CN114656456B - Ratio type near infrared fluorescent probe for monitoring dynamic change of pH value in endoplasmic reticulum of cell, preparation method and application thereof - Google Patents
Ratio type near infrared fluorescent probe for monitoring dynamic change of pH value in endoplasmic reticulum of cell, preparation method and application thereof Download PDFInfo
- Publication number
- CN114656456B CN114656456B CN202210236419.1A CN202210236419A CN114656456B CN 114656456 B CN114656456 B CN 114656456B CN 202210236419 A CN202210236419 A CN 202210236419A CN 114656456 B CN114656456 B CN 114656456B
- Authority
- CN
- China
- Prior art keywords
- endoplasmic reticulum
- reaction
- fluorescent probe
- intermediate product
- stirring
- 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.)
- Active
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 55
- 210000002472 endoplasmic reticulum Anatomy 0.000 title claims abstract description 46
- 210000004027 cell Anatomy 0.000 title claims abstract description 36
- 230000008859 change Effects 0.000 title claims abstract description 36
- 238000012544 monitoring process Methods 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 45
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 238000003756 stirring Methods 0.000 claims abstract description 23
- 239000013067 intermediate product Substances 0.000 claims abstract description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 18
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 18
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims abstract description 18
- 238000010992 reflux Methods 0.000 claims abstract description 18
- 238000010438 heat treatment Methods 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 14
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 claims abstract description 12
- FWPIDFUJEMBDLS-UHFFFAOYSA-L tin(II) chloride dihydrate Chemical compound O.O.Cl[Sn]Cl FWPIDFUJEMBDLS-UHFFFAOYSA-L 0.000 claims abstract description 12
- LDLCZOVUSADOIV-UHFFFAOYSA-N 2-bromoethanol Chemical compound OCCBr LDLCZOVUSADOIV-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000001816 cooling Methods 0.000 claims abstract description 6
- 239000005457 ice water Substances 0.000 claims abstract description 4
- 238000005303 weighing Methods 0.000 claims abstract description 4
- 239000002904 solvent Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000004440 column chromatography Methods 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 8
- 239000012065 filter cake Substances 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000012044 organic layer Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- QXAMGWKESXGGNV-UHFFFAOYSA-N 7-(diethylamino)-1-benzopyran-2-one Chemical compound C1=CC(=O)OC2=CC(N(CC)CC)=CC=C21 QXAMGWKESXGGNV-UHFFFAOYSA-N 0.000 claims 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Substances O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 230000007935 neutral effect Effects 0.000 claims 1
- 238000000967 suction filtration Methods 0.000 claims 1
- -1 7- (diethylamino) coumarin aldehyde Chemical class 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 25
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 230000005284 excitation Effects 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000004044 response Effects 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 9
- 230000003595 spectral effect Effects 0.000 description 8
- 201000007270 liver cancer Diseases 0.000 description 7
- 208000014018 liver neoplasm Diseases 0.000 description 7
- JMJKKMLLEHNELP-UHFFFAOYSA-N ER-Tracke Blue-White DPX dye Chemical compound C1=CC(N(C)C)=CC=C1C1=CN=C(C=2C=CC(=CC=2)S(=O)(=O)NCCNC(=O)C=2C(=C(F)C(F)=C(F)C=2F)F)O1 JMJKKMLLEHNELP-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000000799 fluorescence microscopy Methods 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 239000012064 sodium phosphate buffer Substances 0.000 description 6
- 239000012085 test solution Substances 0.000 description 6
- 230000007423 decrease Effects 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 229920002334 Spandex Polymers 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000004759 spandex Substances 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000001917 fluorescence detection Methods 0.000 description 3
- 238000002073 fluorescence micrograph Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- DANUORFCFTYTSZ-UHFFFAOYSA-N epinigericin Natural products O1C2(C(CC(C)(O2)C2OC(C)(CC2)C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)C)C(C)C(OC)CC1CC1CCC(C)C(C(C)C(O)=O)O1 DANUORFCFTYTSZ-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- DANUORFCFTYTSZ-BIBFWWMMSA-N nigericin Chemical compound C([C@@H]1C[C@H]([C@H]([C@]2([C@@H](C[C@](C)(O2)C2O[C@@](C)(CC2)C2[C@H](CC(O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C)O1)C)OC)[C@H]1CC[C@H](C)C([C@@H](C)C(O)=O)O1 DANUORFCFTYTSZ-BIBFWWMMSA-N 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011155 quantitative monitoring Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000004094 calcium homeostasis Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 125000000332 coumarinyl group Chemical group O1C(=O)C(=CC2=CC=CC=C12)* 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 230000006506 pH homeostasis Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- 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
-
- 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"
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- 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
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a ratio type near infrared fluorescent probe with dynamic change of pH value of an endoplasmic reticulum, a preparation method and application thereof. The method comprises the following steps: weighing 2, 3-trimethyl-5-nitroindole, dissolving in acetonitrile, sequentially adding bromoethanol and potassium iodide, and heating and refluxing; intermediate A is obtained after the reaction; refluxing the intermediate product A, stannous chloride dihydrate and hydrochloric acid solution, stirring and heating, cooling to room temperature, and adjusting the pH to 7 with NaOH solution under ice water bath to obtain an intermediate product B; dissolving an intermediate product B and p-toluenesulfonyl chloride in dichloromethane, dropwise adding triethylamine, and stirring at room temperature for reaction to obtain an intermediate product C; dissolving 7- (diethylamino) coumarin aldehyde and an intermediate C in acetonitrile, and carrying out heating reflux stirring reaction to obtain the ratio type near infrared fluorescent probe for monitoring dynamic change of pH value of an endoplasmic reticulum. The invention develops a novel ratio type near infrared fluorescent probe which is successfully applied to monitoring the micro dynamic change of the pH value in the endoplasmic reticulum of a cell.
Description
Technical Field
The invention relates to the technical field of fluorescence detection, in particular to a ratio type near infrared fluorescent probe for highly sensitively monitoring dynamic change of pH value in a cell endoplasmic reticulum, and a preparation method and application thereof.
Background
The intracellular pH value is involved in various biological processes such as endocytosis, cell proliferation and ion transport, and the function of most proteins can be exerted only in a certain pH range. Minor deviations (0.1 to 0.2 units) of the organelle pH from normal pH may cause cellular dysfunction, ultimately leading to a variety of diseases. Thus, pH homeostasis plays an important role in the biological processes of living cells and in the functioning of cells. The endoplasmic reticulum, the central organelle of the cell secretory pathway, is an important site for protein and lipid synthesis, calcium homeostasis and detoxification of toxic substances (ph=7.2±0.2). The pH of the endoplasmic reticulum is an important parameter that is critical to regulating physiological functions of the endoplasmic reticulum, including the classification and targeting of proteins during secretion. Under normal physiological conditions, the pH of the endoplasmic reticulum is generally considered to be the same as that of the cytoplasm. As the biosynthetic products approach the end of their secretory pathway, the endoplasmic reticulum becomes progressively acidic. In the secretory pathway, any change in pH of the endoplasmic reticulum in the normal range disrupts the activity of the enzyme, the classification and processing of secreted proteins, and the storage of calcium. In recent years, many studies have shown that endoplasmic reticulum stress can activate autophagy, which is closely related to many diseases (neurodegenerative, infectious, cancer, etc.), resulting in a decrease in pH of the endoplasmic reticulum. Therefore, the highly sensitive quantitative monitoring of the minute dynamic changes in pH in the endoplasmic reticulum is of great importance for the deep disclosure of its biological role in physiological events.
Fluorescence imaging has the advantages of high sensitivity, noninvasive detection, in-situ detection and the like, and is a method capable of monitoring the dynamic change of the pH value at the cellular level. In recent years, the small molecular fluorescent probe is widely focused by people because of the characteristics of easy chemical structure modification, convenient emission color adjustment and the like. Currently, only a few small molecule fluorescent probes are capable of detecting changes in endoplasmic reticulum pH, but these probes have only one response site to pH and provide signal output based on one or two sensing mechanisms. The pH of the endoplasmic reticulum is generally not lower than that of lysosomes (4.7-6.5), and the fluctuation of pH change in the endoplasmic reticulum is small, however, the sensitivity of these probes to pH is limited to the range of 5.0-7.2, and is generally not suitable for quantitative measurement of micro dynamic change of pH in the endoplasmic reticulum. In contrast, the fluorescent probe adopting the open-loop and closed-loop double modes and regulating and controlling the fluorescence characteristic through the ICT mechanism is easier to enhance the response signal of the emission wavelength, and has higher sensitivity to monitoring the dynamic change of the pH value.
Here, we reasonably construct a compound LM-1 as a novel near infrared fluorescent probe for highly sensitively monitoring the dynamic change ratio of pH value in the endoplasmic reticulum of cells. Fluorescence imaging shows that the probe can not only target the endoplasmic reticulum, but also can carry out highly sensitive monitoring on tiny pH value changes in the endoplasmic reticulum.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a ratio type near infrared fluorescent probe for sensitively monitoring the dynamic change of the pH value in the endoplasmic reticulum of a cell, and a preparation method and application thereof. The method has the advantages of good selectivity, high sensitivity and simple preparation method, and can be well applied to high-sensitivity monitoring of the micro dynamic change of the pH value in the endoplasmic reticulum.
The invention is realized by the following technical scheme:
a ratio type near infrared fluorescent probe for highly sensitively monitoring dynamic change of pH value in a cell endoplasmic reticulum has the following structure:
the preparation method of the ratio type near infrared fluorescent probe for highly sensitively monitoring the dynamic change of the pH value in the endoplasmic reticulum of the cell comprises the following steps:
step 1, weighing 2, 3-trimethyl-5-nitroindole, dissolving in acetonitrile, adding bromoethanol, stirring uniformly, adding potassium iodide, heating, refluxing and stirring the mixed solution for reaction; after the reaction is finished, cooling to room temperature, removing redundant solvent, filtering the precipitate to obtain a filter cake, and washing the filter cake with acetonitrile to obtain an intermediate product A.
Step 2, the intermediate product a obtained in step 1, stannous chloride dihydrate and 38% hydrochloric acid (volume percentage concentration) were added to a 50mL round bottom flask equipped with a reflux condenser, and the mixture was stirred and heated under reflux. After cooling to room temperature, adjusting pH to neutrality with sodium hydroxide solution in ice water bath, extracting with distilled water and ethyl acetate, separating, recovering organic layer, removing excessive solvent, and purifying by column chromatography to obtain intermediate B.
And 3, dissolving the intermediate product B and the p-toluenesulfonyl chloride obtained in the step 2 in dichloromethane, uniformly stirring, dropwise adding triethylamine, stirring the mixture at room temperature for reaction, removing redundant solvent after the reaction is finished, and purifying by column chromatography to obtain an intermediate product C.
Step 4, adding 7- (diethylamino) coumarin aldehyde and the intermediate product C obtained in the step 3 into acetonitrile, heating, refluxing and stirring the mixture for reaction, and removing redundant solvent after the mixture is cooled to room temperature; the ratio type near infrared fluorescent probe for highly sensitively monitoring the dynamic change of the pH value in the endoplasmic reticulum of the cell can be obtained by purifying through a column chromatography method.
In the step 1, the dosage ratio of the 2, 3-trimethyl-5-nitroindole, acetonitrile, bromoethanol and potassium iodide is 0.5 g-1 g:6 mL-8 mL:192 mu L-200 mu L:0.2 g-0.25 g.
In the step 1, the temperature of the heating reflux stirring reaction is 95-100 ℃ and the time is 23-24 h.
In the step 2, the dosage ratio of the intermediate product A, stannous chloride dihydrate and 38% hydrochloric acid is 0.2 g-0.3 g:0.48 g-0.5 g:6 mL-8 mL.
In the step 2, the temperature of the stirring and heating reaction in the reflux state is 95-100 ℃ and the time is 2-3 h.
In the step 3, the dosage ratio of the intermediate product B, the p-toluenesulfonyl chloride, the methylene dichloride and the triethylamine is 0.3 g-0.5 g:0.26 g-0.4 g:5 mL-6 mL:2 mu L to 3 mu L.
In the step 3, the reaction time at room temperature is 1-2 h, and the solvent removal method is rotary evaporation.
In the step 4, the dosage ratio of the 7- (diethylamino) coumarin aldehyde, the intermediate C and the acetonitrile is 0.5 g-1 g:0.76 g-1 g:5 mL-6 mL.
In the step 4, the temperature of the heating reflux stirring reaction is 95-100 ℃ and the time is 12-13 h.
The ratio type near infrared fluorescent probe prepared by the invention is used for highly sensitively monitoring the dynamic change of the pH value in the endoplasmic reticulum of a cell, and the specific using method is as follows:
(1) The fluorescent probe LM-1 provided by the invention is used for monitoring the dynamic change of the pH value through an open loop mode and a closed loop mode, and the specific method comprises the following steps:
10. Mu.M fluorescent probe test solutions were prepared with 20mM sodium phosphate buffer/DMSO (19:1 v/v, pH=2.02-8.36), followed by determination of the fluorescence emission spectra of probe LM-1 at different pH. It was found by fluorescence spectroscopy that the fluorescence intensity at 408nm gradually decreased as the pH value was lowered from 8.36 to 2.02, and at the same time, new fluorescence centered at 652nm was generated in the near infrared channel and the fluorescence intensity gradually increased. The results show that the nucleophilicity of the hydroxyl groups decreases with decreasing pH. Thus, under acidic conditions, probe LM-1 is converted from the ring-closed to the ring-open form, and the coumarin moiety and indole moiety form a conjugate.
(2) The fluorescent probe LM-1 provided by the invention has reversibility when being used for highly sensitively monitoring the pH value, and the specific method comprises the following steps:
at room temperature, 20mM sodium phosphate buffer/DMSO (19:1 v/v) at pH 7.4 was prepared, the pH was adjusted back and forth between 6.13 and 8.11 with concentrated hydrochloric acid and aqueous sodium hydroxide (10M), and the fluorescence emission spectrum of probe LM-1 was measured for the above pH change.
(3) The fluorescent probe LM-1 provided by the invention can be used for monitoring the micro dynamic change of the pH value in real time and sensitively, and the specific method comprises the following steps:
at room temperature, 20mM sodium phosphate buffer/DMSO (19:1 v/v) was prepared at pH 7.45,7.33,7.21, respectively, for use. The fluorescence intensity of the fluorescent probe LM-1 was monitored at a fluorescence emission wavelength of 652nm with an excitation wavelength of 408nm and a time interval of 100 s. Along with the trace reduction of the pH value by 0.1-0.2 unit, the fluorescence intensity of the fluorescent probe test solution at 652nm is obviously enhanced and is quickly stabilized
(4) The fluorescent probe LM-1 provided by the invention can be used for targeting and positioning an endoplasmic reticulum, and the specific method comprises the following steps:
co-localization experiments: liver cancer cells (SMMC-7721) were placed in 35mm glass bottom dishes and stained with 2. Mu.M probe LM-1 for 25 min. Then, 0.5 mu M ER tracker Blue-White DPX dye was used 18 Dyeing for 25 minutes. Fluorescent images were obtained on a lycra TCS SP5 II confocal scanning microscope using an objective lens. The near infrared channel of probe LM-1 is involved in the emission of the 650-700nm spectral window under 543nm excitation. ER tracker Blue-White DPX dye was recorded on an emission window of 440-490nm under 405nm excitation 18 Is a blue channel of (c).
(5) The fluorescent probe LM-1 provided by the invention can be used for highly sensitively monitoring the micro dynamic change of the pH value in the endoplasmic reticulum, and the specific method comprises the following steps:
cell culture: hepatoma cells (SMMC-7721) were incubated in 35mm glass bottom dishes in DMEM containing 10% fetal bovine serum in an incubator containing 5% carbon dioxide at 37 ℃.
Cell staining: liver cancer cells (SMMC-7721) were placed in 35mm glass bottom dishes and stained with 2. Mu.M LM-1 for 25 minutes. Then, nigericin (5 mg mL) was used -1 ) The cells were treated for 20min. The cells were washed three times with PBS at pH 7.52,7.36 and 7.18, respectively. Subsequently, fluorescence images were obtained on a lycra TCS SP5 II confocal scanning microscope with an objective lens. The near infrared channel of probe LM-1 is involved in the emission of the 650-700nm spectral window under 543nm excitation. The green channel of probe LM-1 was recorded at an emission window of 500-530nm under 405nm excitation.
The beneficial effects of the invention are as follows:
(1) The invention provides a brand new ratio type near infrared fluorescent probe for high sensitivity monitoring of micro dynamic change of pH value, which has simple synthesis method, good selectivity to pH value and Na + 、K + 、Ca 2+ 、Mg 2+ 、NH 4 + 、SO 4 2- 、Cl - The detection is not affected by related interferents such as biomolecules (GSH, cys, hcy); the fluorescence emission peak (652 nm) of the probe is in the near infrared region, contributing to eliminationThe interference of background fluorescence is removed, the detection error is reduced, and the accuracy of analysis and detection is improved; fluorescence intensity of probe solution (I 485 ，I 652 ) Has good linear relation with pH value change within the range of 2.02-8.36, and has extremely high sensitivity to the pH value fluctuating in 0.1-0.2 units;
(2) After the synthesis method is improved, the side reaction can be effectively prevented, and impurities which are difficult to separate in the reaction process are greatly reduced, so that a target product with high purity can be obtained.
(3) The invention develops a novel high-sensitivity ratio type near infrared fluorescent probe for monitoring the dynamic change of the pH value in the endoplasmic reticulum of a cell, and a laser confocal scanning microscope is firstly used for fluorescent imaging to monitor the micro change of the pH value in the endoplasmic reticulum with high sensitivity. It is worth noting that the highly sensitive quantitative monitoring of the minute dynamic changes of pH values in the endoplasmic reticulum is of great importance for the deep disclosure of its biological role in physiological events.
Drawings
FIG. 1 is a synthetic route diagram of a ratio-type near infrared fluorescent probe for monitoring pH dynamic change in endoplasmic reticulum in example 1 of the present invention;
FIG. 2 a is a graph showing fluorescence titration of the fluorescent probe according to example 1 of the present invention for detecting different pH values;
FIG. 2 b shows the fluorescence intensities (I) obtained by detecting the spectral data of different pH values with the fluorescent probe of example 1 652 ) A linear relation graph of pH value change, wherein the abscissa is pH value, and the ordinate is fluorescence intensity;
FIG. 2 c shows the fluorescence intensity (I) constructed by detecting the spectral data of different pH values with the fluorescent probe according to example 1 of the present invention 485 ) A linear relation graph of pH value change, wherein the abscissa is pH value, and the ordinate is fluorescence intensity;
d in FIG. 2 is a kinetic study of the fluorescent probe of example 1 of the present invention, in which the abscissa indicates the reaction time and the ordinate indicates the fluorescence intensity.
FIG. 3 is a graph showing the reversibility of the fluorescent probe according to example 1 of the present invention, wherein the abscissa represents the pH value and the ordinate represents the fluorescence intensity.
FIG. 4 is a selective histogram of a fluorescent probe according to example 1 of the present invention; the abscissa is the addition condition of different ions or molecules, and the ordinate is the ratio value of fluorescence intensity;
FIG. 5 shows the fluorescent probes LM-1 and ER tracker Blue-White DPX dye of example 1 of the present invention 18 Fluorescence imaging of co-localization in hepatoma cells (SMMC-7721).
FIG. 6 is a fluorescence imaging chart of the fluorescent probe LM-1 stained liver cancer cell (SMMC-7721) of example 1 of the present invention, recorded at different pH values (pH= 7.18,7.36,7.52).
Detailed Description
The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
Example 1:
(1) Weighing 0.5g of 2, 3-trimethyl-5-nitroindole, dissolving in 6mL of acetonitrile, adding 192 mu L of bromoethanol, uniformly stirring, adding 0.2g of potassium iodide, heating, refluxing and stirring the mixed solution at 95 ℃ for reaction for 23 hours; after the reaction was completed, the reaction mixture was cooled to room temperature, excess solvent was removed, and the precipitate was suction-filtered to obtain a cake and washed with acetonitrile to obtain 0.32g of an intermediate product A as a brown solid in a yield of 52.6%.
(2) 0.2g of intermediate A from step 1, 0.48g of stannous chloride dihydrate were added to a 25mL round bottom flask containing 6mL of 38% hydrochloric acid and the mixture was heated and stirred at 95℃for 3h. After cooling to room temperature, pH was adjusted to 7 with sodium hydroxide solution in ice water bath, extraction and separation were performed with distilled water and ethyl acetate, the organic layer was recovered, excess solvent was removed, and the residue was purified by column chromatography (dichloromethane: methanol=100:1, v/v) to give 87.7mg of intermediate B as a yellow solid in 49.6% yield.
(3) 0.3g of intermediate B obtained in the step 2 and 0.26g of p-toluenesulfonyl chloride were weighed and dissolved in 5mL of methylene chloride, and stirred well, 2. Mu.L of triethylamine was added dropwise, and then the mixture was stirred at room temperature for 2 hours. After the reaction, the excess solvent was removed, and the residue was purified by column chromatography (dichloromethane: methanol=10:1, v/v) to give 238.8mg of intermediate C as a pale yellow solid in 46.7% yield.
(4) 0.5g of 7- (diethylamino) coumarin aldehyde and 0.76g of intermediate C obtained in step 3 were added to 6mL of acetonitrile, and the mixture was heated at 95℃under reflux with stirring for reaction for 12 hours. After the mixture cooled to room temperature, removing excess solvent; purification by column chromatography (dichloromethane: petroleum ether: methanol=60:10:1, v/v/v) gave 307.4mg of the ratio-type near infrared fluorescent probe for monitoring dynamic changes in pH in the endoplasmic reticulum with a yield of 25.1%.
Example 2:
the high sensitivity monitoring of the fluorescence spectral response of pH dynamically varying fluorescent probe LM-1 to different pH values in the endoplasmic reticulum of a cell obtained in example 1.
The spectral properties were tested in 20mM sodium phosphate buffer/DMSO (19:1 v/v) at a pH in the range of 2.02-8.36. As the pH was lowered from 8.36 to 2.02, the fluorescence intensity at 408nm was gradually decreased (a in FIG. 2), and at the same time, new fluorescence centered at 652nm was generated in the near infrared channel and the fluorescence intensity was gradually increased (a in FIG. 2). In addition, probe LM-1 exhibited good linear response (b in FIG. 2, c in FIG. 2) at pH values in the range of 2.02 to 8.36. The fluorescent probe obtained in example 1 thus has excellent fluorescence response to changes in pH.
Example 3:
kinetic study of the fluorescent probe LM-1 for monitoring dynamic pH change in the endoplasmic reticulum of a cell with high sensitivity, which is obtained in example 1.
The kinetics of the fluorescent probe for monitoring different pH values is to monitor the fluorescence intensity of the fluorescent emission wavelength of 652nm by using 408nm as an excitation wavelength. As shown by d in fig. 2, at different pH values (7.45,7.33,7.21), every 100s, with the slight decrease of the pH value by 0.1-0.2 units, the fluorescence intensity of the fluorescent probe test solution at 652nm is obviously enhanced and quickly reaches a stable state, which indicates that the probe can quickly respond to the slight change of the pH value, thereby being capable of monitoring the dynamic change of the pH value in real time and sensitively.
Example 4:
reversibility of pH fluorescence detection by fluorescent probe LM-1 obtained in example 1.
10. Mu.M of fluorescent probe test solution was prepared with 20mM sodium phosphate buffer/DMSO (19:1 v/v, pH=7.4) for use. The fluorescence emission spectrum of probe LM-1 was measured at room temperature by adjusting the pH between 6.13 and 8.11 with concentrated hydrochloric acid and aqueous sodium hydroxide (10M) and then exciting at 408nm, and the measurement result is shown in FIG. 3.
From the results of FIG. 3, it can be seen that the probe LM-1 has a reversible fluorescence response to a change in pH when the pH of the system is switched between 6.13 and 8.11, indicating that: the fluorescent probe LM-1 of the invention can be used for monitoring the fluctuation of the pH value.
Example 5:
selective investigation of the fluorescent probe LM-1 obtained in example 1 for pH fluorescence detection.
10. Mu.M of fluorescent probe test solution was prepared with 20mM sodium phosphate buffer/DMSO (19:1 v/v, pH=7.31) for use. First, adding other possible interferents to 10 μm fluorescent probe molecule test solution, including Na + 、K + 、Ca 2+ 、Mg 2+ 、NH 4 + 、SO 4 2- 、Cl - And biomolecules (GSH, cys, hcy). After mixing for 2 minutes, fluorescence spectrum test was performed under the same conditions with excitation at 408nm to obtain fluorescence spectra of the respective sets of solutions, and the measurement results are shown in fig. 4.
As can be seen from the results of FIG. 4, when Na is added to the system + 、K + 、Ca 2+ 、Mg 2+ 、NH 4 + 、SO 4 2- 、Cl - After the possible interferents such as biological molecules (GSH, cys, hcy), the fluorescence intensity of each group was not significantly different from that of the blank solution without the interferents. The results show that: the fluorescent probe has high selectivity for pH value fluorescent detection, is not interfered by other coexisting ions or molecules, and is suitable for accurate monitoring of different pH values.
Example 6:
practical application of the fluorescent probe LM-1 obtained in example 1.
(1) Co-localized fluorescence imaging of fluorescent probe LM-1 obtained in example 1.
Liver cancer cells (SMMC-7721) were placed in 35mm glass bottom dishes and stained with 2. Mu.M probe LM-1 for 25 min. Then, 0.5 mu M ER tracker Blue-White DPX dye was used 18 Dyeing for 25 minutes. Fluorescent images were obtained on a lycra TCS SP5 II confocal scanning microscope using an objective lens. The near infrared channel of probe LM-1 is involved in the emission of the 650-700nm spectral window under 543nm excitation. The blue channel of ER tracker blue-White DPX dye18 was recorded on an emission window of 440-490nm under 405nm excitation.
FIG. 5 shows the probe LM-1 and ER tracker Blue-White DPX dye 18 Confocal fluorescence image of co-stained hepatoma cells (SMMC-7721). (a) is an image of a blue channel (b) is an image of a red channel, (c) is an image obtained by superimposing (a) and (b), and (d) is a ROIs intensity distribution indicated by a line segment in (c) in a co-stained liver cancer cell (SMMC-7721). The results indicate that probe LM-1 can be located in the endoplasmic reticulum of the cell.
(2) Confocal fluorescence imaging of fluorescent probes obtained in example 1 stained liver cancer cells (SMMC-7721) at different pH values (ph= 7.52,7.36,7.18) intervals.
Cell culture: hepatoma cells (SMMC-7721) were incubated in 35mm glass bottom dishes in DMEM containing 10% fetal bovine serum in an incubator containing 5% carbon dioxide at 37 ℃.
Cell staining: liver cancer cells (SMMC-7721) were placed in 35mm glass bottom dishes and stained with 2. Mu.M LM-1 for 25 minutes. Then, nigericin (5 mg mL) was used -1 ) The cells were treated for 20min. The cells were washed three times with PBS at pH 7.52,7.36 and 7.18, respectively. Subsequently, fluorescence images were obtained on a lycra TCS SP5 II confocal scanning microscope with an objective lens. The near infrared channel of probe LM-1 is involved in the emission of the 650-700nm spectral window under 543nm excitation. The green channel of probe LM-1 was recorded at an emission window of 500-530nm under 405nm excitation.
As can be seen from m in fig. 6 and n in fig. 6, with a slight decrease in pH (about 0.1 to 0.2 pH units), fluorescence in the near infrared channel increases significantly, while fluorescence in the green channel decreases. These results indicate that LM-1 shows a highly sensitive fluorescent response to small pH changes in the endoplasmic reticulum.
In summary, we have reasonably developed a novel fluorescent probe LM-1 for highly sensitively monitoring the micro dynamic changes of pH value in the endoplasmic reticulum of cells. The probe has good reversibility and selectivity, and can monitor the tiny fluctuation of the pH value and generate specific fluorescence response. The probe LM-1 has near infrared fluorescence, has ratio response to different pH values, effectively reduces background fluorescence and experimental errors, and has deeper penetrating capacity to tissues.
Fluorescence imaging experiments show that the probe LM-1 not only can target and locate the endoplasmic reticulum, but also has a pKa value which tends to the normal pH value of the endoplasmic reticulum. When the pH value is slightly reduced by about 0.1 to 0.2 pH units, the fluorescence in the near infrared channel is significantly enhanced, while the fluorescence in the green channel is reduced. Therefore, the novel ratio type near infrared fluorescent probe LM-1 designed by us can be used as a multipurpose tool for accurately and highly sensitively monitoring the micro dynamic change of the pH value in the endoplasmic reticulum of the cell.
Claims (9)
1. A ratio-type near infrared fluorescent probe for monitoring dynamic change of pH value in an endoplasmic reticulum, which is characterized by comprising the following structure:
2. a method for preparing a ratio near infrared fluorescent probe for monitoring dynamic changes of pH in the endoplasmic reticulum of a cell according to claim 1, comprising the steps of:
step 1, weighing 2, 3-trimethyl-5-nitroindole, dissolving in acetonitrile, then adding 2-bromoethanol, uniformly stirring, then adding potassium iodide, heating, refluxing and stirring the mixed solution for reaction; cooling to room temperature after the reaction is finished, removing redundant solvent, carrying out suction filtration on the precipitate to obtain a filter cake, and washing the filter cake with acetonitrile to obtain an intermediate product A;
step 2, stirring and heating the mixture of the intermediate product A, stannous chloride dihydrate and hydrochloric acid solution obtained in the step 1 in a reflux state, cooling to room temperature, regulating the pH to be neutral by using a sodium hydroxide solution under ice water bath, extracting and separating liquid by using distilled water and ethyl acetate, recovering an organic layer, removing redundant solvent, and purifying by column chromatography to obtain an intermediate product B;
step 3, dissolving the intermediate product B and the p-toluenesulfonyl chloride obtained in the step 2 in dichloromethane, uniformly stirring, dropwise adding triethylamine, stirring the mixture at room temperature for reaction, removing redundant solvent after the reaction is finished, and purifying by column chromatography to obtain an intermediate product C;
step 4, adding 7- (diethylamino) coumarin-3-formaldehyde and the intermediate product C obtained in the step 3 into acetonitrile, heating, refluxing and stirring the mixture for reaction, and removing redundant solvent after the mixture is cooled to room temperature; the ratio type near infrared fluorescent probe for monitoring the dynamic change of the pH value in the endoplasmic reticulum of the cell can be obtained after purification by a column chromatography method.
3. The preparation method according to claim 2, wherein in the step 1, the dosage ratio of the 2, 3-trimethyl-5-nitroindole, acetonitrile, 2-bromoethanol and potassium iodide is 0.5g to 1g:6 mL-8 mL:192 mu L-200 mu L:0.2 g-0.25 g.
4. The preparation method according to claim 2, wherein in the step 1, the temperature of the heating reflux stirring reaction is 95-100 ℃ and the time is 23-24 h.
5. The preparation method according to claim 2, wherein in the step 2, the ratio of the intermediate product a, stannous chloride dihydrate to hydrochloric acid solution is 0.2g to 0.3g:0.48 g-0.5 g:6 mL-8 mL; the volume percentage concentration of the hydrochloric acid solution is 38%.
6. The preparation method according to claim 2, wherein in step 2, the temperature of stirring and heating in the reflux state is 95 to 100 ℃ for 2 to 3 hours.
7. The preparation method according to claim 2, wherein in the step 3, the dosage ratio of the intermediate product B, the p-toluenesulfonyl chloride, the methylene dichloride and the triethylamine is 0.3g to 0.5g:0.26 g-0.4 g:5 mL-6 mL:2 mu L to 3 mu L.
8. The preparation method according to claim 2, wherein in the step 3, the room temperature reaction time is 1-2 hours, and the solvent removal method is rotary evaporation.
9. The preparation method according to claim 2, wherein in the step 4, the dosage ratio of the 7- (diethylamino) coumarin-3-formaldehyde, the intermediate C and the acetonitrile is 0.5 g-1 g:0.76 g-1 g:5 mL-6 mL; the temperature of the heating reflux stirring reaction is 95-100 ℃ and the time is 12-13 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210236419.1A CN114656456B (en) | 2022-03-10 | 2022-03-10 | Ratio type near infrared fluorescent probe for monitoring dynamic change of pH value in endoplasmic reticulum of cell, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210236419.1A CN114656456B (en) | 2022-03-10 | 2022-03-10 | Ratio type near infrared fluorescent probe for monitoring dynamic change of pH value in endoplasmic reticulum of cell, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114656456A CN114656456A (en) | 2022-06-24 |
| CN114656456B true CN114656456B (en) | 2024-03-19 |
Family
ID=82029215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210236419.1A Active CN114656456B (en) | 2022-03-10 | 2022-03-10 | Ratio type near infrared fluorescent probe for monitoring dynamic change of pH value in endoplasmic reticulum of cell, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114656456B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113683604A (en) * | 2021-08-09 | 2021-11-23 | 江苏大学 | Ratio type near-infrared fluorescent probe for detecting sulfur dioxide derivatives in crop mitochondria and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7625758B2 (en) * | 2005-01-26 | 2009-12-01 | Berkelman Thomas R | Coumarin-based cyanine dyes for non-specific protein binding |
-
2022
- 2022-03-10 CN CN202210236419.1A patent/CN114656456B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113683604A (en) * | 2021-08-09 | 2021-11-23 | 江苏大学 | Ratio type near-infrared fluorescent probe for detecting sulfur dioxide derivatives in crop mitochondria and preparation method and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| A colorimetric and ratiometric fluorescent pH probe based on ring opening/closing approach and its applications in monitoring cellular pH change;Qi-Hua You等;RSC Adv.;第5卷;第4099-4102页 * |
| Construction of highly fluorescent N-O seven-membered heterocyclesvia thermo-oxidation of oxazolidines;Qiaonan Chen等;Journal of Materials Chemistry C;第7卷;第8045-8052页 * |
| Highlighting Cancer Cells with Halochromic Switches;Sicheng Tang等;ACS Sens.;第2卷;第92-101页 * |
| Substituent Effects on the Photochromism of Bichromophoric Oxazines;Erhan Deniz等;J. Phys. Chem. C;第113卷(第19期);第8491-8497页 * |
| 新型pH荧光探针的合成及其性能和生物学应用的研究;宋文辉;济南大学硕士学位论文;第35-37页 * |
| 生物活性小分子响应的光学成像探针研究;杨明旺;大连理工大学博士学位论文;第32-60页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114656456A (en) | 2022-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | A two-photon ESIPT based fluorescence probe for specific detection of hypochlorite | |
| CN110143966B (en) | Spiropyran-naphthalimide derivative and synthesis method and application thereof | |
| CN113603654B (en) | Difunctional fluorescent probe for detecting lipid droplets and/or protein aggregates as well as preparation method and application thereof | |
| CN108383823B (en) | Naphthopyran carbazone derivatives, and preparation method and application thereof | |
| CN103382189B (en) | One class cyanine compound, its preparation method and application | |
| CN113292582A (en) | Synthesis and application of bifunctional fluorescent probe capable of distinguishing hydroxyl free radicals and hydrogen peroxide simultaneously | |
| CN114437013A (en) | Coumarin-benzopyrylium salt derivative MI-BP-CC and synthetic method and application thereof | |
| CN107286173A (en) | Rhodol analog derivatives and its preparation method and application | |
| CN112939963B (en) | Benzopyranoate derivative and synthesis method and application thereof | |
| CN114163463A (en) | Near-infrared fluorescent two-photon fluorescent probe design aiming at real-time change of hydrogen peroxide in tumor process and synthetic method thereof | |
| CN111040465B (en) | Near-infrared fluorescent probe for bimodal detection of sulfur dioxide and preparation method and application thereof | |
| CN110105376B (en) | Fluorescein derivative and synthetic method and application thereof | |
| CN106749142B (en) | A kind of SO32-/HSO3-Detection reagent and its synthetic method and application | |
| CN114656456B (en) | Ratio type near infrared fluorescent probe for monitoring dynamic change of pH value in endoplasmic reticulum of cell, preparation method and application thereof | |
| CN110357896B (en) | Compound, preparation and application thereof in detecting divalent copper ions and strong acid pH | |
| CN117164575A (en) | Single excitation detection ONOO - Near infrared ratio fluorescent probe of (2), preparation method and application thereof | |
| CN113999159B (en) | Viscosity-sensitive fluorescent probe, and preparation method and application thereof | |
| CN112457249B (en) | Novel cell membrane specific formaldehyde fluorescent probe, preparation method and application | |
| CN115141145A (en) | Fluorescence probe for detecting lysosome hypobromous acid, preparation method and application | |
| CN112920157B (en) | Benzene coupling benzopyran derivative and synthesis method and application thereof | |
| CN111087362B (en) | Fluorescent probe for detecting formaldehyde with high selectivity, and synthetic method and application thereof | |
| CN111574506B (en) | Preparation method of ratio-type acidic pH fluorescent probe | |
| CN109134483B (en) | Hydrogen sulfide fluorescent probe and preparation method and application thereof | |
| CN113200988A (en) | Synthesis and application of bifunctional fluorescent probe for simultaneously detecting hydroxyl free radicals and viscosity | |
| CN113234111A (en) | Fluorescent probe for simultaneously detecting beta-galactosidase and lysosome pH, and 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 |