CN109824571A - A kind of fluorescence probe and the preparation method and application thereof - Google Patents
A kind of fluorescence probe and the preparation method and application thereof Download PDFInfo
- Publication number
- CN109824571A CN109824571A CN201910286087.6A CN201910286087A CN109824571A CN 109824571 A CN109824571 A CN 109824571A CN 201910286087 A CN201910286087 A CN 201910286087A CN 109824571 A CN109824571 A CN 109824571A
- Authority
- CN
- China
- Prior art keywords
- fluorescence probe
- formula
- fluorescence
- preparation
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention relates to a kind of for detecting the difunctional fluorescence probe and the preparation method and application thereof of hydroxyl radical free radical and viscosity.The structure of the fluorescence probe is shown in formula I, and wherein n is the integer of 1-6, R C1‑6Alkyl.Difunctional fluorescence probe provided by the invention can carry out highly sensitive, highly selective detection to hydroxyl radical free radical and viscosity in two independent analytical wavelength ranges simultaneously.In addition, difunctional fluorescence probe property provided by the invention is stablized, preparation method is easy, can be mass-produced, there is huge application prospect in the fields such as food, environment, biology.
Description
Technical field
The present invention relates to biochemical analysis technical fields, and in particular to a kind of fluorescence probe and the preparation method and application thereof.
Background technique
Hydroxyl radical free radical is a kind of active oxygen species that reactivity is high, in the cell, it can with injury protein matter,
The important biomolecules molecule such as DNA, lipid, so influence cell normal physiological function, cause a series of diseases, as artery sclerosis,
Neurodegenerative disease, cancer etc..Hydroxyl radical free radical has reactivity high, and the service life is short (nanosecond), the short (nanometer of diffusion length
Grade) the features such as.Therefore, the sensitivity for analysis of hydroxyl radical free radical probe and selectivity are required in actual application high.Mesh
Preceding hydroxyl radical free radical probe in the market mainly has: the APF based on oxidation reaction, HPF, CM-H2DCFDA etc.；Based on free radical
Proxyl fluorescamine, TEMPO-9-AC of spin trapping etc..But these probes suffer from other active oxygen objects
The oxidisability interference of kind (such as hydrogen peroxide, hypochlorous acid, Peroxynitrite), or need additional be added to the toxic work of organism
Dimethyl sulfoxide (DMSO).In addition, cytoplasm viscosity is the important indicator for influencing cell function, it can be with regulating cell inside
The rate of physiological activity species dispersal and signal transmitting.Therefore the change of the generation and viscosity that detect intracellular hydroxyl radical free radical is equal
Have great importance.
Summary of the invention
The difunctional fluorescence probe that the purpose of the present invention is to provide a kind of for detecting hydroxyl radical free radical and viscosity and its
Preparation method and application.
For this purpose, in a first aspect, structure is shown in formula I the present invention provides a kind of fluorescence probe,
Wherein, n is the integer of 1-6, R C1-6Alkyl.
Further, n is the integer of 2-4.Further, R is-CH3Or-CH2CH3。
Further, the structure of the fluorescence probe is as shown in Formula II,
Second aspect, the present invention provides the preparation methods of the fluorescence probe, including, change as shown in formula III and formula IV
It closes object in organic solvent, through base catalyzed condensation, obtains fluorescence probe shown in Formulas I,
Wherein, the definition of n and R is identical as the definition in the fluorescence probe.
Further, the concretely compound shown in Formula V of compound shown in formula III, the concretely formula of compound shown in formula IV
Fluorescence probe shown in Formula II is prepared in compound shown in VI,
Further, in the preparation method, the organic solvent can at least one of for acetic anhydride, ethyl alcohol and toluene,
Preferably acetic anhydride.
Further, the alkali can be at least one of sodium acetate, potassium carbonate and pyridine, preferably sodium acetate.
Further, the reaction temperature of the condensation reaction can be 20-100 DEG C, concretely 60 DEG C；The condensation reaction
Reaction time can be 1-5h, concretely 3h.
The third aspect, the present invention provide application of the fluorescence probe in hydroxyl radical free radical detection.
Specifically, by detecting fluorescence response intensity of the fluorescence probe at 652nm wavelength to measure hydroxyl free
Base.
Fourth aspect, the present invention provide application of the fluorescence probe in k value detection.
Specifically, by detecting fluorescence response intensity of the fluorescence probe at 520nm wavelength to measure viscosity number.
5th aspect, the present invention provide the fluorescence probe and detect application in hydroxyl radical free radical and viscosity number at the same time.
Specifically, by detecting fluorescence response intensity of the fluorescence probe at 652nm wavelength to measure hydroxyl free
Base；With by detecting the fluorescence response intensity of the fluorescence probe at 520nm wavelength to measure viscosity number.
The difunctional fluorescence probe of hydroxyl radical free radical and viscosity provided by the invention have the advantages that following characteristics and:
1) probe shows hypofluorescence, maximum emission wavelength 520nm in low-viscosity medium；
2) fluorescence of the probe at 520nm can enhance with the increase of medium viscosity, while not by medium polarity and pH
The influence of value；
3) probe can open response in the fluorescence that the near-infrared wavelength region (652nm) carries out specificity to hydroxyl radical free radical,
Not by the interference of other active oxygen species or physiological activity species；
4) probe can be carried out while be detected to hydroxyl radical free radical and viscosity in two independent wavelength periods；
5) good water solubility can avoid in use introducing the organic solvent to the toxic effect of organism；
6) preparation method is simple, can be mass-produced, and easily stored.
Detailed description of the invention
By reading the following detailed description of the preferred embodiment, various other advantages and benefits are common for this field
Technical staff will become clear.The drawings are only for the purpose of illustrating a preferred embodiment, and is not considered as to the present invention
Limitation.In the accompanying drawings:
Fig. 1 is fluorescence response figure of the fluorescence probe shown in Formula II to various concentration Fenton reagent.
Fig. 2 is fluorescence response intensity and Fenton of the fluorescence probe to Fenton reagent at 652nm wavelength shown in Formula II
The linear relationship chart of reagent concentration.
Fig. 3 is fluorescence response figure of the fluorescence probe shown in Formula II to different k values.
Fig. 4 is the logarithm and viscosity of fluorescence response intensity of the fluorescence probe to k value at 520nm wavelength shown in Formula II
The linear relationship chart of the logarithm of value, relationship meet- Hoffmann equation.
Fig. 5 is fluorescence imaging figure of the fluorescence probe shown in Formula II to HT-1080 cell.
Specific embodiment
The illustrative embodiments of the disclosure are more fully described below with reference to accompanying drawings.Although showing this public affairs in attached drawing
The illustrative embodiments opened, it being understood, however, that may be realized in various forms the disclosure without the reality that should be illustrated here
The mode of applying is limited.It is to be able to thoroughly understand the disclosure on the contrary, providing these embodiments, and can be by this public affairs
The range opened is fully disclosed to those skilled in the art.
The preparation of fluorescence probe shown in 1 Formula II of embodiment
The preparation method of fluorescence probe shown in Formula II is present embodiments provided, synthetic route is as follows:
Specific step is as follows:
By compound (164mg, 1mmol) and sodium acetate shown in compound shown in Formula V (562mg, 2mmol), Formula IV
(100mg) is dissolved in 5mL anhydrous acetic acid acid anhydride, stirs 3h at 60 DEG C.After cooling, by this reaction solution evaporated under reduced pressure, by what is obtained
Crude product uses column chromatography (silica G, 200-300 mesh), CH2Cl2/ MeOH/TFA (100:15:0.5, v/v/v) makees eluant, eluent,
Purification obtains end product (586mg, 85%), and product is yellow solid, as fluorescence probe shown in Formula II, by what is be prepared
Fluorescence probe is used for
In embodiment 2-4.
1H NMR(300MHz,298K,CD3OD):δ8.67-8.58(m,4H),8.06-8.01(m,4H),7.84-7.81
(m, 2H), 7.70-7.67 (m, 4H), 7.64-7.59 (m, 1H), 4.97 (t, 4H, J=7.5Hz), 4.09 (s, 3H), 3.06 (t,
4H, J=7.5Hz), 2.45 (s, 4H), 1.92 (s, 12H) .13C NMR (75MHz, 298K, DMSO-d6): δ 182.3,160.9,
145.9,144.4,141.3,134.5,130.4,129.8,129.0,126.3,123.6,116.2,115.8,65.5,63.3,
52.9,47.7,26.5,25.4.HR-ESI-MS:m/z calcd.for probe 1(C35H42N2O7S2,[M-H]-),
689.2360；found,689.2361.
Fluorescence probe shown in 2 Formula II of embodiment measures hydroxyl radical free radical in phosphate buffer
The phosphate buffer of 3mL 20mM is added in the testing tube of 5mL, is subsequently added into the fluorescence probe water of appropriate volume
Solution (1mM) makes 10 μM of final concentration and probe concentration, and a series of Fenton reagent for being eventually adding various concentrations (can produce hydroxyl
Base free radical).After reacting 30min, the fluorescence intensity of reaction solution is measured on sepectrophotofluorometer, excitation light wave is a length of
590nm。
Fig. 1 is fluorescence response figure of the fluorescence probe shown in Formula II to various concentration Fenton reagent, as shown in Figure 1, with
The increase of Fenton reagent concentration, the fluorescence at 652nm wavelength gradually increase；It is vertical seat that Fig. 2, which is with the fluorescence intensity at 652nm,
Mark, Fenton reagent concentration are abscissa mapping, obtain the relationship of fluorescence intensity Yu Fenton reagent concentration, as shown in Figure 2,
Within the scope of 0-30 μM, fluorescence intensity and Fenton reagent concentration show good linear relationship.
Methanol-glycerol mixed liquor viscosity of the measurement different volumes ratio of fluorescence probe shown in 3 Formula II of embodiment
A series of methanol of different volumes ratios-glycerol mixed liquor is added in testing tube, the fluorescence of appropriate volume is added
Probe methanol solution (1mM) makes 10 μM of final concentration and probe concentration.Above-mentioned solution is placed on shaking table, continues at the uniform velocity to rock 1h
It is sufficiently mixed solution, standing 30min again later eliminates bubble.The fluorescence that solution is measured on sepectrophotofluorometer is strong
Degree, a length of 400nm of excitation light wave.
Fig. 3 is fluorescence response figure of the fluorescence probe shown in Formula II to different k values, from the figure 3, it may be seen that with k value
Increase, the fluorescence at wavelength 520nm gradually increases；Fig. 4 is using the logarithm of the fluorescence intensity at 520nm as ordinate, and solution is glutinous
The logarithm of angle value be abscissa mapping, obtain the relationship of fluorescence intensity and k value, as shown in Figure 4, the logarithm of fluorescence intensity with
The logarithm of solution viscosity value shows good linear relationship, meets- Hoffmann equation.
Hydroxyl radical free radical level and cytoplasm in the detection cell iron death process of fluorescence probe shown in 4 Formula II of embodiment is glutinous
Degree variation
1) in the dedicated culture vessel with glass bottom of laser confocal fluorescence microscope, 37 DEG C and 5% (v/v) CO2Atmosphere item
Under part, with containing 10% (v/v) newborn bovine serum, 100U/mL penicillin, 100 μ g/mL streptomysin RPMI 1640 cultivate
Base culture HT-1080 cell for 24 hours, makes cell sufficiently be attached to culture dish bottom.
2) DMSO solution (1mM) of 10 μ L drug erastin is added in above-mentioned Tissue Culture Dish, makes final drug concentration
It is 10 μM, it is dead with inducing cell iron cultivates 4,6 or 8h；The cell of control group is then that 10 μ L DMSO are added to cultivate 4,6 or 8h；
In order to inhibit iron dead, while drug erastin cultivation is added, it is also added into 100 μM of iron death inhibitor Deferoxamine
(DFO)。
3) after the cell culture in step 2, the culture solution in culture dish is siphoned away, cleans cell with PBS buffer solution,
It is subsequently added into the RPMI 1640 culture medium that 1mL contains 20 μM of probes, continues to cultivate 30min.
4) after the cell culture in step 3, by culture dish be placed in confocal fluorescent microscopic carry out fluorescence at
Picture.Wherein, green channel excitation light source is 405nm laser, and fluorescence signal collection range is 480-540nm, and signal strength becomes
Change represents the variation of cytoplasm viscosity；Red channel excitation light source is 559nm laser, and fluorescence signal collection range is 600-
700nm, change in signal strength represent the variation of hydroxyl radical free radical level.
Fig. 5 is fluorescence imaging figure of the fluorescence probe shown in Formula II to HT-1080 cell, as shown in Figure 5, with erastin
The extension of time is cultivated, the fluorescence signal of either green channel or red channel enhances, this shows iron death process companion
With cytoplasm viscosity increase and hydroxyl radical free radical level rise, and this variation can the fluorescence probe shown in Formula II supervise simultaneously
It measures.
Finally it should be noted that above-described embodiment only enumerates the preparation and application of fluorescence probe shown in formula II, remaining fluorescence
Probe since structure and properties are close, do not list one by one by preparation method, test condition etc..
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
In the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of the claim
Subject to enclosing.
Claims (10)
1. a kind of fluorescence probe, which is characterized in that the structure of the fluorescence probe is shown in formula I,
Wherein, n is the integer of 1-6, R C1-6Alkyl.
2. fluorescence probe as described in claim 1, which is characterized in that the structure of the fluorescence probe as shown in Formula II,
3. the preparation method of fluorescence probe as described in claim 1, including, the compound as shown in formula III and formula IV is organic molten
In agent, through base catalyzed condensation, fluorescence probe shown in Formulas I is prepared,
Wherein, n is the integer of 1-6, R C1-6Alkyl.
4. preparation method as claimed in claim 3, which is characterized in that compound shown in formula III is compound shown in Formula V, formula
Compound shown in IV is compound shown in Formula IV, and fluorescence probe shown in Formula II is prepared,
5. preparation method as described in claim 3 or 4, which is characterized in that the organic solvent is acetic anhydride, ethyl alcohol and toluene
At least one of；The alkali is at least one of sodium acetate, potassium carbonate and pyridine.
6. preparation method as described in claim 3 or 4, which is characterized in that the organic solvent is acetic anhydride, and the alkali is second
Sour sodium.
7. preparation method as described in claim 3 or 4, which is characterized in that the reaction temperature of the condensation reaction is 20-100
DEG C, reaction time 1-5h.
8. application of the fluorescence probe as claimed in claim 1 or 2 in hydroxyl radical free radical detection.
9. application of the fluorescence probe as claimed in claim 1 or 2 in k value detection.
10. fluorescence probe as claimed in claim 1 or 2 detects the application in hydroxyl radical free radical and k value at the same time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910286087.6A CN109824571B (en) | 2019-04-10 | 2019-04-10 | Fluorescent probe and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910286087.6A CN109824571B (en) | 2019-04-10 | 2019-04-10 | Fluorescent probe and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109824571A true CN109824571A (en) | 2019-05-31 |
| CN109824571B CN109824571B (en) | 2020-07-07 |
Family
ID=66874385
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910286087.6A Active CN109824571B (en) | 2019-04-10 | 2019-04-10 | Fluorescent probe and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109824571B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999159A (en) * | 2021-11-09 | 2022-02-01 | 山东第一医科大学(山东省医学科学院) | Viscosity-sensitive fluorescent probe, and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103597039A (en) * | 2011-03-15 | 2014-02-19 | 雷蒙特亚特特拉维夫大学有限公司 | Activatable fluorogenic compounds and uses thereof as near infrared probes |
-
2019
- 2019-04-10 CN CN201910286087.6A patent/CN109824571B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103597039A (en) * | 2011-03-15 | 2014-02-19 | 雷蒙特亚特特拉维夫大学有限公司 | Activatable fluorogenic compounds and uses thereof as near infrared probes |
Non-Patent Citations (3)
| Title |
|---|
| HONGYU LI ET AL.: "Rationally Designed Fluorescence •OH Probe with High Sensitivity and Selectivity for Monitoring the Generation of •OH in Iron Autoxidation without Addition of H2O2", 《ANGEW. CHEM. INT. ED.》 * |
| XIANGBIN CAO ET AL.: "Styrylcyanine-based fluorescent probes with red-emission and large Stokes shift for the detection of viscosity", 《JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A: CHEMISTRY》 * |
| 曹相斌: "苯并噻唑半菁染料荧光探针的设计合成及应用", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999159A (en) * | 2021-11-09 | 2022-02-01 | 山东第一医科大学(山东省医学科学院) | Viscosity-sensitive fluorescent probe, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109824571B (en) | 2020-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | An AIE+ ESIPT ratiometric fluorescent probe for monitoring sulfur dioxide with distinct ratiometric fluorescence signals in mammalian cells, mouse embryonic fibroblast and zebrafish | |
| CN105542756B (en) | A kind of fluorescence probe for detecting formaldehyde and preparation method and application | |
| Yan et al. | A ratiometric fluorescence probe based on a novel recognition mechanism for monitoring endogenous hypochlorite in living cells | |
| CN106946773B (en) | Ratio type two-photon formaldehyde fluorescent probe and preparation method and application thereof | |
| CN108164450B (en) | A kind of carboxy-lesterase fluorescence probe and its preparation method and application | |
| CN105572095B (en) | A kind of detection reagent and quantitative detecting method of human serum albumins | |
| CN106967102B (en) | A kind of enhanced fluorescence probe of hydrogen peroxide based on Rhodamine Derivatives | |
| CN109836394B (en) | Near-infrared fluorescent probe for identifying hydrogen sulfide and preparation method and application thereof | |
| CN104673278A (en) | Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe | |
| CN106841128B (en) | Application of high-specificity fluorescent probe for detecting human serum albumin | |
| US9791451B2 (en) | Squaraine based fluorescent probe and a process for the preparation thereof | |
| CN106281304B (en) | A kind of fluorescence probe and preparation method thereof that can be used for malonaldehyde in living cells and be imaged | |
| CN109438326A (en) | A kind of fluorescence probe for detecting carboxy-lesterase and preparation method thereof and dedicated test kit | |
| CN106905310A (en) | Hypochlorous fluorescence probe of one kind detection and its preparation method and application | |
| CN109336815A (en) | Hypochlorous two-photon fluorescence probe in a kind of detection endocytoplasmic reticulum | |
| CN106588966B (en) | A kind of preparation and application of open form hydrogen peroxide fluorescent probe compounds | |
| CN109651249A (en) | A kind of fluorescence probe detecting endocytoplasmic reticulum cysteine and its synthesis and application | |
| CN106117241A (en) | A kind of detect the fluorescent probe of lysosomal pH in cancerous cell | |
| CN109776564A (en) | The ferrous ion fluorescence probe and its synthetic method of a kind of xanthene structure and application | |
| CN114539183B (en) | Lipid-droplet targeting and biological thiol-sensitive fluorescent probe for cancer cell tissue diagnosis and preparation and application thereof | |
| CN109180716B (en) | Multi-signal ratio type distinguishing detection H2O2And H2Design, synthesis and application of fluorescent probe of S | |
| CN105985299B (en) | A kind of fluorescence probe of highly selective hypersensitive analysis ferrous ion | |
| Zhou et al. | Water-soluble meso-ester substituted BODIPY with aggregation-induced emission property for ratiometric detection of carboxylesterases in living hepatoma cell | |
| CN110092771A (en) | A kind of fluorescence probe and preparation method thereof for human serum albumins detection, Fluorescence kit | |
| CN109824571A (en) | A kind of fluorescence probe and the preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |