CN109053710A - 近红外、高信噪比的Aβ荧光成像剂及其制备方法和应用 - Google Patents
近红外、高信噪比的Aβ荧光成像剂及其制备方法和应用 Download PDFInfo
- Publication number
- CN109053710A CN109053710A CN201811069366.9A CN201811069366A CN109053710A CN 109053710 A CN109053710 A CN 109053710A CN 201811069366 A CN201811069366 A CN 201811069366A CN 109053710 A CN109053710 A CN 109053710A
- Authority
- CN
- China
- Prior art keywords
- formula
- ratio
- imaging agent
- fluorescence imaging
- infrared
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000799 fluorescence microscopy Methods 0.000 title claims abstract description 19
- 239000012216 imaging agent Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000003384 imaging method Methods 0.000 claims abstract description 13
- 230000002776 aggregation Effects 0.000 claims abstract description 12
- 238000004220 aggregation Methods 0.000 claims abstract description 12
- 210000005013 brain tissue Anatomy 0.000 claims abstract description 11
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 15
- 150000001875 compounds Chemical class 0.000 claims description 15
- JLTDJTHDQAWBAV-UHFFFAOYSA-N N,N-dimethylaniline Chemical compound CN(C)C1=CC=CC=C1 JLTDJTHDQAWBAV-UHFFFAOYSA-N 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical class CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 claims description 8
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 7
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- -1 hydrocarbyl sulfonate Chemical compound 0.000 claims description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 3
- 229940043376 ammonium acetate Drugs 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- CMEWLCATCRTSGF-UHFFFAOYSA-N N,N-dimethyl-4-nitrosoaniline Chemical compound CN(C)C1=CC=C(N=O)C=C1 CMEWLCATCRTSGF-UHFFFAOYSA-N 0.000 claims description 2
- 239000007850 fluorescent dye Substances 0.000 claims description 2
- 238000010992 reflux Methods 0.000 claims description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 claims 6
- 238000004440 column chromatography Methods 0.000 claims 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 1
- 239000003960 organic solvent Substances 0.000 claims 1
- 238000006467 substitution reaction Methods 0.000 claims 1
- 239000000975 dye Substances 0.000 abstract description 56
- 238000010175 APPswe/PSEN1dE9 Methods 0.000 abstract description 13
- 210000004556 brain Anatomy 0.000 abstract description 8
- 238000011830 transgenic mouse model Methods 0.000 abstract description 8
- 241000699660 Mus musculus Species 0.000 abstract description 7
- 239000003550 marker Substances 0.000 abstract description 7
- 238000012360 testing method Methods 0.000 abstract description 7
- 230000008499 blood brain barrier function Effects 0.000 abstract description 4
- 210000001218 blood-brain barrier Anatomy 0.000 abstract description 4
- 238000011065 in-situ storage Methods 0.000 abstract 1
- JADVWWSKYZXRGX-UHFFFAOYSA-M thioflavine T Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C1=[N+](C)C2=CC=C(C)C=C2S1 JADVWWSKYZXRGX-UHFFFAOYSA-M 0.000 description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 7
- 208000024827 Alzheimer disease Diseases 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 239000012046 mixed solvent Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 210000003710 cerebral cortex Anatomy 0.000 description 2
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 238000006000 Knoevenagel condensation reaction Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical group C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 1
- 241000256856 Vespidae Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000048 toxicity data Toxicity 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- 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
- C09K2211/1003—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
- C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom
-
- 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
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Materials Engineering (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Psychiatry (AREA)
- Hospice & Palliative Care (AREA)
- Optics & Photonics (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明提供了近红外、高信噪比的Aβ荧光成像剂及其制备方法和应用。所述聚集诱导发光染料具有式Ⅰ所示结构。另外，本发明还提供了所述Aβ荧光成像剂在脑组织切片荧光标记和活体成像上的应用。测试表明，本发明提供的聚集诱导发光染料具有近红外发射、光稳定性强、生物相容性好以及信噪比高等诸多优点，能够实现对APP/PS1转基因小鼠脑组织切片中Aβ斑块的精确定位，从而高保真地获取关于Aβ斑块数目、实时形态等原位信息。特别值得指出的是，该类成像剂具有良好的血脑屏障穿透性，可应用于APP/PS1转基因小鼠脑部Aβ斑块的在体实时成像。
Description
技术领域
本发明属于精细化工技术领域，具体涉及一类基于近红外、高信噪比的Aβ荧光成像剂及其制备方法和在脑组织切片上的荧光标记应用。
背景技术
阿尔茨海默症(AD)是一种神经退行性疾病，是造成老年痴呆最主要的原因之一。截止目前为止，仍没有一种经过临床试验的药物对其具有显著疗效，因此阿尔茨海默症(AD)被认为是一种无法治愈的疾病。研究表明，大脑中Aβ斑块的形成和沉积在AD的神经病理学中扮演着极为重要的角色，被认为是该疾病早期诊断和预测的一种生物标记物。因此，发展高性能的Aβ斑块成像剂对阿耳茨海默症的早期诊断和治疗具有非常重要的意义。
荧光成像技术作为一种非侵入性的早期诊断方法，可以实时监测生物分子在生物体内的作用，近年来被广泛运用于生物医学成像领域。众所周知，刚果红(CR)和硫黄素T(ThT)作为市售染料，为Aβ斑块的标记提供了一种标准化的方法。
然而，现有市售Aβ斑块荧光标记染料的发射波长较短(主要位于400-600nm区间)、斯托克斯位移小、以及光稳定性差，导致其成像时背景信号高，并且容易发生光漂白，很难实现对Aβ斑块的准确定位；值得注意的是，大多数商业Aβ斑块荧光成像剂在处于较大浓度时，由于π-π堆积作用的影响，造成染料分子严重的自淬灭。由于其固有的聚集荧光淬灭(aggregation-caused quenching,ACQ)现象，不能够真实反映Aβ斑块的位置和浓度。除此之外，市售染料无法有效穿透血脑屏障到达斑块区域，进一步限制了其在生物活体成像上的应用。
发明内容
针对现有Aβ荧光成像技术存在的瓶颈问题，本发明提供了一类基于聚集诱导发光(aggregation-induced emission,AIE)近红外、高信噪比的Aβ荧光成像剂。该类聚集增强型近红外荧光染料以N,N-二甲基苯胺结构单元为Aβ斑块的识别、结合基团，通过引入噻吩基团有效拓展其共轭体系，并修饰磺酸基增加染料的亲水性以及调控其聚集态，降低背景荧光，使其具有长发射波长(位于近红外区域)、大斯托克斯位移、光稳定性优异和生物相容性好等诸多优点。特别是由于该类成像剂在水溶液中完全没有荧光，而仅当高选择性与Aβ斑块特异结合后，其初始的聚集状态发生改变，伴随荧光的显著增强变化，使得该成像剂在检测Aβ斑块时表现为极高的信噪比。利用激光共聚焦显微镜，实现了对APP/PS1转基因小鼠脑组织切片中Aβ斑块的精确定位和长时间示踪，从而高保真地获取关于Aβ斑块数目、实时形态等原位信息。特别值得指出的是，该类成像剂具有良好的血脑屏障穿透性，可应用于APP/PS1转基因小鼠脑部Aβ斑块的在体实时成像。
本发明通过下述方案实现：
本发明提出了一种近红外、高信噪比的Aβ荧光成像剂，其结构为式Ⅰ所示：
式Ⅰ中，R1独立选自：氰基(-CN)，羧基(-COOH)和式Ⅱ所示基团(其中曲线标记处为取代位，下同)中的任意一种；
R2为C1～C4的烃基或烃基磺酸盐；A为N,N-二甲基苯胺，其结构如式Ⅲ所示；
本发明还提供了上述近红外、高信噪比的Aβ荧光成像剂的制备方法，其主要步骤包括：由式IV所示化合物与式V所示化合物经Knoevenagel缩合反应，制得目标化合物。
其中，A和R1，R2的定义与前文所述相同。
本发明还提供了上述近红外、高信噪比的Aβ荧光成像剂在脑组织切片荧光标记和活体成像上的应用。
附图说明
图1.式I-1所示化合物(详见实施例1)在四氢呋喃和水混合溶剂中不断增加水含量的荧光发射光谱图(10-5mol·L-1)；
其中，横坐标为波长(nm)，纵坐标为荧光强度(a.u.)。
图2.式I-2所示化合物(详见实施例1)在水和乙醇混合溶剂中不断增加乙醇含量的荧光发射光谱图(10-5mol·L-1)；
其中，横坐标为波长(nm)，纵坐标为荧光强度(a.u.)。
图3.染料I-2(浓度为10-5mol·L-1)随着加入不同浓度的Aβ42蛋白纤维(0-9μM)后荧光光谱的变化；
其中，横坐标为波长(nm)，纵坐标为荧光强度(a.u.)。
图4.商业染料ThT，染料I-1，染料I-2(浓度为10-6mol·L-1)分别与Aβ42蛋白纤维(3μM)在PBS缓冲液中孵育30min前后荧光强度的变化。
其中，横坐标为三种染料分子，纵坐标为荧光强度(a.u.)。
图5.染料I-2与商业化染料ThT在相同激光强度下光稳定性曲线。
图6.染料I-2生物相容性表征。
图7.染料I-2与商业化染料ThT在APP/PS1转基因鼠相邻脑组织切片上的荧光共定位标记图。
其中，图7(A)与(E)分别为市售染料ThT与染料I-2染色的APP/PS1脑切片海马区域；图7(B)与(F)分别为市售染料ThT与染料I-2染色的APP/PS1脑切片大脑皮层区域；图7(C)与(G)为对大脑皮层区域染色后放大40倍的图片；图7(D)与(H)为野生型(阴性)小鼠脑切片图片。
图8.染料I-2在野生型(阴性)小鼠与APP/PS1转基因小鼠上的活体成像应用。
具体实施方式
在本发明一个优选的技术方案中：
R1独立选自氰基，羧基或式Ⅱ所示基团的任意一种；
R2为C1～C4的直链或支链烷基，C2～C4的直链或支链炔基，C1-C4的直链烃基磺酸盐；
A为N,N-二甲基苯胺；
在进一步优选的技术方案中，R1独立选自氰基或式Ⅱ所示基团的任意一种，R2为乙基，正丁基和丙磺酸盐；
更进一步优选的R1独立选自氰基，R2为乙基、丙磺酸盐中的一种；
更进一步优选的R2为丙磺酸盐；
本发明所提供的，一种制备式Ⅰ所示化合物的方法，所述方法的主要具体步骤是：在有催化剂(如醋酸铵等)存在条件下，由式IV所示化合物与相应的醛V于甲苯或乙腈等有机溶剂(反应介质)中，在回流状态保持20分钟至2小时，制得目标物(式Ⅰ所示化合物)。
下面通过实施例对本发明作进一步的阐述，其目的仅在于更好地理解本发明的内容。因此，所举之例并不限制本发明的保护范围：
实施例1
(1)染料I-1的合成：
在100mL圆底烧瓶中，分别加入1-乙基-2-甲基-4-(α,α-二氰基)亚甲基-1,4-二氢喹啉(116.4mg,0.50mmol)，5-(N,N-二甲基苯胺)-2-噻吩醛(104mg,0.50mmol)和甲苯30mL，滴加0.5mL醋酸铵溶液(2M)，氩气保护下150℃反应2h。冷却至室温后，旋蒸除去甲苯，柱色谱分离(200-300目硅胶，PE:CH2Cl2＝5:2)，旋蒸得红色固体50mg，产率38％。
1H NMR(400MHz,DMSO-d6,ppm)δ＝1.42(t,J＝8.0Hz,3H,NCH2CH3-H),δ＝2.97(s,6H,CH3-H),δ＝4.54(q,J＝8.0Hz,2H,NCH2CH3-H),δ＝6.77(d,J＝8.0Hz,2H,Ph-H),δ＝7.03(s,1H,Ph-H),δ＝7.07(d,J＝16Hz,1H,alkene-H),δ＝7.38(d,J＝4.0Hz,1H,Ph-H),δ＝7.54(d,2H,J＝4.0Hz,thiophene-H),δ＝7.57(s,1H,Ph-H),7.62(t,J＝8.0Hz,1H,Ph-H),δ＝7.66(d,J＝16Hz,1H,alkene-H),7.93(t,J＝4.0Hz,1H,Ph-H),δ＝8.09(d,J＝8.0Hz,1H,Ph-H),δ＝8.93(d,J＝8.0Hz,1H,Ph-H).
Mass spectrometry(ESI-MS,m/z):[M+H]+calcd.for[C28H24N4S+H]+449.1800；found449.1806.
(2)染料I-2的合成：
在50mL单口烧瓶中，分别加入4-二氰亚甲基-2-甲基喹啉腈丙磺酸钠盐(50mg，0.15mmol)、甲苯(10mL)、5-(N,N-二甲基苯胺)-2-噻吩醛(34mg，0.15mmol)，滴加0.5mL醋酸铵溶液(2M)，氩气保护，150℃下反应2h。冷却，旋蒸溶剂，柱色谱分离(300-400目硅胶，DCM:MeOH＝1:2)，旋蒸得深红色固体15mg，产率30％。
1H NMR(400MHz,DMSO-d6,ppm)δ＝2.10(m,2H,-CH2SO3 -),δ＝2.67(m,2H,-CH2CH2CH2-),δ＝2.97(s,6H,-N(CH3)2),δ＝4.70(t,J＝8.0Hz,2H,-NCH2CH2-),δ＝6.78(d,J＝8.0Hz,2H,phenyl-H),δ＝7.07(s,1H,pyrroleapp:addword:pyrrole-H),δ＝7.31(d,J＝16.0Hz,1H,alkene-H),δ＝7.38(d,J＝4.0Hz,1H,thiophene-H),δ＝7.51(t,J＝8.0Hz,1H,phenyl-H),δ＝7.62(d,J＝4.0Hz,2H,thiophene-H,Ph-H),δ＝7.67(d,J＝16.0Hz,1H,alkene-H),δ＝7.70(s,1H,pyrroleapp:addword:pyrrole-H),δ＝7.89(t,J＝8.0Hz,1H,phenyl-H),δ＝8.24(d,J＝8.0Hz,1H,phenyl-H),δ＝8.91(d,J＝8.0Hz,1H,phenyl-H).
Mass spectrometry(ESI-MS,m/z):[M]-calcd.for[C29H25N4O3S2]-541.1368；found541.1372.
实施例2
染料I-2在聚集态的吸收和荧光光谱
取实施例1制备的染料I-2溶于分析纯二甲基亚砜中，制成1.0×10-3M的储备液。然后制备乙醇(EtOH)含量为95％的EtOH/H2O混合溶剂2mL。取20μL上述储备液加入到已制备的EtOH/H2O混合溶剂中，混合均匀后转移至光学石英比色皿(10×10mm)中测试其荧光光谱。如图2所示，以500nm作为激发波长，染料I-2的最大发射峰大约位于720nm处位于近红外区域，斯托克斯位移达到200nm；并且染料I-2在混合溶剂中聚集导致其荧光增强，具有典型的聚集荧光增强特性。
实施例3
染料I-2对Aβ42纤维的光谱响应
取实施例1制备的染料I-2溶于分析纯二甲基亚砜中，制成1.0×10-3M的储备液。然后制备PBS缓冲液2.91mL。取3μL上述储备液加入到已制备的PBS缓冲液中，再加入90μL Aβ42纤维储存液(1×10-4M)。混合液在37℃下孵育30min，混合均匀后转移至光学石英比色皿(10×10mm)中测试其吸收和荧光光谱。如图4所示，未加入Aβ42纤维时，染料I-2基本没有荧光信号；当加入Aβ42纤维后，以500nm作为激发波长，在近红外区域660nm处出现最大发射峰，荧光强度增加50倍左右，表现出极高的信噪比。
实施例4
染料I-2光稳定性测试
光稳定性是评价染料在长时间成像等实际应用中重要的性能指标之一。我们对染料进行连续光照然后监测其荧光强度的方法来评价染料的光稳定性，同时选取商业化染料ThT作为参比染料。如图5所示，随着光照时间增长，ThT荧光强度急剧下降，当光照18min左右时，ThT产生严重的光漂白，表明其光稳定性差；然而，在相同光照条件下，染料I-2荧光强度缓慢下降，当光照时间达到40min时，其荧光强度为初始荧光强度的50％，表明染料I-2具有优异的光稳定性。
实施例5
染料I-2生物相容性测试
我们采用标准的MTT方法测试聚集诱导发光染料I-2的细胞毒性。具体实验步骤如下：首先将HeLa细胞铺到96孔板中，细胞密度为1×104细胞/孔，并在细胞培养箱中培养过夜。不同浓度的染料I-2(0-32μM，100μL/孔)分别加入到不同的孔中，培养基稀释的DMSO(0.5％，100μL/孔)作为阴性对照组。细胞在细胞培养箱中培养24h，然后在每孔中加入10μLMTT溶液(5mg/mL)。孵育4小时后，除去96孔板中的液体，并加入DMSO(100μL/孔)，最后用酶标仪进行测定。由图6中细胞毒性数据所示，当染料I-2的浓度增大到32μM时，细胞存活率大约为95％，表明染料I-2具有良好的生物相容性。
实施例6
染料I-2对APP/PS1转基因小鼠脑组织切片的荧光标记
为了研究染料I-2对阿尔茨海默小鼠脑组织切片是否具有荧光标记能力，我们选取APP/PS1转基因小鼠脑组织切片作为切片模型，同时选取商业化市售染料硫黄素T(ThT)作为金标染料。具体实验步骤如下：取实施例2中染料I-2储备液或者硫黄素T(ThT)储备液(1.0×10-3M)在室温条件下滴加到相邻的脑组织切片上孵育30min，(染料I-2最终浓度为100μM，染料ThT最终浓度为200μM)，接着用40％的乙醇溶液浸泡2min，洗涤两次，再用去离子水冲洗一次。最后用荧光共聚焦显微镜进行组织切片成像。成像结果如图7所示，发现染料I-2能够清楚地对Aβ斑块进行荧光标记，定位结果与市售染料硫黄素T(ThT)标记的相邻切片基本一致。证明了染料I-2能够准确的与Aβ斑块结合；相比于市售染料硫黄素T(ThT)，染料I-2具有更高的灵敏度和信噪比。
实施例7
本发明中所有活体实验均遵守实验室动物饲养和使用的规章制度，并得到华东理工大学大学动物饲养和使用委员会批准。实验用带瘤裸鼠购自上海斯莱克动物实验有限公司，饲养在无菌室中层流通风橱内的无菌鼠笼中，使用高压蒸汽处理过的食物和水进行喂食。
对APP/PS1转基因鼠和对照组野生型黑鼠进行一次剂量为2mg/kg的I-2的尾静脉注射。注射后，使用Perkin Elmer In-Vivo Professional Imaging System对APP/PS1转基因鼠进行整体的光学成像，激发波长和接收波长分别为500nm和660nm。成像实验前，对裸鼠进行腹腔注射1％戊巴比妥钠盐溶液(45mg/kg)使之麻醉。
从图8中看出，尾静脉注射化合物I-2后，APP/PS1转基因小鼠的大脑区域出现明显的荧光信号。而对照组野生型黑鼠脑部无明显的荧光信号。表明化合物I-2具有很好的血脑屏障穿透性和活体成像特性，可进一步应用于生物活体测试中。
Claims (10)
1.近红外、高信噪比的Aβ荧光成像剂，其结构为式Ⅰ所示：
式Ⅰ中，R1独立选自：氰基(-CN)，羧基(-COOH)和式Ⅱ所示基团(其中曲线标记处为取代位，下同)中的任意一种；
R2为C1～C4的烃基或烃基磺酸盐；A为N,N-二甲基苯胺，其结构如式Ⅲ所示；
2.根据权利要求1所述的近红外、高信噪比的Aβ荧光成像剂，其特征在于，所述R1为氰基，羧基和式Ⅱ所示基团中的任意一种，R2为乙基，正丁基和丙磺酸盐中的任意一种。
3.根据权利要求1所述的近红外、高信噪比的Aβ荧光成像剂，其特征在于，所述R1为氰基，式Ⅱ所示基团中的任意一种，R2为乙基、丙磺酸盐中的一种。
4.根据权利要求1所述的近红外、高信噪比的Aβ荧光成像剂，其特征在于，所述R1为氰基，R2为乙基、丙磺酸盐中的一种。
5.根据权利要求1所述的近红外、高信噪比的Aβ荧光成像剂，其特征在于，R1为氰基，R2为丙磺酸盐。
6.一种如权利要求4所述的近红外、高信噪比的Aβ荧光成像剂制备方法，其特征在于，步骤如下：
在有催化剂存在条件下，由式IV所示化合物与相应的醛V于甲苯或乙腈等有机溶剂反应介质中，在回流状态保持20分钟至2小时，制得目标物式Ⅰ所示化合物。
其中，式中：
R1独立选自氰基；R2独立选自乙基和丙磺酸盐的任意一种；
A为N,N-二甲基苯胺。
7.根据权利要求6所述的聚集诱导发光近红外、高信噪比的Aβ荧光成像剂的制备方法，其特征在于，所述步骤中将1当量的式IV所示结构的化合物与1.5当量的式V所示结构的化合物溶于乙腈中，醋酸铵作为催化剂，回流条件下反应20min～2h；反应结束后，柱层析分离得到式Ⅰ所示结构的荧光染料。
8.一种权利要求1所述的近红外、高信噪比的Aβ荧光成像剂在脑组织切片上作为荧光标记的应用。
9.一种权利要求1所述的近红外、高信噪比的Aβ荧光成像剂在活体成像上的应用。
10.一种权利要求1所述的近红外、高信噪比的Aβ荧光成像剂在诊断或治疗阿尔茨海默症的产品上的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811069366.9A CN109053710B (zh) | 2018-09-13 | 2018-09-13 | 近红外、高信噪比的Aβ荧光成像剂及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811069366.9A CN109053710B (zh) | 2018-09-13 | 2018-09-13 | 近红外、高信噪比的Aβ荧光成像剂及其制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109053710A true CN109053710A (zh) | 2018-12-21 |
| CN109053710B CN109053710B (zh) | 2020-08-07 |
Family
ID=64761626
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811069366.9A Expired - Fee Related CN109053710B (zh) | 2018-09-13 | 2018-09-13 | 近红外、高信噪比的Aβ荧光成像剂及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109053710B (zh) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111574438A (zh) * | 2020-06-02 | 2020-08-25 | 华东理工大学 | 红/近红外AIE探针及其制备方法和在检测Aβ聚集体及其纤维化斑块的应用 |
| CN111978248A (zh) * | 2020-08-07 | 2020-11-24 | 华东理工大学 | 一种多位点可修饰聚集诱导发光喹啉腈衍生物、制备方法及其应用 |
| CN112113934A (zh) * | 2019-06-19 | 2020-12-22 | 泰州医药城国科化物生物医药科技有限公司 | 一种用于提高细胞钙流荧光检测的方法 |
| CN112710838A (zh) * | 2020-11-26 | 2021-04-27 | 华东理工大学 | 喹啉腈衍生物在蛋白质印记法检测蛋白质的应用及其制备方法 |
| CN114656447A (zh) * | 2022-03-03 | 2022-06-24 | 华东理工大学 | 一类基于高时空分辨率的近红外荧光、磁共振Aβ双模式成像探针及其制备方法和应用 |
| CN115974874A (zh) * | 2023-02-07 | 2023-04-18 | 上海健康医学院 | 一种基于咪唑并吡啶的pH荧光探针及其制备方法和应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104311477A (zh) * | 2014-09-29 | 2015-01-28 | 华东理工大学 | 含氮杂环化合物及其用途 |
-
2018
- 2018-09-13 CN CN201811069366.9A patent/CN109053710B/zh not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104311477A (zh) * | 2014-09-29 | 2015-01-28 | 华东理工大学 | 含氮杂环化合物及其用途 |
Non-Patent Citations (2)
| Title |
|---|
| ANDONG SHAO: "Insight into aggregation-induced emission characteristics of red-emissive quinoline-malononitrile by cell tracking and real-time trypsin detection", 《CHEMICAL SCIENCE》 * |
| BI-YUE ZHU: "Synthesis and evaluation of styrylpyran fluorophores for noninvasive detection of cerebral β-amyloid deposits", 《BIOORGANIC & MEDICINAL CHEMISTRY》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112113934A (zh) * | 2019-06-19 | 2020-12-22 | 泰州医药城国科化物生物医药科技有限公司 | 一种用于提高细胞钙流荧光检测的方法 |
| CN111574438A (zh) * | 2020-06-02 | 2020-08-25 | 华东理工大学 | 红/近红外AIE探针及其制备方法和在检测Aβ聚集体及其纤维化斑块的应用 |
| CN111574438B (zh) * | 2020-06-02 | 2022-10-18 | 华东理工大学 | 红/近红外AIE探针及其制备方法和在检测Aβ聚集体及其纤维化斑块中的应用 |
| CN111978248A (zh) * | 2020-08-07 | 2020-11-24 | 华东理工大学 | 一种多位点可修饰聚集诱导发光喹啉腈衍生物、制备方法及其应用 |
| CN112710838A (zh) * | 2020-11-26 | 2021-04-27 | 华东理工大学 | 喹啉腈衍生物在蛋白质印记法检测蛋白质的应用及其制备方法 |
| CN114656447A (zh) * | 2022-03-03 | 2022-06-24 | 华东理工大学 | 一类基于高时空分辨率的近红外荧光、磁共振Aβ双模式成像探针及其制备方法和应用 |
| CN114656447B (zh) * | 2022-03-03 | 2024-08-06 | 华东理工大学 | 一类基于高时空分辨率的近红外荧光、磁共振Aβ双模式成像探针及其制备方法和应用 |
| CN115974874A (zh) * | 2023-02-07 | 2023-04-18 | 上海健康医学院 | 一种基于咪唑并吡啶的pH荧光探针及其制备方法和应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109053710B (zh) | 2020-08-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109053710A (zh) | 近红外、高信噪比的Aβ荧光成像剂及其制备方法和应用 | |
| Song et al. | A highly sensitive near-infrared ratiometric fluorescent probe for imaging of mitochondrial hydrazine in cells and in mice models | |
| CN113444046B (zh) | 一种荧光探针及其制备方法和用途 | |
| JP5688899B2 (ja) | 生物試料用標識剤並びに該標識剤を用いた標識方法及びスクリーニング方法 | |
| Shi et al. | In vivo ratiometric tracking of endogenous β-galactosidase activity using an activatable near-infrared fluorescent probe | |
| Sun et al. | A rosamine-based red-emitting fluorescent sensor for detecting intracellular pH in live cells | |
| JP5997288B2 (ja) | ナフタレンを基本骨格とする二光子蛍光プローブ、その製造方法及びその利用方法 | |
| US9745318B2 (en) | Fluorescent red emitting functionalizable calcium indicators | |
| CN109180680A (zh) | 一种紫外光触发交联型近红外分子探针及其制备方法与应用 | |
| CN110234735A (zh) | 双模态生物成像用探针 | |
| WO2023029638A1 (zh) | N,N二苯基氨基修饰的β-咔啉吲哚鎓盐、制备方法与应用 | |
| Yu et al. | A new near-infrared ratiometric fluorescent probe based on quinoline-fused rhodamine dye for sensitive detection of cysteine and homocysteine in mitochondria | |
| Li et al. | A lysosomal probe for monitoring of pH in living cells and ovarian tumour | |
| CN108864056A (zh) | 具有aie性能的近红外荧光化合物及其制备方法和应用 | |
| Jin et al. | Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring | |
| Ni et al. | Convenient construction of fluorescent markers for lipid droplets with 1, 8-naphthalimide unit | |
| CN115385851A (zh) | 具有不对称二乙腈基结构的近红外聚集诱导发光型超高效光敏剂、制备方法及应用 | |
| Varvuolytė et al. | Synthesis and photodynamic properties of pyrazole-indole hybrids in the human skin melanoma cell line G361 | |
| Romero et al. | N1-aryl-2-(trifluoromethyl) benzo [b][1, 8] naphthyridin-4 (1H)-one as convenient platform to design high photostable and long-lived dyad fluorophore with potential application in live-cell imaging | |
| US20150369812A1 (en) | Flavonoid compounds of low toxicity for biological imaging applications | |
| CN111303111B (zh) | 一类黄菁近红外二区染料、制备方法及荧光成像应用 | |
| Patel et al. | Impact of Substituents in Tumor Uptake and Fluorescence Imaging Ability of Near‐Infrared Cyanine‐like Dyes | |
| Dore et al. | Chromophores for the delivery of bioactive molecules with two-photon excitation | |
| CN114656447B (zh) | 一类基于高时空分辨率的近红外荧光、磁共振Aβ双模式成像探针及其制备方法和应用 | |
| CN115557878A (zh) | 一种吲哚菁类衍生物及其制备和应用 |
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 |
Granted publication date: 20200807 Termination date: 20210913 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |