CN117024395A - Be used for detecting H 2 S flavonol derivative fluorescent probe and preparation method and application thereof - Google Patents
Be used for detecting H 2 S flavonol derivative fluorescent probe and preparation method and application thereof Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 52
- 150000002216 flavonol derivatives Chemical class 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 16
- 235000013305 food Nutrition 0.000 claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 claims description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 5
- 238000006073 displacement reaction Methods 0.000 claims description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 3
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- 210000004027 cell Anatomy 0.000 description 20
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 238000012360 testing method Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 9
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-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
- 238000003384 imaging method Methods 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
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- QSQDXWXPRUIQMY-UHFFFAOYSA-N 1-(3-hydroxynaphthalen-2-yl)ethanone Chemical compound C1=CC=C2C=C(O)C(C(=O)C)=CC2=C1 QSQDXWXPRUIQMY-UHFFFAOYSA-N 0.000 description 2
- 102100034976 Cystathionine beta-synthase Human genes 0.000 description 2
- 108010073644 Cystathionine beta-synthase Proteins 0.000 description 2
- 102000020018 Cystathionine gamma-Lyase Human genes 0.000 description 2
- 108010045283 Cystathionine gamma-lyase Proteins 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
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- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 description 2
- 235000011957 flavonols Nutrition 0.000 description 2
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000020995 raw meat Nutrition 0.000 description 2
- UPHOPMSGKZNELG-UHFFFAOYSA-N 2-hydroxynaphthalene-1-carboxylic acid Chemical compound C1=CC=C2C(C(=O)O)=C(O)C=CC2=C1 UPHOPMSGKZNELG-UHFFFAOYSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
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- 241000238557 Decapoda Species 0.000 description 1
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- 235000013878 L-cysteine Nutrition 0.000 description 1
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- 239000007864 aqueous solution Substances 0.000 description 1
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- KCGOXHUBDPEPGG-UHFFFAOYSA-N benzaldehyde 4-methoxybenzaldehyde Chemical compound O=CC1=CC=CC=C1.COC1=CC=C(C=O)C=C1 KCGOXHUBDPEPGG-UHFFFAOYSA-N 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
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- 238000003745 diagnosis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 150000007946 flavonol Chemical class 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/92—Naphthopyrans; Hydrogenated naphthopyrans
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- 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
- G01N21/6428—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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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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Abstract
The invention discloses a method for detecting H 2 S flavonol derivative fluorescent probe and its preparation method and application. Belongs to the technical field of fluorescence detection, and the structural formula of the probe is shown as formula I:the synthesis process of the probe molecule is easy to control, the reaction condition is mild, and the probe molecule is specific to H 2 S has high selectivity and sensitivity, low detection limit, excellent anti-interference capability, and H in environmental water sample, food spoilage and cancer cells 2 The S detection process shows a good detection effect, has important practical application prospect and has potential application value in the field of drug development.
Description
Technical Field
The invention relates to the technical field of fluorescence detection (bioanalytical detection), in particular to a flavonol derivative fluorescent probe with large Stokes displacement, a preparation method thereof and a detection method thereof in H detection 2 Practical application of S aspect.
Background
H 2 S is a colorless and odorous gas with high toxicity, and is mixed with CO and NOTogether considered as endogenous gas signaling molecules in the living system. In a mammal, H 2 S catalyzes the production of L-cysteine and homocysteine by enzymes such as cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE) and the like, and plays a vital role in the regulation and protection of the cardiovascular system and the nervous system in pathological and physiological processes. Intracellular H is reported 2 Abnormal fluctuations in S concentration are closely related to a variety of diseases including diabetes, cardiovascular disease, alzheimer' S disease and even cancer. In addition, H 2 S is also one of the common components of gases generated during food spoilage, and is produced by bacterial degradation of various sulfides, the release level of which is an important indicator for monitoring the freshness and quality of food. Thus, H is detected efficiently and accurately 2 The S content is very important.
A number of assays and assays for H have been developed in the prior art 2 S, such as electrochemical method, gas chromatography, liquid chromatography, colorimetry, methylene blue analysis, and the like. Unfortunately, these methods have the disadvantages of complex operation, expensive equipment, poor selectivity, and the like, and even have a certain damage to cells, which greatly limits their practical application. In contrast, the fluorescent probe has high selectivity, high sensitivity and timely imaging performance, and is simple to operate and low in cost, so that the fluorescent probe is used in H 2 S qualitative and quantitative detection is of great interest.
Currently, many are used to detect H 2 S fluorescent probes were reported, while H synthesized using flavonol derivatives 2 S fluorescent probes are reported less, and H in the prior art 2 S fluorescent probes are mostly used in living cell imaging for food spoilage process H 2 Fluorescent probes for S detection are more rare. Therefore, a rapid and convenient detection method for H in organisms and foods is developed 2 S-fluorescence probes are of great research value. Furthermore, in addition to the property of flavonols to release CO under light induction, the flavonol-based fluorescent probe can be further designed as a dual-functional platform, namely by biomarker H 2 The guidance of S can release CO molecule under light induction, which has a role in the research and development of anticancer drugsThe fixed guiding significance and development prospect.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects existing in the prior art, the invention provides a novel H with excellent performance and larger Stokes displacement 2 S fluorescent probe molecule, and provides a synthesis method and application of the probe. The probe has the advantages of short response time, good selectivity, high sensitivity, simple preparation method and good application to H in the process of cell and food spoilage 2 S, detection. The invention also provides the probe developed into H 2 S tracks and directs the use of CO to release bifunctional drug molecules.
(II) technical scheme
In order to achieve the above purpose, the invention adopts the following technical scheme: a flavonol derivative fluorescent probe with large Stokes displacement is characterized in that the structural formula I of the probe Fla-DNP is as follows:
preferably, the preparation method of the flavonol derivative fluorescent probe with large Stokes shift is characterized by comprising the following steps of:
s1, placing 4-dimethylamino benzoflavonol, 2, 4-dinitrofluorobenzene and potassium carbonate into a round-bottom flask, adding DMF (dimethyl formamide) as a solvent, stirring for 2 hours at room temperature, pouring the mixed solution after the reaction into ice water after the reaction is finished, filtering, washing and drying to obtain a crude product after solid precipitation;
s2, performing column chromatography separation by using ethyl acetate and hexane as eluent, and performing spin-drying to obtain an orange solid, namely the compound shown in the formula I.
Preferably, a preparation method of a flavonol derivative fluorescent probe with large Stokes shift comprises the following reaction preparation routes:
preferably, one of the compounds of formula I above is used for detecting H generated by food spoilage in the preparation of a fluorescent probe Fla-DNP 2 And S, loading the fluorescent probe on a paper strip to prepare a fluorescent test paper strip, placing the fluorescent test paper strip and raw meat in a closed culture dish, and monitoring the freshness of the raw meat according to the color change of the fluorescent test paper strip under a 365nm ultraviolet lamp.
Preferably, one of the compounds of formula I above is used for detecting H in living cells in the preparation of a fluorescent probe Fla-DNP 2 The fluorescent probe provided by the invention has low toxicity and better biocompatibility, and can be used for detecting endogenous and exogenous H in cancer cells 2 S, according to whether fluorescence appears in a green channel of a cell imaging experiment, detecting H in cells 2 S。
Preferably, one of the compounds of formula I is designed as H in the preparation of a fluorescence detection probe Fla-DNP 2 The fluorescent probe provided by the invention has the characteristic that the selected fluorophores have light-induced CO release property, and can be designed to be H by utilizing the characteristic 2 S activated CO releaser, which can be used for intracellular H 2 S levels regulate and direct CO release.
Preferably, the molar ratio of 4-dimethylamino benzoflavonol, 2, 4-dinitrofluorobenzene and potassium carbonate is 1: (1-2): (1-2).
(III) beneficial effects
The invention has the beneficial effects that:
the flavonol derivative-based hydrogen sulfide fluorescent probe is simpler in synthesis, excellent in hydrogen sulfide recognition performance in complex environment, high in sensitivity and low in detection limit, and can be used for rapidly and conveniently detecting H generated in the food spoilage process through the change of fluorescent signals of test strips 2 S, is used for monitoring food freshness and detecting H in cancer cells 2 S, even can utilize H 2 The S detection function locates the CO molecule release position, and has wide application prospect in the technical field of fluorescence detection and the field of targeted anticancer drug development.
Drawings
FIG. 1 is a synthetic route diagram of a fluorescent probe Fla-DNP of the present invention;
FIG. 2 is a bar graph of the selectivity and tamper resistance of fluorescent probes Fla-DNP of the present invention for different analytes;
FIG. 3 shows a fluorescent probe Fla-DNP recognition H according to the present invention 2 S time response diagram;
FIG. 4 shows a fluorescent probe Fla-DNP recognition H according to the present invention 2 A sensitivity map of S;
FIG. 5 shows the detection of H in food spoilage by the fluorescent probe Fla-DNP of the present invention designed as a fluorescent strip 2 S is a change chart;
FIG. 6 shows the fluorescence probe Fla-DNP of the present invention against H in living cells 2 S, fluorescence imaging diagram;
FIG. 7 shows that the fluorescent probe Fla-DNP of the present invention is designed to be H 2 S activated CO releaser in vitro releases the fluorescence emission spectrum chart of CO;
FIG. 8 shows that the fluorescent probe Fla-DNP of the present invention is designed to be H 2 S-activated CO releasers were applied to the fluorescence change patterns of living cells.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
this example provides a flavonol derivative H with a large Stokes shift 2 The preparation method of the S fluorescent probe comprises the following steps:
1. synthesis of 1- (3-hydroxynaphthalen-2-yl) ethanone
At N 2 1.88g of 2-hydroxynaphthoic acid was dissolved in 40mL of redistilled THF under protection and cooled to 0℃with an ice bath. 18.7mL LiCH was slowly added dropwise to the system using a closed syringe 3, Reacting at 0deg.C for 3 hr, adding HCl dropwise for quenching until no bubbles are generated. THF was drained under reduced pressure, 0.5mol/L HCl was added dropwise to the residue, and CH was used 2 Cl 2 The acidic aqueous solution was extracted three times, the organic phases were combined, dried, filtered, and the solvent was evaporated to dryness to give bright yellow solid product 2 (fig. 1). The yield thereof was found to be 68%.
Nuclear magnetic resonance hydrogen spectrum: 1 H NMR(400MHz,Chloroform-d)δ8.37(s,1H),7.83(d,J＝8.3Hz,1H),7.76–7.64(m,2H),7.57–7.48(m,1H),7.35(d,J＝8.1Hz,1H),7.29(s,1H),2.81(s,3H)。
2. synthesis of 4-dimethylaminobenzoflavonol
The NaOH solution was added to a suspension of 3.90g of 1- (3-hydroxynaphthalen-2-yl) ethanone in ethanol and stirred at room temperature for 30min. 2.55mL of benzaldehyde p-methoxybenzaldehyde was added to the reaction mixture and stirred for 5h to obtain a black-red solution. The reaction is ice-bathed to 0 ℃ and a proper amount of H is added dropwise 2 O 2 Stir at room temperature overnight. Acidifying the solution with HCl to ph=6.5, obtaining a bright yellow precipitate, filtering, washing with ethanol, and drying to obtain the target product Fla. The yield thereof was found to be 30%.
Nuclear magnetic resonance hydrogen spectrum: 1 H NMR(600MHz,Chloroform-d)δ9.75(s,1H),8.83(s,1H),8.32–8.26(m,2H),8.06(d,J＝8.4Hz,1H),8.02(s,1H),7.92(d,J＝8.4Hz,1H),7.59(ddd,J＝8.2,6.6,1.2Hz,1H),7.50(ddd,J＝8.0,6.6,1.2Hz,1H),6.85–6.83(m,2H),3.10(s,6H)。
3. synthesis of fluorescent Probe Fla-DNP
61.4mg of 1-fluoro-2, 4-dinitrobenzene was added to a mixture containing Fla (100 mg) and anhydrous K 2 CO 3 Is stirred for 2 hours in 3mL of DMF solution. After the reaction was completed, the mixture was poured into ice water, and then solid was precipitated. The crude product obtained was filtered with hexane: ethyl acetate = 3:1 (v/v) was further purified by column chromatography as eluent to give Fla-DNP as an orange solid. Yield: 67%.
Nuclear magnetic resonance hydrogen spectrum: 1 h NMR (400 mhz, dmso-D6) delta 8.93 (D, j=2.7 hz, 1H), 8.73 (s, 1H), 8.42 (s, 1H), 8.32 (dd, j=9.4, 2.7hz, 1H), 8.26 (D, j=8.4 hz, 1H), 8.14 (D, j=8.2 hz, 1H), 8.04 (D, j=9.1 hz, 2H), 7.73 (t, j=7.6 hz, 1H), 7.61 (dd, j=12.5, 8.4hz, 2H), 6.86 (D, j=9.1 hz, 2H), 3.05 (s, 6H). HR-MS (ESI-TOF) calculated Fla-DNPm/z＝520.1115[M+Na] + The method comprises the steps of carrying out a first treatment on the surface of the Actual value m/z= 520.1112.
Example 2:
1. fluorescent probe Fla-DNP vs H 2 S selectivity test:
to 20. Mu. Mol/L fluorescent probe Fla-DNP in PBS buffer (pH=7.4, 10% DMSO and 3mM CTAB) was added 0.8mL (1 mmol/L) of each analyte (0-34, na + ,Ni 2+ ,Al 3+ ,Ca 2+ ,NH 4 + ,Fe 3+ ,Zn 2+ ,K + ,Co 2+ ,Mn 2+ ,SO 3 2- ,NO 3 - ,SO 4 2- ,Cl - ,Br - ,HCO 3 - ,NO 2 - ,CO 3 2- ,SCN - ,F - ,I - ,Trp,Leu,Try,Gly,Phe,Lys,Arg,Val,Pro,Cys,Hcy,GSH,H 2 S), the change in fluorescence intensity of the solution at 566nm was detected. As shown in FIG. 2, fla-DNP vs. H 2 S shows a clear fluorescence response, while in the presence of other analytes, the increase in fluorescence intensity is limited, almost negligible, indicating that the fluorescent probe Fla-DNP is H-directed 2 The identification of S has good selectivity.
2. Fluorescent probe Fla-DNP vs H 2 S, testing of anti-interference performance:
to 20. Mu. Mol/L fluorescent probe Fla-DNP in PBS buffer (pH=7.4, 10% DMSO and 3mM CTAB) was added 0.8mL (1 mmol/L) of each analyte (0-34, na + ,Ni 2+ ,Al 3+ ,Ca 2+ ,NH 4 + ,Fe 3+ ,Zn 2+ ,K + ,Co 2+ ,Mn 2+ ,SO 3 2- ,NO 3 - ,SO 4 2- ,Cl - ,Br - ,HCO 3 - ,NO 2 - ,CO 3 2- ,SCN - ,F - ,I - ,Trp,Leu,Try,Gly,Phe,Lys,Arg,Val,Pro,Cys,Hcy,GSH,H 2 S) and then 0.1mL (8 mmol/L) of H was added to the above mixed solution containing the other analytes, respectively 2 S, detecting fluorescence emission spectrum at 566 nm. As shown in FIG. 2, when the above-mentioned is dryFla-DNP vs H in the presence of interfering analytes 2 S is still effective, and it is known that the presence of other ions does not affect the fluorescence probe Fla-DNP to H 2 S has good anti-interference performance.
3. Fluorescent probe Fla-DNP vs H 2 S response time test:
to 20. Mu. Mol/L fluorescent probe Fla-DNP in PBS buffer (pH=7.4, 10% DMSO and 3mM CTAB) was added 0.8mL (1 mmol/L) H 2 Fluorescence intensity was measured after S, once every 1 minute. As shown in FIG. 3, the fluorescence intensity gradually increased with time, and the fluorescence intensity reached a substantially saturated state at about 6 minutes, indicating that the probe Fla-DNP was sensitive to H 2 The identification of S can be completed within about 6 minutes, and the fluorescence intensity is linearly increased within 0 to 4 minutes, so that the rapid response capability is realized.
4. Fluorescent probe Fla-DNP vs H 2 S sensitivity test:
to 20. Mu. Mol/L fluorescent probe Fla-DNP in PBS buffer (pH=7.4, 10% DMSO and 3mM CTAB) was added different concentrations of H 2 S (0. Mu.M, 2. Mu.M, 4. Mu.M, 6. Mu.M, 8. Mu.M, 10. Mu.M, 12. Mu.M, 14. Mu.M, 16. Mu.M, 18. Mu.M, 20. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M, 100. Mu.M, 120. Mu.M, 140. Mu.M, 180. Mu.M, 200. Mu.M, 220. Mu.M, 240. Mu.M, 260. Mu.M, 280. Mu.M, 300. Mu.M, 320. Mu.M, 340. Mu.M, 360. Mu.M, 380. Mu.M, 400. Mu.M), the fluorescence emission spectrum is as shown in FIG. 4, it can be seen that the fluorescence intensity at 566nm gradually increases when 10 times H is added 2 At S, the fluorescence intensity substantially reached a saturation state, and the detection limit was calculated to be 0.834. Mu.M.
Example 3:
1. fluorescent probe Fla-DNP for detecting H generated by food spoilage 2 S：
Fla-DNP is prepared into a DMSO solution with the concentration of 40 mu mol/L, 1cm multiplied by 2cm paper strips are immersed into the solution, and the fluorescent test paper strips can be obtained by drying at normal temperature. The fluorescent test strips and food samples (pork and shrimp) are placed in a culture dish to be stored in a sealed manner for 48 hours, and the fluorescent change of the test strips is observed every 12 hours. As shown in FIG. 5, the orange fluorescence of the test strip increases gradually with time, indicating H 2 The concentration of S also gradually increased, fluorescent probe Fla-DNP with a design to monitor food freshness for portable toolsPotential applications of the degree.
2. Fluorescent probe Fla-DNP for detecting H in living cells 2 S：
The cytotoxicity of fluorescent probe Fla-DNP was tested by MTT method first, and cell viability was still above 90% after incubation with 4T1 cells for 24 hours at all concentrations (0. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M), indicating that the probe had good biocompatibility. Subsequently, four sets of cell imaging experiments were performed, one set being a control set, the remaining three sets of 4T1 cells were incubated with probe Fla-DNP (30. Mu.M) for 30 minutes in a cell incubator, washed three times with PBS buffer, and two of the sets were added with 5. Mu.M and 20. Mu.M Na, respectively 2 The S solution was incubated for an additional 30 minutes. As shown in FIG. 6, fla-DNP can detect endogenous H in cells 2 S, and green fluorescence with added exogenous H 2 The S concentration increases to enhance. The results show that the Fla-DNP has good cell membrane penetrability and can better detect H in living cells 2 S, has potential application in disease diagnosis.
3. Fluorescent probe Fla-DNP was designed as H 2 S-activated CO releaser:
to 60. Mu. Mol/L fluorescent probe Fla-DNP in PBS buffer (pH=7.4, 10% DMSO and 3mM CTAB) was added 200. Mu.M H 2 S, after the identification is completed, the light is used for irradiation, the emission spectrum is tested every 1 minute, as shown in FIG. 7, the fluorescence intensity gradually decreases along with the irradiation time, and the fluorescence intensity is almost reduced to 0 in about 10 minutes, which indicates that the CO is basically completely released. At the H 2 In the presence of S activated CO releaser Fla-DNP, 4T1 cells were cultured for 30min and irradiated with light, as shown in FIG. 8, fla-DNP can also complete the CO release process in living cells, and is expected to be developed into a small-molecule anticancer drug targeting cancer cells.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A flavonol derivative fluorescent probe with large Stokes displacement is characterized in that the structural formula I of the probe Fla-DNP is as follows:
2. the method for preparing a flavonol derivative fluorescent probe with large Stokes shift according to claim 1, which is characterized by comprising the following steps:
s1, placing 4-dimethylamino benzoflavonol, 2, 4-dinitrofluorobenzene and potassium carbonate into a round-bottom flask, adding DMF (dimethyl formamide) as a solvent, stirring for 2 hours at room temperature, pouring the mixed solution after the reaction into ice water after the reaction is finished, filtering, washing and drying to obtain a crude product after solid precipitation;
s2, performing column chromatography separation by using ethyl acetate and hexane as eluent, and performing spin-drying to obtain an orange solid, namely the compound shown in the formula I.
3. The method for preparing the flavonol derivative fluorescent probe with large Stokes shift according to claim 2, which comprises the following reaction route preparation:
。
4. the method for preparing a fluorescent probe Fla-DNP for detecting H generated by food spoilage according to the compound of the formula I as defined in claim 1 2 S application.
5. The method for preparing a fluorescent probe Fla-DNP for detecting H in living cells by using a compound of formula I as defined in claim 1 2 S application.
6. The method for preparing and detecting fluorescent probe Fla-DNP designed as H by using the compound of the formula I as shown in claim 1 2 Use of an S-activated CO releaser in living cells.
7. The method for preparing the flavonol derivative fluorescent probe with large Stokes shift according to claim 2, which is characterized in that: the molar ratio of the 4-dimethylamino benzoflavonol, the 2, 4-dinitrofluorobenzene and the potassium carbonate is 1: (1-2): (1-2).
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| CN104419401A (en) * | 2013-08-28 | 2015-03-18 | 苏州罗兰生物科技有限公司 | Fluorescent probe for detecting hydrogen sulfide by virtue of fluorescence enhancement as well as synthetic method and application of fluorescent probe |
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| CN104419401A (en) * | 2013-08-28 | 2015-03-18 | 苏州罗兰生物科技有限公司 | Fluorescent probe for detecting hydrogen sulfide by virtue of fluorescence enhancement as well as synthetic method and application of fluorescent probe |
Non-Patent Citations (2)
| Title |
|---|
| STACEY N. ANDERSON ET AL: "A Structurally-Tunable 3-Hydroxyflavone Motif for Visible Light-Induced Carbon Monoxide-Releasing Molecules (CORMs)", 《CHEMISTRYOPEN》, no. 4, 31 December 2015 (2015-12-31), pages 590 * |
| YAO LIU ET AL: "A visible light excitable colorimetric and fluorescent ESIPT probe for rapid and selective detection of hydrogen sulfide", 《ORG. BIOMOL. CHEM》, no. 12, 31 December 2014 (2014-12-31), pages 438 * |
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