CN111777603B - Fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaque and peroxynitrite and preparation method and application thereof - Google Patents
Fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaque and peroxynitrite and preparation method and application thereof Download PDFInfo
- Publication number
- CN111777603B CN111777603B CN202010542302.7A CN202010542302A CN111777603B CN 111777603 B CN111777603 B CN 111777603B CN 202010542302 A CN202010542302 A CN 202010542302A CN 111777603 B CN111777603 B CN 111777603B
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- probe
- peroxynitrite
- onoo
- beta
- 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.)
- Expired - Fee Related
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 49
- 102000013455 Amyloid beta-Peptides Human genes 0.000 title claims abstract description 22
- 108010090849 Amyloid beta-Peptides Proteins 0.000 title claims abstract description 22
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 title claims abstract 10
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 230000008021 deposition Effects 0.000 title abstract description 6
- 239000000523 sample Substances 0.000 claims abstract description 54
- 238000003384 imaging method Methods 0.000 claims abstract description 19
- 238000001514 detection method Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- 210000004556 brain Anatomy 0.000 claims description 12
- 241001465754 Metazoa Species 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 9
- 238000010226 confocal imaging Methods 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 230000000638 stimulation Effects 0.000 claims description 4
- 210000000683 abdominal cavity Anatomy 0.000 claims description 3
- 239000007928 intraperitoneal injection Substances 0.000 claims description 3
- 230000004936 stimulating effect Effects 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims 5
- 201000010099 disease Diseases 0.000 claims 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 5
- 238000006243 chemical reaction Methods 0.000 abstract description 11
- 238000010172 mouse model Methods 0.000 abstract description 7
- 230000004044 response Effects 0.000 abstract description 4
- WOTMJKVBSMZEEQ-UHFFFAOYSA-N 1,3-benzothiazole naphthalene Chemical class C1=CC=CC2=CC=CC=C12.S1C=NC2=C1C=CC=C2 WOTMJKVBSMZEEQ-UHFFFAOYSA-N 0.000 abstract description 3
- 150000003951 lactams Chemical group 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 16
- 238000000799 fluorescence microscopy Methods 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- 230000005284 excitation Effects 0.000 description 10
- CMFNMSMUKZHDEY-UHFFFAOYSA-M peroxynitrite Chemical compound [O-]ON=O CMFNMSMUKZHDEY-UHFFFAOYSA-M 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- 229940125904 compound 1 Drugs 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000004043 dyeing Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 229960000583 acetic acid Drugs 0.000 description 5
- 238000000151 deposition Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000012362 glacial acetic acid Substances 0.000 description 5
- 230000006933 amyloid-beta aggregation Effects 0.000 description 4
- DBKKFIIYQGGHJO-UHFFFAOYSA-N diethyl 2-oxopropanedioate Chemical compound CCOC(=O)C(=O)C(=O)OCC DBKKFIIYQGGHJO-UHFFFAOYSA-N 0.000 description 4
- 239000003642 reactive oxygen metabolite Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 208000037259 Amyloid Plaque Diseases 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 229940087646 methanolamine Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- VRVRGVPWCUEOGV-UHFFFAOYSA-N 2-aminothiophenol Chemical compound NC1=CC=CC=C1S VRVRGVPWCUEOGV-UHFFFAOYSA-N 0.000 description 1
- NZTPZUIIYNYZKT-UHFFFAOYSA-N 6-aminonaphthalene-2-carboxylic acid Chemical compound C1=C(C(O)=O)C=CC2=CC(N)=CC=C21 NZTPZUIIYNYZKT-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000024683 calcium ion homeostasis Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Inorganic materials O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920000137 polyphosphoric acid Polymers 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000007845 reactive nitrogen species Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000000482 two photon fluorescence microscopy Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
-
- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a preparation method and application of a fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaques and peroxynitrite, wherein the structural formula of the fluorescent probe is as follows:
the fluorescent probe is named as BTNPO, and the fluorescent probe takes a naphthalene-benzothiazole derivative as a fluorophore and takes a substituted lactam functional group as ONOO‑By reaction of recognition groups, other than with ONOO‑Besides quick response, the probe can be combined with the A beta deposited plaque, and has high sensitivity and good selectivity. Can be used as an excellent imaging tool for living cells and AD model mice to treat A beta deposited plaque and ONOO‑While simultaneously detecting.
Description
Technical Field
The invention belongs to the technical field of fluorescence imaging detection, and particularly relates to a fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaques and peroxynitrite, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Current studies indicate that plaque deposits formed by amyloid beta (a β) aggregation are one of the key factors leading to a decline in cognitive function. Neurotoxicity of a β -deposited plaques can alter mitochondrial calcium ion homeostasis, induce inflammation, and increase Reactive Oxygen Species (ROS) production. Studies have demonstrated that A β plaque deposits can stimulate nerve cells to produce excess peroxynitrite (ONOO)-) Accelerating damage to neurons. ONOO-As a ROS, irreversible damage can be caused to lipids, proteins, and DNA in the organism. Researchers have found high levels of nitrated proteins in neurons of the brain in patients with Alzheimer's Disease (AD). Evidence of Abeta plaque deposits and ONOO-Is closely related to the occurrence and development of AD. Thus, many small molecule fluorescent probes have been developed and used to target A β deposited plaques or ONOO-Detection of (3). However, the inventors found that: capable of simultaneously detecting Abeta plaque deposits and ONOO-The number of the small molecular fluorescent probes is very small.
Disclosure of Invention
In order to overcome the above problems, it is an object of the present invention to provide a method for simultaneously detecting A β deposited plaque and ONOO-The fluorescent probe and the preparation method and the application thereof. The fluorescent probe is named as BTNPO, and the probe takes naphthalene-benzothiazole derivative as a fluorophore and takes substituted lactam as ONOO-Reactive groups capable of reacting with ONOO-And the probe is capable of binding to a β deposited plaque resulting in an increase in fluorescence. The fluorescent probe can be used for depositing plaque and ONOO (oriented organic oxygen radical) on Abeta (beta-amyloid) through a double-fluorescence channel-The fluorescence detection is carried out simultaneously, has the characteristics of high sensitivity and good selectivity, and can realize simultaneous fluorescence imaging of the cell and the living body.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a use of a fluorescent probe for simultaneously detecting β amyloid plaque deposits and peroxynitrite, the fluorescent probe having a structural formula:
the probe has the characteristics of high sensitivity and good selectivity, can realize simultaneous fluorescence imaging of the two on the cellular and living levels, and can be used for researching A beta deposited plaque and ONOO-The interrelationship between them.
In a second aspect of the present invention, there is provided a use of a fluorescent probe for simultaneously imaging amyloid-beta plaque deposits and peroxynitrite at a living cell level, the fluorescent probe having a structural formula:
the probe of the invention can be used for treating A beta deposited plaque and ONOO at living cell level through double channels-And (3) fluorescence imaging is carried out, the dyeing effect is good, the dyeing time is short (30min), and the dyeing efficiency is high.
In a third aspect of the present invention, there is provided a use of a fluorescent probe for simultaneous imaging of amyloid-beta plaque deposits and peroxynitrite in a living animal, the fluorescent probe having the structural formula:
the probe can be used for treating A beta deposited plaques and ONOO in brain slices of AD model mice through two channels-Fluorescence imaging is performed.
In a fourth aspect of the invention, there is provided a method for simultaneous imaging detection of amyloid-beta plaque deposits and peroxynitrite at a viable cell level, comprising:
stimulating living cells by using an Abeta aggregate, and then adding the living cells into a solution containing a fluorescent probe for incubation;
after incubation is finished, laser confocal imaging is carried out, and the compound is obtained;
wherein, the structural formula of the fluorescent probe is as follows:
the fluorescence of the probe is very weak, and after the probe is combined with an Abeta deposited plaque, the fluorescence at the position of-418 nm is obviously enhanced; probe and ONOO-After the reaction, the fluorescence at the position of 506nm is obviously enhanced. Thereby realizing the deposition of the plaque and the ONOO of the Abeta through two channels-Simultaneous detection is performed.
In a fifth aspect of the invention, there is provided a method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite in a living animal comprising:
injecting a fluorescent probe into the abdominal cavity of the living animal, taking out the brain of the living animal, slicing, and carrying out two-photon confocal imaging to obtain the fluorescent probe;
wherein, the structural formula of the fluorescent probe is as follows:
the probe BTNPO provided by the invention is used as an excellent fluorescence imaging tool, and can realize the effects of A beta deposited plaque and ONOO in cell level and AD mouse models-While simultaneously imaging.
In a sixth aspect of the present invention, a fluorescent probe capable of simultaneously detecting β amyloid plaque deposits and peroxynitrite is provided, which has the structural formula:
the invention names the fluorescent probe BTNPO, the probe takes naphthalene-benzothiazole derivative as a fluorophore and takes substituted lactam as ONOO-Reactive groups capable of reacting with ONOO-And the probe is capable of binding to a β deposited plaque resulting in an increase in fluorescence.
In a seventh aspect of the present invention, there is provided a method for preparing a fluorescent probe capable of simultaneously detecting β amyloid plaque deposits and peroxynitrite, comprising:
dissolving the compound 1 in glacial acetic acid, adding ketomalonic acid diethyl ester, and uniformly mixing to obtain a mixed solution;
reacting the mixed solution to obtain a fluorescent probe;
wherein the structural formula of the compound 1 is
The preparation method is simple, low in cost, strong in practicability and easy to popularize.
The invention has the beneficial effects that:
(1) the fluorescence of the probe is very weak, and after the probe is combined with an Abeta deposited plaque, the fluorescence at the position of-418 nm is obviously enhanced; probe and ONOO-After the reaction, the fluorescence at the position of 506nm is obviously enhanced. Thereby realizing the deposition of the plaque and the ONOO of the Abeta through two channels-Simultaneous detection is performed.
(2) The probe can be used for treating A beta deposited plaque and ONOO (open cell amyloid) at living cell level through double channels-And (3) fluorescence imaging is carried out, the dyeing effect is good, the dyeing time is short (30min), and the dyeing efficiency is high.
(3) The probe can be used for treating A beta deposited plaque and ONOO in brain slices of AD model mice through two channels-Fluorescence imaging is performed.
(4) The method is simple, low in cost, strong in practicability and easy to popularize.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 shows the fluorescent probes of example 1 of the present invention for different concentrations of ONOO-(A) And a fluorescence response spectrum of the a β aggregate (B); wavelength (nm) on the abscissa and fluorescence emission on the ordinateStrength.
FIG. 2 shows fluorescence intensities at 418nm (A) and 506nm (B) of the fluorescent probe of the present invention in the presence of different substances in example 1 of the present invention. The abscissa is the intensity of the fluorescence emission of the probe at the corresponding wavelength, and the ordinate is the intensity of the different species.
FIG. 3 is a sequence of addition of ONOO to the fluorescent probe of example 1 of the present invention-And a β aggregate, a fluorescence intensity quantification map of the probe; the abscissa is the sample for different conditions and the ordinate is the fluorescence emission intensity of the probe at 418nm (A) or 506nm (B).
FIG. 4 is the alignment of the fluorescent probes of example 1 of the present invention at the PC12 cell level to ONOO-And fluorescence imaging of Α β deposited plaques.
FIG. 5 is a plot of the fluorescence probe of example 1 of the present invention against ONOO in brain sections from AD model mice of different ages of months ( months 2, 3, 4, 5 and 6)-And fluorescence imaging of Α β deposited plaques.
FIG. 6 is a three-dimensional fluorescence scanning image of the fluorescent probe of example 1 of the present invention for the brain of a 6-month-old AD mouse.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The invention utilizes the existing raw material intermediate compound 1 to prepare the product probe BTNPO through one-step simple and efficient synthesis and finally silica gel chromatographic column separation. Compound 1 is obtained from 6-amino-2-naphthoic acid and o-aminothiophenol in polyphosphoric acid, heated to 150 deg.C, and reacted overnight.
Can detect A beta deposits plaque and ONOO simultaneously-The structural formula of the fluorescent probe is as follows:
the preparation method of the fluorescent probe comprises the following steps:
the reaction equation is:
1) dissolving the compound 1 in glacial acetic acid, adding diethyl ketomalonate into the glacial acetic acid, and uniformly stirring to obtain a mixed solution;
2) and (3) reacting the mixed solution obtained in the step 1) for a set time and temperature to obtain the catalyst.
In some embodiments, in step 1), the ratio of the amounts of the substance of compound 1 and the ketomalonic acid diethyl ester is 1:1-3 to synthesize the target probe BTNPO.
In some embodiments, the reaction temperature in step 2) is 30-118 ℃ and the reaction time is 8-18h, so as to improve the reaction efficiency.
In some examples, the reaction temperature in step 2) is 90-118 ℃ and the reaction time is 12-15h, so that higher reaction rate and yield can be obtained.
In some embodiments, step 2) further comprises a step of spin-drying and purifying the reaction product solution to obtain a high-purity probe, so as to enhance the subsequent fluorescence imaging effect.
In some embodiments, in step 2), the reaction product is purified by silica gel column chromatography, which is simple in operation, large in sample carrying capacity and high in separation purity.
In some embodiments, the eluent for silica gel column chromatography is a mixed solution of dichloromethane, methanol and triethylamine, and the volume ratio of dichloromethane, methanol and triethylamine for reaction product purification is 120-90:1-1.5: 1; preferably 100:1:1, to improve desorption efficiency.
The fluorescent probe can be used for treating A beta deposited plaque and ONOO at living cell level-Application to imaging.
The method for imaging the PC12 cells by the fluorescent probe comprises the following steps: stimulating the cultured PC12 cells by using an Abeta aggregate, adding a dimethyl sulfoxide solution of a probe for incubation, and directly carrying out two-photon confocal imaging after the incubation is carried out for a set time.
Wherein the A beta aggregate is A beta oligomer, fibril, fiber, plaque and the like prepared by adopting A beta monomer.
The stimulation time of the a β aggregates and the probe incubation time are not particularly limited in this application, and in some embodiments, the stimulation time of the a β aggregates is 12h, and the probe incubation time is 30min, which can be adjusted by those skilled in the art according to actual situations.
The fluorescent probe is used as a detection tool for detecting Abeta deposition plaques and ONOO in Alzheimer disease model mice-The simultaneous imaging detection comprises the following steps:
AD model mice with different ages in months are selected, a probe is injected into the abdominal cavity, and then the brain of the mouse is taken out and sliced to carry out two-photon confocal imaging.
The dosage of the intraperitoneal injection probe is not particularly limited in the present application, and in some embodiments, the dosage of the intraperitoneal injection probe is (20mg kg-1), and the injection amount can be adjusted by a person skilled in the art according to the characteristics of the test animal.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Example 1: synthesis of fluorescent probes
Compound 1(274mg, 1mmol) was dissolved in 3mL of glacial acetic acid, and the mixture was heated to reflux. Then, a solution of ketomalonic acid diethyl ester (348mg, 2mmol) in glacial acetic acid (2mL) was added dropwise to the above mixed solution, followed by reaction for 15 hours. After completion of the reaction, the reaction solution was cooled to room temperature, the solvent was concentrated and dried, and the solid was subjected to column chromatography (eluent, dichloromethane: methanol: triethylamine: 100:1:1, V/V, respectively) to give the product solids as 324mg (yield 65%), respectively).
Mass spectrum and nuclear magnetism characterization:
BTNPO：HRMS(ESI):calculated forC22H15N2O4S-(M-H)-403.0747,found403.0747。
1H NMR(400MHz,DMSO-d6)δ10.92(s,1H),8.69(s,1H),8.22(t,J＝8.3Hz,2H),8.17(d,J＝8.0Hz,1H),8.09(d,J＝8.1Hz,1H),7.96(d,J＝8.8Hz,1H),7.57(t,J＝7.6Hz,1H),7.48(t,J＝7.5Hz,1H),7.33(d,J＝8.6Hz,1H),7.24(s,1H),4.18–4.10(m,1H),4.10–4.02(m,1H),1.01(t,J＝8.0Hz,3H)ppm。
13C NMR(100MHz,DMSO)δ176.22,169.54,167.74,154.11,143.66,134.94,133.08,130.57,129.69,129.23,128.54,127.15,126.11,125.94,123.29,123.21,122.80,121.02,113.46,78.83,61.97,14.38ppm。
effect experiment:
probes against Abeta aggregates and ONOO-Fluorescence response experiment of (2):
probe stock solution (1.0mM) was added to PBS (pH 7.4) buffer solution, diluted with water, and different concentrations of ONOO were added-Or Abeta aggregate, with a final concentration of 2.0. mu.M, PBS concentration of 50mM, Abeta aggregate concentration of 0-25. mu.M, ONOO-The concentration is 0-20. mu.M (0-10 eq). As shown in A in FIG. 1, when the probe is excited at 340nm, weak fluorescence exists at 418nm, and after the probe is incubated with different concentrations of A beta aggregates (the concentrations of the A beta aggregates sequentially correspond to 0 mu M, 4 mu M, 8 mu M, 12 mu M, 16 mu M, 20 mu M and 25 mu M from bottom to top), the fluorescence at 418nm is obviously enhanced; in FIG. 1, panel B, the probe had essentially no fluorescence emission at 506nm upon excitation at 380nm, with different concentrations of ONOO-(ONOO corresponding in sequence from bottom to top-At a concentration of 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), a new emission peak at 506nm appeared and fluorescence gradually increased.
Subsequently, probe pairs of Α β aggregates and ONOO were investigated-Selectivity of (2). In summary, the selection of different excitation wavelengths can achieve the targeting of A β aggregates and ONOO-Specific response, while other bioactive substances do not interfere. In FIG. 2, the reference numeral 1 denotes 20. mu. MA. beta. aggregates (A in FIG. 2) or ONOO-(B in FIG. 2), 2 represents blank control, 3 represents 20. mu.M ONOO-(A in FIG. 2) or Abeta aggregates (B in FIG. 2), 4 represents 100. mu.MH2O2And 5 represents 100. mu. MClO-And 6 represents 100. mu. M O2 ·-And 7 represents 100. mu.M·OH, 8 represents 100. mu.M1O29 for 100. mu. M t-BuOOH, 10 for 100. mu.M NO, 11 for 5mM GSH, 12 for 5mMCys, 13 for 100. mu. MHcy, 14 for 100. mu. M H2S, 15 represents 100. mu.M NO3 -And 16 represents 100. mu. MNO2 -And 17 represents 100. mu. MAcO -18 represents 100. mu.M SO4 2-And 19 represents 100. mu.M SO3 2-20 stands for 100. mu.M CO 3 2-21 represents 100. mu.M PO4 3-And 22 represents 100. mu.M Na +23 represents 100. mu. M K+And 24 represents 100. mu. MCa2+And 25 represents 100. mu.M Mg 2+26 represents 100. mu.M Zn2+And 27 represents 100. mu.M Cu2+And 28 represents 100. mu. MFe2+And 29 represents 100. mu. MFe3+. As can be seen from FIG. 2, none of the other potentially interfering substances is able to cause a change in fluorescence of the probe, including reactive oxygen species/reactive nitrogen species (H)2O2，ClO-，O2 ·-，·OH，1O2t-BuOOH, and NO), active sulfur (GSH, Cys, Hcy, and H)2S), anion (NO)3 -，NO2 -，AcO-，SO4 2-，SO3 2-，CO3 2-And PO4 3-) Cation (Na)+，K+，Ca2 +，Mg2+，Zn2+，Cu2+，Fe2+And Fe3+). In contrast, the probe was on the Abeta aggregates or ONOO-(corresponding to reference numeral 1) exhibits a significant increase in fluorescence in the presence ofIs strong. In summary, the probe BTNPO pairs with the A beta aggregate and ONOO-Has high specificity, and can be used for treating A beta aggregate and ONOO in physiological environment by selecting different excitation wavelengths-And (4) fluorescent visualization research.
Next, studies were made on A.beta.aggregates and ONOO-When present, the probe responds to the fluorescence of both. In FIG. 3, excess probe (4. mu.M) was added followed by the addition of A.beta.aggregates (20. mu.M) and ONOO-(20 μ M), setting the excitation wavelength to 340nm, and detecting the change of fluorescence at 418 nm; or sequentially adding ONOO-(20. mu.M) and A.beta.aggregates (20. mu.M), under the same conditions, the excitation wavelength was set at 380nm, and the change in fluorescence emission at 506nm was detected. As shown in FIG. 3A, fluorescence at 418nm is significantly enhanced under excitation at 340nm as long as A β aggregates are present, ONOO-Whether the detection of the A beta aggregate by the probe is influenced or not; as shown in B of FIG. 3, only in ONOO under 380nm excitation-When existing, a new emission peak appears at 506nm, and the existence of Abeta aggregates does not influence the probe pair ONOO-And (6) responding. Therefore, the selection of different excitation wavelengths can realize the aim of A beta aggregate and ONOO-And simultaneously detecting the two, and the two do not interfere with each other.
Probes at the PC12 cell level on A β plaque deposits and ONOO-Simultaneous imaging experiments of (1):
as shown in FIG. 4, we verified the probe BTNPO on the A β plaque deposits and ONOO at the cellular level-While simultaneously detecting. Stimulation of PC12 cells to produce ONOO Using different concentrations of A β aggregates (0 μ M, 5 μ M, 10 μ M, 20 μ M, 30 μ M and 40 μ M)-12h later, incubating for 30min with 10 μ M probe, and selecting different excitation wavelengths and emission and reception ranges to treat A β deposition plaque (Ex:700 nm; Em:380--(Ex:750 nm; Em:490-550nm) was subjected to two-photon fluorescence imaging. The results of the experiment are shown in FIG. 4, and PC12 intracellular ONOO is generated along with the increase of the concentration of the A beta aggregate-The concentration of the A beta-amyloid is increased, and the probe can realize the effect of the A beta-amyloid deposition plaque and the ONOO by selecting different excitation wavelengths and signal receiving ranges-The two channels of (2) are imaged simultaneously.
Probes against A β plaque deposits and ONOO in Alzheimer's disease model mice (APPswe/PS1-dE9)-Simultaneous imaging experiments of (1):
in FIG. 5, AD model mice of different months of age (2, 3, 4, 5 and 6 months) were selected and injected intraperitoneally with a probe (20mg kg)-1) Taking out the mouse brain after 2h, slicing, carrying out two-photon confocal imaging, exciting at 700nm, collecting at 380-450nm, and detecting A beta deposited plaques; excitation at 750nm, collection at 490-550nm, detection of ONOO-. The results are shown in FIG. 5, which shows the ONOO in the brains of AD mice with increasing age-The concentration gradually increases; plaques of Α β deposits began to appear in the brains of 4-month-old AD mice and gradually increased with increasing age of the month.
In FIG. 6, three-dimensional fluorescence scanning of the 6-month-old AD mouse brain sections of FIG. 5 shows that the probes detect Abeta plaque deposits and ONOO-Is about 100 μm. The upper left is ONOO-The upper right of the fluorescence scan of the a β plaque deposits, and the lower side of the fluorescence scan of the a β plaque deposits is a superimposed image of the two.
In conclusion, the probe BTNPO can be used as an excellent fluorescence imaging tool to realize the effect of A beta deposition plaque and ONOO in a cell level and AD mouse model-While simultaneously imaging.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (9)
1. The application of the fluorescent probe in simultaneously detecting the amyloid beta plaque and the peroxynitrite does not include the diagnosis and treatment of diseases, and is characterized in that the structural formula of the fluorescent probe is as follows:
2. use of a fluorescent probe for simultaneous imaging of amyloid-beta plaque deposits and peroxynitrite at the level of viable cells, said use not including diagnosis and treatment of disease, characterized in that the structural formula of said fluorescent probe is:
3. the use of claim 2, wherein the living cells are PC12 cells.
4. Use of a fluorescent probe for simultaneous imaging of amyloid-beta plaque deposits and peroxynitrite in a living animal, said use not including diagnosis and treatment of disease, characterized in that the structural formula of said fluorescent probe is:
5. a method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite at a viable cell level, said method not including diagnosis and treatment of disease, comprising:
stimulating living cells by using an Abeta aggregate, and then adding the living cells into a solution containing a fluorescent probe for incubation;
after incubation is finished, laser confocal imaging is carried out, and the compound is obtained;
wherein, the structural formula of the fluorescent probe is as follows:
6. the method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite at the viable cell level of claim 5, wherein said stimulation time is 12 hours.
7. The method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite at the viable cell level of claim 5, wherein said incubation time is 30 min.
8. A method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite in a living animal, said method not including diagnosis and treatment of disease, comprising:
injecting a fluorescent probe into the abdominal cavity of the living animal, taking out the brain of the living animal, slicing, and carrying out two-photon confocal imaging to obtain the fluorescent probe;
wherein, the structural formula of the fluorescent probe is as follows:
9. the method for simultaneous imaging and detection of amyloid-beta plaques and peroxynitrite in a living animal according to claim 8, wherein the intraperitoneal injection of the probe is at a dose of 20mg kg-1。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010542302.7A CN111777603B (en) | 2020-06-15 | 2020-06-15 | Fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaque and peroxynitrite and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010542302.7A CN111777603B (en) | 2020-06-15 | 2020-06-15 | Fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaque and peroxynitrite and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111777603A CN111777603A (en) | 2020-10-16 |
| CN111777603B true CN111777603B (en) | 2021-10-01 |
Family
ID=72756473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010542302.7A Expired - Fee Related CN111777603B (en) | 2020-06-15 | 2020-06-15 | Fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaque and peroxynitrite and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111777603B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113088281B (en) * | 2021-03-13 | 2022-07-29 | 山东师范大学 | Fluorescent probe for detecting beta-amyloid protein and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104220438A (en) * | 2012-01-30 | 2014-12-17 | 香港大学 | Diarylamine-based fluorogenic probes for detection of peroxynitrite |
| CN105622657A (en) * | 2016-03-09 | 2016-06-01 | 中国药科大学 | Curcumin derivative and preparation method and application thereof |
| CN106905235A (en) * | 2017-01-19 | 2017-06-30 | 北京师范大学 | Peroxynitrite detection probe, preparation method and applications |
-
2020
- 2020-06-15 CN CN202010542302.7A patent/CN111777603B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104220438A (en) * | 2012-01-30 | 2014-12-17 | 香港大学 | Diarylamine-based fluorogenic probes for detection of peroxynitrite |
| CN105622657A (en) * | 2016-03-09 | 2016-06-01 | 中国药科大学 | Curcumin derivative and preparation method and application thereof |
| CN106905235A (en) * | 2017-01-19 | 2017-06-30 | 北京师范大学 | Peroxynitrite detection probe, preparation method and applications |
Non-Patent Citations (2)
| Title |
|---|
| "Mitochondrial Peroxynitrite Mediation of Anthracycline-Induced Cardiotoxicity as Visualized by a Two-Photon Near-Infrared Fluorescent Probe";Xilei Xie et al.;《Analytical Chemistry》;20180910;第90卷;第11629-11635页 * |
| "基于甲酰胺和内酰胺识别基团构建过氧亚硝酸根荧光探针";刘光照;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20190915(第9期);第B014-950页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111777603A (en) | 2020-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111423423B (en) | Application of ratiometric fluorescent probe in detecting peroxynitrite anion | |
| KR101180695B1 (en) | Methods for Preparing high purity chlorophyll and Chlorin e6 from Chlorophyll extracts | |
| CN108299438B (en) | PH-responsive near-infrared fluorescent probe compound and preparation method and application thereof | |
| CN108219780B (en) | Near-infrared fluorescent probe and preparation method and application thereof | |
| CN113264904A (en) | Detection of HOCl/ClO-Fluorescent probe, preparation method and application thereof | |
| CN111777603B (en) | Fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaque and peroxynitrite and preparation method and application thereof | |
| CN116444509A (en) | DCM derivative near infrared fluorescent molecular probe for specifically detecting hypochlorous acid/hypochlorite ions and preparation method and application thereof | |
| CN114394977B (en) | Fluorescent probe for detecting hydrogen sulfide and carbon monoxide respectively and simultaneously, and preparation method and application thereof | |
| CN113603654A (en) | Difunctional fluorescent probe for detecting lipid droplets and/or protein aggregates and preparation method and application thereof | |
| CN112500386A (en) | Near-infrared HClO fluorescent probe based on pyrrazone oxime, and preparation and application thereof | |
| Wang et al. | Highly selective recognition of intramolecular parallel G-quadruplex using a chiral supramolecular probe | |
| CN112812103A (en) | Double-emission fluorescent probe for detecting beta-amyloid plaque and preparation method and application thereof | |
| CN110655510B (en) | Sulfite ratiometric fluorescent probe targeting lipid droplets and application thereof | |
| CN115677554A (en) | 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission, and preparation method and application thereof | |
| CN113651828B (en) | Near-infrared fluorescent probe for detecting chromium ions and aluminum ions as well as preparation method and application of near-infrared fluorescent probe | |
| CN112552901B (en) | Ratio type zinc ion fluorescent probe and preparation and application thereof | |
| CN113666937B (en) | Near-infrared fluorescent probe for detecting zinc ions and preparation method and application thereof | |
| CN114437053A (en) | Nano probe and application thereof in detecting superoxide anion in Golgi apparatus | |
| CN113444099B (en) | Reaction type hydrogen sulfide fluorescent probe and preparation and application thereof | |
| CN114380856B (en) | Silorhodamine derivative for detecting brain hydrogen sulfide and preparation method and application thereof | |
| CN118047719B (en) | Be used for Abeta1-42Preparation and application of quinolinium photosensitizer for protein imaging and viscosity imaging | |
| CN112521373A (en) | Multi-modal probe and preparation method and application thereof | |
| CN116769470B (en) | HTCP fluorescent probe and application thereof | |
| CN113444067B (en) | Preparation method and application of benzothiophene carboxylate | |
| CN116425737A (en) | Fluorescent molecular probe for detecting hydroxyl free radicals 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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20211001 |