CN110372590A - 一种检测溶酶体pH的荧光探针及其制备方法和应用 - Google Patents
一种检测溶酶体pH的荧光探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN110372590A CN110372590A CN201910687350.2A CN201910687350A CN110372590A CN 110372590 A CN110372590 A CN 110372590A CN 201910687350 A CN201910687350 A CN 201910687350A CN 110372590 A CN110372590 A CN 110372590A
- Authority
- CN
- China
- Prior art keywords
- probe
- preparation
- fluorescence probe
- fluorescence
- lysosome
- 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
- 239000000523 sample Substances 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 230000002132 lysosomal effect Effects 0.000 title claims abstract description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 8
- JKNCOURZONDCGV-UHFFFAOYSA-N 2-(dimethylamino)ethyl 2-methylprop-2-enoate Chemical compound CN(C)CCOC(=O)C(C)=C JKNCOURZONDCGV-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910021589 Copper(I) bromide Inorganic materials 0.000 claims abstract description 6
- UKODFQOELJFMII-UHFFFAOYSA-N pentamethyldiethylenetriamine Chemical compound CN(C)CCN(C)CCN(C)C UKODFQOELJFMII-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 8
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 238000001291 vacuum drying Methods 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 239000004411 aluminium Substances 0.000 claims description 3
- 229910052782 aluminium Inorganic materials 0.000 claims description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 2
- 210000003712 lysosome Anatomy 0.000 abstract description 16
- 230000001868 lysosomic effect Effects 0.000 abstract description 16
- 238000003786 synthesis reaction Methods 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 230000004044 response Effects 0.000 abstract description 3
- 230000002378 acidificating effect Effects 0.000 abstract description 2
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000003595 spectral effect Effects 0.000 abstract description 2
- 230000000007 visual effect Effects 0.000 abstract description 2
- 125000001246 bromo group Chemical group Br* 0.000 abstract 1
- 238000012544 monitoring process Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 17
- 239000000243 solution Substances 0.000 description 16
- 238000003384 imaging method Methods 0.000 description 7
- 230000005284 excitation Effects 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 2
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical compound C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- -1 dimethylaminoethyl Chemical group 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 241000736199 Paeonia Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 229910001626 barium chloride Inorganic materials 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 229960003677 chloroquine Drugs 0.000 description 1
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000007849 functional defect Effects 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明提供了一种检测溶酶体pH的荧光探针,其化学结构式为:其中,n=25‑40。由于该探针高分子链上具有弱碱性的二甲氨基,其在溶酶体的酸性环境中很容易发生聚集,因此探针具有溶酶体靶向性。并且灵敏度高、特异性高、抗干扰性强、较好的水溶性等特点,具有良好的荧光发射光谱特性(380‑550 nm),能实现对溶酶体中pH变化的快速荧光信号响应及其实时可视化监测。可由DMAEMA、Nap‑Br、PMDETA,异丙醇,CuBr在氮气保护下反应制得,合成工艺简单易行,原料廉价易得,制备成本低,易于推广。
Description
技术领域
本发明属于分析化学技术领域,具体涉及一种基于聚甲基丙烯酸二甲氨基乙酯的检测溶酶体pH的荧光探针及其应用。
背景技术
细胞生长、黏附、内吞和离子转运等过程在很大程度上依赖于细胞内pH值。细胞内pH值异常可导致严重的功能障碍,如细胞的坏死、凋亡以及炎症、癌症等疾病。溶酶体是膜封闭的细胞器,其存在于所有哺乳动物细胞中的。它含有一系列可降解的酶,可以分解各种碳水化合物、核酸、生物聚合物、蛋白质和脂类。在活细胞中,溶酶体保持酸性pH值在4.5-5.5之间,这是酶水解的最佳状态,同时也对细胞内物质的消化和清除中起着关键作用。溶酶体pH值异常可导致溶酶体功能缺陷和许多溶酶体储存紊乱。因此,追踪溶酶体的pH变化对于了解溶酶体相关的生物学过程和疾病是至关重要的。
近年来,荧光探针因其无损伤、高灵敏度、实时和原位检测等优点,成为研究溶酶体pH变化的有效工具。萘酰亚胺作为一种经典的荧光染料,已被广泛用于开发用于pH值监测和生物分子检测的荧光探针。现有的pH探针多是小分子的荧光探针,它们稳定性不好并且容易被清除,为了提高探针的水溶性和光稳定性,开发基于萘酰亚胺染料和生物相容聚合物的溶酶体pH探针具有重要意义。
发明内容
针对现有技术中的问题,本发明提供一种检测溶酶体pH的荧光探针,响应速度快、抗干扰能力强,稳定性好。
本发明的另一目的是提供一种上述荧光探针在检测溶液中或生物细胞内溶酶体pH的应用。
为实现上述目的,本发明采用如下技术方案。
一种检测溶酶体pH的荧光探针,简称NapBr-PDM,其化学结构式如式(I)所示:
式(I);
其中,n=25-40。
上述荧光探针的制备方法,包括以下步骤:
(1)在氮气保护下,DMAEMA(甲基丙烯酸二甲氨基乙酯)、Nap-Br和PMDETA(N-五甲基二亚乙基三胺)于异丙醇中混匀后在液氮(-196℃)和25℃水浴中反复冻融,得反应液1;
(2)向反应液1中加入CuBr,混匀后,于液氮(-196℃)和25℃水浴中反复冻融,解冻后,混匀反应液,分离提纯得荧光探针。
所述物料DMAEMA: Nap-Br: PMDETA: CuBr的物质的量比为4:0.2:0.15:0.1。
步骤(1)中,所述冻融过程为冷冻-抽真空-解冻-通高纯N2,重复3次,冷冻和解冻每次各5 min。
步骤(2)中,所述冻融过程为冷冻-抽真空-解冻-通高纯N2,重复3次,冷冻和解冻每次各5 min。
步骤(2)中,所述反应时间为6 h。
步骤(2)中,所述反应温度为50 ℃。
步骤(2)中,所述分离提纯步骤为向反应液中加入无水四氢呋喃,使用中性氧化铝柱去除铜盐。滤液在石油醚中沉淀,搅拌至澄清,过滤,抽真空干燥,所得固体溶解于无水四氢呋喃中,使用正己烷再次过氧化铝柱沉淀,真空干燥。
一种上述荧光探针在检测溶液、细胞或生物体中溶酶体pH的应用。
本发明的机理如下:
本发明所述的荧光探针由于高分子链上具有弱碱性的二甲氨基,其在溶酶体的酸性环境中很容易发生聚集,因此探针具有溶酶体靶向性。
本发明具有以下优点:
本发明所述的荧光探针具有溶酶体靶向性、较好的水溶性、特异性高、抗干扰性强等优点,实现对溶酶体中pH变化的快速荧光信号响应及其实时可视化监测;灵敏度高,具有良好的荧光发射光谱特性(380-550 nm);同时,本发明所述的荧光探针可经化学合成获得,合成工艺简单易行,原料廉价易得,制备成本低,易于推广。
附图说明
图1是探针NapBr-PDM的氢谱;
图2是探针NapBr-PDM在水相中的选择性,探针的浓度为60 µg/mL,分析物的浓度为100µM;
图3是探针NapBr-PDM在不同pH值得PBS溶液中的荧光光谱,探针的浓度为60 µg/mL;
图4是探针NapBr-PDM在HeLa细胞中的共定位成像,探针的浓度为60 µg/mL;
图5是探针NapBr-PDM在HeLa细胞以及经100 µM氯喹处理1 h后的细胞成像,探针的浓度为60 µg/mL。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 荧光探针NapBr-PDM的合成
(1)向20 mL聚合管中加入0.7 mL(4 mmol)DMAEMA,100 mg(0.2 mmol)Nap-Br,30 μL(0.15 mmol) PMDETA,再加入1.6 mL (21 mmol) 异丙醇,在N2保护下,于液氮(-196℃)和25℃水浴中反复冻融,完成三次冷冻-抽真空-解冻-通高纯N2过程,冷冻和解冻每次各5min,得反应液1;
(2)向反应液1中后加入15 mg(0.1 mmol)CuBr,搅拌均匀后于液氮(-196℃)和25℃水浴中反复冻融,完成三次冷冻-抽真空-解冻-通高纯N2过程,冷冻和解冻各每次5 min。解冻后,搅拌均匀,置于50 ℃油浴中反应6 h;反应结束分别加入3 mL无水四氢呋喃(THF),使用中性氧化铝柱去除铜盐。滤液在石油醚中沉淀,搅拌至澄清,过滤,抽真空干燥,所得固体溶解于3 mL THF中,使用正己烷再次过氧化铝柱沉淀,真空干燥得到黄色固体,即为荧光探针NapBr-PDM,其1H NMR图谱如图1,经计算n平均值为25。
实施例2 荧光探针NapBr-PDM对不同检测物的选择性
配制5 mL浓度为10 mM的金属盐、氧化性物质、还原性物质及氨基酸的PBS水溶液,配制浓度为6 mg/mL的实施例1所得荧光探针NapBr-PDM母液作为备用。加入20 μL探针母液、200μL DMSO和10当量的各离子(或活性氧),用磷酸缓冲液PBS定容至2 mL,摇匀后进行荧光检测(λex=345 nm,λem=424 nm),建立荧光强度与各离子(或活性氧)的柱状图,如图2,其中,1-19加入的检测物分别为:PBS溶液、氯化钾、氯化钙、氯化钡、氯化钠、氯化镁、氯化铝、氯化锌、氯化铁、硝酸银、氯化钴、碘化钾、氟化钠、次氯酸钠、过氧化氢、维生素C、半胱氨酸、同型半胱氨酸、谷胱甘肽。由图2可以发现,常规的离子(或活性氧)对荧光探针NapBr-PDM的荧光几乎没有影响。
实施例3 荧光探针NapBr-PDM在不同pH值PBS溶液中的荧光光谱
配制pH值为3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5和9的PBS溶液及浓度为6 mg/mL的实施例1所得荧光探针NapBr-PDM母液作为备用。将探针母液稀释至浓度为60 µg/mL,分别加入不同pH值的PBS溶液中,并进行荧光检测(λex=345 nm,λem=424 nm),其荧光谱图如图3所示。由图3可以发现,随着pH的增大,荧光探针NapBr-PDM的荧光逐渐减弱。
实施例4 荧光探针NapBr-PDM与溶酶体在HeLa细胞中共定位的荧光成像图
将HeLa细胞放在培养基(DMEM培养液和10%胎牛血清)中,放置于条件为37℃、5% CO2和20% O2的培养箱中培养24-48 h。将实施例1所得荧光探针(浓度为60 µg/mL)和商用溶酶体定位染料Lyso-Tracker Deep Red(浓度为1 μM)加入到HeLa细胞中,培养30 min后,进行激光共聚焦成像。蓝色通道的激发波长是405 nm,收集的波长范围是425-475 nm;绿色通道的激发波长是405 nm,收集的波长范围是500-550 nm;深红色色通道的激发波长是647 nm,收集的波长范围是675-730 nm。成像结果如图4所示。由图4可知,荧光探针NapBr-PDM与商用溶酶体在细胞中的荧光信号重叠系数高达0.91,表明该荧光探针能够定位溶酶体。
实施例5 探针NapBr-PDM在癌细胞中对溶酶体成像以及追踪其pH变化
配置1mL浓度为60 µg/mL的实施例1所得荧光探针的PBS溶液,然后加入到HeLa细胞中孵育30 min成像,激发波长为405 nm,发射波长为425-475 nm和500-550 nm;加入100 µM氯喹孵育30 min再次成像,激发波长为405 nm,发射波长为425-475和500-550 nm。结果如图5所示,a和e为明场成像;b和c为HeLa细胞经60 μg/mL探针孵育30 min后的荧光场;f和g为HeLa细胞经60 μg/mL探针孵育30 min以及经过100 μM氯喹处理30 min后的荧光场;d与h为叠加场。由图5可以看出,由于氯喹的刺激使得细胞溶酶体pH增大,因此探针的蓝色和绿色荧光都变弱。
Claims (8)
1.一种检测溶酶体pH的荧光探针,其化学结构式如式(I)所示:
式(I);
其中,n=25-40。
2.一种如权利要求1所述荧光探针的制备方法,其特征在于,包括以下步骤:
(1)在氮气保护下,DMAEMA、Nap-Br和PMDETA(于异丙醇中混匀后在液氮和25℃水浴中反复冻融,得反应液1;
(2)向反应液1中加入CuBr,混匀后,于液氮和25℃水中反复冻融,解冻后,混匀反应液,分离提纯得荧光探针。
3.根据权利要求2所述的制备方法,其特征在于,物料DMAEMA:Nap-Br:PMDETA: CuBr的物质的量比为4:0.2:0.15: 0.1。
4.根据权利要求2所述的制备方法,其特征在于,步骤(1)和步骤(2)中,冻融过程为冷冻-抽真空-解冻-通高纯N2,重复3次,冷冻和解冻每次各5 min。
5.根据权利要求2所述的制备方法,其特征在于,步骤(2)中反应时间为6 h。
6.根据权利要求2所述的制备方法,其特征在于,步骤(2)中反应温度为50 ℃。
7.根据权利要求2所述的制备方法,其特征在于,步骤(2)中,所述分离提纯步骤为向反应液中加入无水四氢呋喃,使用中性氧化铝柱去除铜盐;
滤液在石油醚中沉淀,搅拌至澄清,过滤,抽真空干燥,所得固体溶解于无水四氢呋喃中,使用正己烷再次过氧化铝柱沉淀,真空干燥。
8.一种如权利要求1所述的荧光探针在检测溶液、细胞或生物体中溶酶体pH的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910687350.2A CN110372590B (zh) | 2019-07-29 | 2019-07-29 | 一种检测溶酶体pH的荧光探针及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910687350.2A CN110372590B (zh) | 2019-07-29 | 2019-07-29 | 一种检测溶酶体pH的荧光探针及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110372590A true CN110372590A (zh) | 2019-10-25 |
CN110372590B CN110372590B (zh) | 2021-10-26 |
Family
ID=68256709
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910687350.2A Expired - Fee Related CN110372590B (zh) | 2019-07-29 | 2019-07-29 | 一种检测溶酶体pH的荧光探针及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110372590B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113281397A (zh) * | 2021-05-19 | 2021-08-20 | 中国科学技术大学 | 追踪单溶酶体中亲溶酶体内容物的方法 |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103408493A (zh) * | 2013-07-05 | 2013-11-27 | 东南大学 | 一种多臂萘酰亚胺荧光分子及其制备方法 |
CN107226783A (zh) * | 2017-06-20 | 2017-10-03 | 武汉工程大学 | 一种溶酶体靶向荧光探针及其制备方法 |
CN108148573A (zh) * | 2018-02-01 | 2018-06-12 | 济南大学 | 一种检测溶酶体pH的荧光探针及其合成方法和应用 |
CN108181288A (zh) * | 2018-02-10 | 2018-06-19 | 郑州大学 | 一种检测细胞内pH值的聚合物纳米荧光探针及其制备方法以及应用 |
WO2018136794A1 (en) * | 2017-01-20 | 2018-07-26 | Arizona Board Of Regents On Behalf Of Arizona State University | Fluorescent ph sensors and methods of preparing and using them |
CN108727265A (zh) * | 2018-06-07 | 2018-11-02 | 浙江工业大学 | 一种检测甲醛和pH的双功能荧光探针及其制备方法和应用 |
CN108752275A (zh) * | 2018-06-07 | 2018-11-06 | 浙江工业大学 | 一种pH荧光探针及其制备方法和应用 |
CN109096189A (zh) * | 2018-09-14 | 2018-12-28 | 济南大学 | 一种检测细胞内质网内pH的双光子荧光探针 |
CN109824592A (zh) * | 2018-06-07 | 2019-05-31 | 浙江工业大学 | 一种检测甲醛和pH的双功能荧光探针中间体及其制备方法和应用 |
-
2019
- 2019-07-29 CN CN201910687350.2A patent/CN110372590B/zh not_active Expired - Fee Related
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103408493A (zh) * | 2013-07-05 | 2013-11-27 | 东南大学 | 一种多臂萘酰亚胺荧光分子及其制备方法 |
WO2018136794A1 (en) * | 2017-01-20 | 2018-07-26 | Arizona Board Of Regents On Behalf Of Arizona State University | Fluorescent ph sensors and methods of preparing and using them |
CN107226783A (zh) * | 2017-06-20 | 2017-10-03 | 武汉工程大学 | 一种溶酶体靶向荧光探针及其制备方法 |
CN108148573A (zh) * | 2018-02-01 | 2018-06-12 | 济南大学 | 一种检测溶酶体pH的荧光探针及其合成方法和应用 |
CN108181288A (zh) * | 2018-02-10 | 2018-06-19 | 郑州大学 | 一种检测细胞内pH值的聚合物纳米荧光探针及其制备方法以及应用 |
CN108727265A (zh) * | 2018-06-07 | 2018-11-02 | 浙江工业大学 | 一种检测甲醛和pH的双功能荧光探针及其制备方法和应用 |
CN108752275A (zh) * | 2018-06-07 | 2018-11-06 | 浙江工业大学 | 一种pH荧光探针及其制备方法和应用 |
CN109824592A (zh) * | 2018-06-07 | 2019-05-31 | 浙江工业大学 | 一种检测甲醛和pH的双功能荧光探针中间体及其制备方法和应用 |
CN109096189A (zh) * | 2018-09-14 | 2018-12-28 | 济南大学 | 一种检测细胞内质网内pH的双光子荧光探针 |
Non-Patent Citations (7)
Title |
---|
BEI-YU LIU,等: "Novel biocompatible fluorescent polymeric micelles based on 1,8-naphthalimide derivatives for cell imaging", 《POLYM. CHEM》 * |
CHAO WANG,等: "Dual site‐controlled two‐photon fluorescent probe for the imaging of lysosomal pH in living cells", 《LUMINESCENCE.》 * |
HAITAO ZHANG,等: "Hydrogen Sulfide Triggered Charge-Reversal Micelles for Cancer-Targeted Drug Delivery and Imaging", 《ACS APPL. MATER. INTERFACES》 * |
NAN ZHANG,等: "A two-photon endoplasmic reticulum-targeting fluorescent probe for the imaging of pH in living cells and zebrafish", 《ANAL. METHODS》 * |
XUEJUAN WAN,等: "Fluorescent pH-Sensing Organic/Inorganic Hybrid Mesoporous Silica Nanoparticles with Tunable Redox-Responsive Release Capability", 《LANGMUIR》 * |
于法祺: "刺激响应性聚合物/无机杂化材料的制备及其性能研究", 《济南大学硕士学位论文》 * |
戚裕: "含罗丹明基团聚合物的荧光性能及其应用研究", 《苏州大学博士学位论文》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113281397A (zh) * | 2021-05-19 | 2021-08-20 | 中国科学技术大学 | 追踪单溶酶体中亲溶酶体内容物的方法 |
Also Published As
Publication number | Publication date |
---|---|
CN110372590B (zh) | 2021-10-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110283583B (zh) | γ-谷氨酰转肽酶响应型分子探针及其应用 | |
CN108982447B (zh) | 一种用于检测肼的比率式荧光探针的制备方法及应用 | |
CN104357044B (zh) | 一种荧光探针及其制备方法和应用 | |
CN108129428B (zh) | 一种检测亚硫酸氢根的比率荧光探针及其应用 | |
CN110563650B (zh) | 一种硫酸酯酶的比率型双光子荧光探针及其合成方法和应用 | |
CN112608734B (zh) | 一种检测碱性磷酸酶的复合荧光探针及其制备方法与应用 | |
CN106565966B (zh) | 一种铕基配位聚合物纳米球及其制备方法与应用 | |
Chen et al. | Quantitative image analysis method for detection of nitrite with cyanine dye-NaYF4: Yb, Tm@ NaYF4 upconversion nanoparticles composite luminescent probe | |
Das et al. | Turn on ESIPT based chemosensor for histidine: application in urine analysis and live cell imaging | |
CN110372590A (zh) | 一种检测溶酶体pH的荧光探针及其制备方法和应用 | |
Zhao et al. | A FRET-based ratiometric fluorescent probe for Hg2+ detection in aqueous solution and bioimaging in multiple samples | |
CN110438115A (zh) | 一种提高铅DNAzyme稳定性的固定化酶方法及应用 | |
Yin et al. | Reversible AIE self-assembled nanohybrids coordinated by La3+ for ratiometric visual acid phosphatase monitoring and intracellular imaging | |
CN101551382A (zh) | 细胞活力检测试剂盒及其制备方法、应用 | |
Chen et al. | Phosphorescence, fluorescence, and colorimetric triple-mode sensor for the detection of acid phosphatase and corresponding inhibitor | |
CN108426867A (zh) | 在水中检测Fe3+和抗生素头孢曲松钠的MOF-Cd探针及其制备方法和应用 | |
CN106518800A (zh) | 一种基于氢离子激活的双响应检测ClO‑/H2S荧光分子探针的制备方法及应用 | |
CN110143931A (zh) | 一种靶向溶酶体检测硫化氢的荧光探针及其应用 | |
CN114605376A (zh) | 一种检测半胱氨酸和粘度的双功能荧光探针及其制备 | |
CN108997255A (zh) | 一种乙烯基醚类Hg2+荧光探针及其制备方法和应用 | |
CN110229203B (zh) | 一种氨基己糖酶荧光探针及其制备方法和应用 | |
CN111072011B (zh) | 一种线粒体-核仁可逆迁移荧光碳点的制备及在监测细胞活性中的应用 | |
CN110105377B (zh) | 一种检测结核分枝杆菌β-内酰胺酶的探针化合物、制备方法、一种荧光探针 | |
CN113788821A (zh) | 近红外联氨化合物、制备方法以及甲醛检测试剂盒和应用 | |
CN109082271B (zh) | 一种基于苯并吡喃腈衍生物的荧光探针及其制备方法和应用 |
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: 20211026 |