(c(cc(-c1nc(C(*)=C(*)C*)c(C*)[s]1)c(O)c1*)c1OC1=C2)C1=CC(*)C2N(CC)CC 0.000 description 1
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The present invention relates to the purposes of high selection hypersensitive peroxynitrite ratio fluorescent probe.Specifically, high selection hypersensitive peroxynitrite ratio fluorescent probe of the invention can be used as detection of the peroxynitrite ratio fluorescent probe for peroxynitrite.This kind of probe can realize following at least one of technique effect：Peroxynitrite is identified with high selectivity；Quickly peroxynitrite can be realized and be responded；The hypersensitive analysis to peroxynitrite may be implemented；The ratio quantitative analysis to peroxynitrite may be implemented；Stablize with property, can be used with long-term preservation.
Description
Technical field
Use the present invention relates to benzothiazole-rhodamine compound as peroxynitrite ratio fluorescent probe
On the way, peroxynitrite high selection hypersensitive can be analyzed or its can in determination sample peroxynitrite it is dense
Degree.
Background technology
Active oxygen and reactive nitrogen species participate in various physiological processes in life system, including signal transduction, inflammation and antioxygen
Change damage.Peroxynitrite as a kind of oxidizing species in active oxygen and active nitrogen, it be by nitric oxide and
Superoxide radical reaction generates, and has high a reactivity and unstability, signal transduction in life system and antibacterial
It plays an important role in effect.However since it damages the nitrification of protein, nucleic acid, lipid etc., peroxynitrite
It is considered harmful, the abnormal accumulation of peroxynitrite will lead to many diseases, including ischemia-reperfusion damage in cell
Wound, diseases associated with inflammation, neurodegenerative disease, even cancer.Due to lacking the peroxynitrite of detection physiological level
Robust techniques, therefore the mechanism of action of peroxynitrite not yet completely illustrated.Therefore, seek it is a kind of specific and
The technology of peroxynitrite in Sensitive Detection life entity is vital.
In recent years, the method for the detection peroxynitrite reported for work has spectrophotometric Colo, high-efficient liquid phase color
Spectrometry, chemiluminometry, fluorescence probe analytic approach etc., wherein fluorescence probe are simple etc. because of its high selection, hypersensitive, synthesis
Unique advantage and become researcher's focus of attention.There are still certain to lack for the fluorescence probe analysis method reported at present
Fall into, for example, sensitivity is low, poor selectivity, poorly water-soluble, synthesis complexity etc..Other active oxygens and active nitrogen and peroxide in life entity
It is similar to change nitrite property, it can constitute the detection of peroxynitrite potential interference, and the mistake in physiological environment
Nitrite oxidation content is relatively low, and therefore, developing highly selective highly sensitive fluorescence probe becomes the project for being badly in need of solving.In addition,
Ratiometric fluorescent probe can eliminate the interference of environmental factor, therefore receive significant attention.In short, development is highly selective, high
Sensitivity, synthesis be simple, good water solubility ratio fluorescent probe is those skilled in the art's urgent problem
Invention content
This field is badly in need of preparing a kind of simple high selection hypersensitive peroxynitrite ratio fluorescent probe, so as to
Enough effectively detection peroxynitrites.It is visited for this purpose, the present invention has synthesized a kind of novel peroxynitrite ratio fluorescent
Needle, synthesis is simple, selectivity is good, high sensitivity, can identify peroxynitrite.Specifically, the present invention provides
A kind of purposes of peroxynitrite ratio fluorescent probe, the probe are benzothiazole-rhodamine compound, structure
It is as follows：
Preferably, fluorescence probe of the invention is：
The present invention also provides the probes of the present invention to prepare the system for detecting peroxynitrite concentration in sample
Purposes in agent or kit.
The peroxynitrite ratio fluorescent probe of the present invention can be acted on peroxynitrite, generate fluorescence
The variation of spectrum, to realize the quantitative detection to peroxynitrite.
Specifically, the present invention peroxynitrite ratio fluorescent probe not with potassium ion, calcium ion, sodium ion,
Magnesium ion, ferric ion, ferrous ion, nitrate anion, nitrite anions, chlorion, sulfate radical, hydrogen peroxide, surpasses zinc ion
The reactions such as potassium oxide, to realize the Selective recognition to peroxynitrite.
The peroxynitrite ratio fluorescent probe of the present invention reacts very sensitive with peroxynitrite, to have
Conducive to the detection to peroxynitrite.
Selectively, the stability of peroxynitrite fluorescence probe of the invention is good, so can long-term preservation make
With.
Further, peroxynitrite ratio fluorescent probe of the invention is highly selective hypersensitive peroxidating nitrous
Acid salt ratio fluorescence probe, and synthesize simply, be conducive to commercialized popularization and application.
Description of the drawings
Fig. 1 a are (5 μM) fluorescence spectrums being added before and after peroxynitrite (0-60 μM) of probe；
Fig. 1 b are the working curves of probe (5 μM) quantitative analysis various concentration peroxynitrite (0-20 μM)；
Fig. 2 is that (5 μM) of probe is added peroxynitrites (50 μM) spectrum changes with time situation afterwards；
Fig. 3 is the influence of substance common in human body to probe (5 μM) fluorescence intensity.Wherein number 1-24 is respectively sky
In vain, potassium ion, calcium ion, sodium ion, magnesium ion, zinc ion, ferric ion, ferrous ion, nitrate anion, nitrite anions,
Chlorion, sulfate radical, cysteine (500 μM), homocysteine (500 μM), glutathione (5mM), tertbutanol peroxide,
Hydroxyl radical free radical, tertbutanol peroxide free radical, hydrogen peroxide, potassium superoxide, nitric oxide, singlet oxygen, sodium hypochlorite, mistake
Nitrite oxidation (50 μM) (in addition to special indicate, other analyte concentrations are 100 μM).What block diagram represented is different points
The ratio of probe fluorescence intensity at 581nm and 454nm in the presence of analysis object.
Specific implementation mode：
The present invention provides the synthetic route of above-mentioned highly selective highly sensitive peroxynitrite fluorescence probe, method and
Its spectrum property.
The peroxynitrite ratio fluorescent probe of the present invention is benzothiazole-rhodamine compound, tool
There is following general structure
In above formula：R1, R2, R3, R4, R5, R6, R7, R8, R9, R10For hydrogen atom, linear or branched alkyl group, linear chain or branched chain alkane
Oxygroup, sulfonic group, ester group, carboxyl；R1, R2, R3, R4, R5, R6, R7, R8, R9And R10It can be identical or different.
The synthetic route and method of such peroxynitrite ratio fluorescent probe are as follows：
Specifically, ratio fluorescent probe of the invention can be prepared via a method which, by certain mol proportion (such as 1: 1-
1: 20) benzothiazole-rhodamine compound is dissolved in cooling in absolute ethyl alcohol with hydrazine hydrate (such as 1: 1-1: 20), the two
Molar ratio is (1: 20), is then refluxed for a period of time (such as 6h), reduced pressure backspin solvent evaporated.If obtained purer
Product, can by the mixed system of crude product dichloromethane and methanol (such as v/v, 100: 1) carrying out pillar layer separation and obtain
Sterling.
Therefore, the present invention also provides hydrazine hydrates in preparing the ratio fluorescent probe for detecting peroxynitrite
Purposes.
The present invention also provides benzothiazole-rhodamine compounds to prepare the ratio for detecting peroxynitrite
Purposes in rate fluorescence probe.
The present invention peroxynitrite ratio fluorescent probe be noteworthy characterized by can high selection hypersensitive identified
Accurately peroxynitrite quantitatively can be divided in the presence of nitrite oxidation and other ions in human body
Analysis.
It below will be by the way that the present invention be described in more detail by following embodiment.Following embodiment is merely illustrative,
It should be understood that the present invention is not limited by following embodiments.
Embodiment 1
200mg (0.38mmol) benzothiazole-rhodamine compound is dissolved in 15mL absolute ethyl alcohols, is added
380mg (7.6mmol) hydrazine hydrate reflux 6h, reduced pressure backspin solvent evaporated.It, can will be thick if obtaining purer product
Product dichloromethane and methanol mixed system (such as v/v, 100: 1) carrying out pillar layer separation and obtain sterling, obtain yellowish
Colour purity net products 180mg, yield 89%.
1H NMR demonstrate the correctness of product structure.
Embodiment 2
Fig. 1 a are (5 μM) fluorescence spectrums being added before and after peroxynitrite (0-60 μM) of probe.Fig. 1 b are different dense
Spend linear relationship chart of the nitrite oxidation (0-20 μM) to probe (5 μM).
The parallel sample that multiple concentration and probe concentrations are 5 μM is configured in 10mL colorimetric cylinders, then by the peroxidating of various concentration
Nitrite is added in test system, is stood after rocking uniformly.Said determination is in dimethyl sulfoxide (DMSO): water=3: 7 (20mM
PBS, pH 7.4) it carries out in system, used probe is in embodiment 1 prepared probe, and all spectrum tests are all
It is measured at 25 DEG C.
Its fluorescence intensity change is tested with Fluorescence Spectrometer, can clearly be found out from Fig. 1 a, with peroxinitrites
The increase of salinity, the fluorescence intensity at 454nm reduce, and the fluorescence intensity at 581nm gradually increases.Also, it can be with by Fig. 1 b
Find out that (5 μM) of fluorescence probe is added after peroxynitrite (0-20 μM), the ratio and mistake of fluorescence intensity at two wavelength
Good linear relationship is presented between the concentration of nitrite oxidation, this proof can be to peroxidating by means of the fluorescence probe
Nitrite carries out ratio quantitative analysis.
Embodiment 3
Fig. 2 is (5 μM) response times being added after peroxynitrite (50 μM) of probe.50 are taken out from probe mother liquor
μ L are placed in the test system of 10mL, and then 50 μM of peroxynitrite is added in test system, vertical after rocking uniformly
Its fluorescence intensity change is tested with Fluorescence Spectrometer.Said determination is in dimethyl sulfoxide (DMSO): water=3: 7 (20mM PBS, pH
7.4) it is carried out in system, used probe is probe prepared in embodiment 1, and all spectrum tests are all at 25 DEG C
Under measure.
By scheming it will be clear that after peroxynitrite is added, 6min or so fluorescence intensities reach after testing
Maximum value simultaneously remains unchanged, this illustrates that the probe is swift in response with peroxynitrite, can be peroxynitrite
It measures and quick analysis method is provided.
Embodiment 4
Fig. 3 is influence of the different analytes to probe (5 μM) fluorescence spectrum.
Analyte is respectively blank, potassium ion, calcium ion, sodium ion, magnesium ion, zinc ion, ferric ion, ferrous iron
Ion, nitrate anion, nitrite anions, chlorion, sulfate radical, cysteine (500 μM), homocysteine (500 μM), gluathione
Peptide (5mM), tertbutanol peroxide, hydroxyl radical free radical, tertbutanol peroxide free radical, hydrogen peroxide, potassium superoxide, an oxidation
Nitrogen, singlet oxygen, sodium hypochlorite, peroxynitrite (50 μM), (in addition to special indicate, other analyte concentrations are 100
μM).What block diagram represented is fluorescence intensity ratio of the probe at 581nm and at 454nm in the presence of different analytes.Above-mentioned survey
Surely it is in dimethyl sulfoxide (DMSO): water=3: is carried out in 7 (20mM PBS, pH7.4) systems, used probe is in embodiment 1
Prepared probe, and all spectrum tests all measure at 25 DEG C.Specifically, it is 5 μM to configure multiple concentration and probe concentrations
Then parallel sample is added a certain amount of analyte, shakes up in 10mL colorimetric cylinders, wait for measuring after ten minutes.It can be clear by Fig. 3
Clear finds out, which has high selectivity to peroxynitrite.
Although with above embodiments describe the present invention, it should be appreciated that before the spirit without departing substantially from the present invention
It puts, the present invention further can be modified and be changed, and these modifications and variation all belong to the scope of protection of the present invention it
It is interior.
Claims (6)
1. compound is being prepared for detecting the preparation of peroxynitrite in sample or the purposes in kit, wherein described
Compound has following structure：
Wherein：R1, R2, R3, R4, R5, R6, R7, R8, R9And R10For independently selected from by hydrogen atom, linear or branched alkyl group, straight chain
Or the group of branched alkoxy, sulfonic group, ester group and hydroxyl composition；And R therein1, R2, R3, R4, R5, R6, R7, R8, R9And R10It can
With identical or different.
2. purposes according to claim 1, wherein the compound has the following structure：
3. purposes according to claim 1 or 2, wherein the sample is water or blood.
4. purposes according to claim 1 or 2, wherein the kit also includes buffer.
5. purposes according to claim 1 or 2, wherein the kit also includes operation instructions.
6. purposes according to claim 1 or 2, wherein the detection is qualitative detection or quantitative detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108586473A (en) * | 2018-05-30 | 2018-09-28 | 济南大学 | A kind of high selection hypersensitive peroxynitrite ratio fluorescent probe |
Citations (2)
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|---|---|---|---|---|
| CN104031039A (en) * | 2013-03-05 | 2014-09-10 | 西北大学 | Xanthene dye, and preparation method and application thereof |
| CN105086998A (en) * | 2015-09-08 | 2015-11-25 | 济南大学 | Lysosome targeted fluorescence probe for detecting hypochlorous acid molecules, as well as preparations method and application thereof |
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2018
- 2018-05-30 CN CN201810543675.9A patent/CN108717055A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104031039A (en) * | 2013-03-05 | 2014-09-10 | 西北大学 | Xanthene dye, and preparation method and application thereof |
| CN105086998A (en) * | 2015-09-08 | 2015-11-25 | 济南大学 | Lysosome targeted fluorescence probe for detecting hypochlorous acid molecules, as well as preparations method and application thereof |
Non-Patent Citations (4)
| Title |
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| BAOCUN ZHU等: "A highly specific far-red fluorescent probe for imaging endogenous peroxynitrite in the mitochondria of living cells", 《SENSORS AND ACTUATORS B》 * |
| CHUNLEI FAN等: "Optimization of an analytical method for the spectrophotometric determination of copper in tea and water samples after ultrasonic assisted cloud point extraction using a benzothiazole fluorescein derivative complexing agent", 《RSC ADVANCES》 * |
| HONGYU LI等: "Observation of the Generation of ONOO− in Mitochondria under Various Stimuli with a Sensitive Fluorescence Probe", 《ANALYTICAL CHEMISTRY》 * |
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| CN108586473A (en) * | 2018-05-30 | 2018-09-28 | 济南大学 | A kind of high selection hypersensitive peroxynitrite ratio fluorescent probe |
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