CN113582985A - 一种线粒体靶向的pH和粘度双通道检测荧光探针及其制备方法和用途 - Google Patents
一种线粒体靶向的pH和粘度双通道检测荧光探针及其制备方法和用途 Download PDFInfo
- Publication number
- CN113582985A CN113582985A CN202110979379.5A CN202110979379A CN113582985A CN 113582985 A CN113582985 A CN 113582985A CN 202110979379 A CN202110979379 A CN 202110979379A CN 113582985 A CN113582985 A CN 113582985A
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- viscosity
- channel detection
- cmpv
- dual
- 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
- 210000003470 mitochondria Anatomy 0.000 title claims abstract description 33
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 19
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 12
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 10
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000005457 ice water Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- 235000010299 hexamethylene tetramine Nutrition 0.000 claims description 5
- 239000004312 hexamethylene tetramine Substances 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- -1 benzothiazole iodomethane onium compound Chemical class 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 claims description 3
- 230000009977 dual effect Effects 0.000 claims description 2
- 239000012043 crude product Substances 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 238000003756 stirring Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 33
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 7
- 238000001218 confocal laser scanning microscopy Methods 0.000 abstract description 4
- 231100000263 cytotoxicity test Toxicity 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- 230000035440 response to pH Effects 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 239000000523 sample Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 230000003834 intracellular effect Effects 0.000 description 9
- 230000002438 mitochondrial effect Effects 0.000 description 8
- 230000003247 decreasing effect Effects 0.000 description 7
- 229960000988 nystatin Drugs 0.000 description 7
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 7
- 239000012452 mother liquor Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000008045 co-localization Effects 0.000 description 4
- 238000002073 fluorescence micrograph Methods 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 210000003463 organelle Anatomy 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- DANUORFCFTYTSZ-UHFFFAOYSA-N epinigericin Natural products O1C2(C(CC(C)(O2)C2OC(C)(CC2)C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)C)C(C)C(OC)CC1CC1CCC(C)C(C(C)C(O)=O)O1 DANUORFCFTYTSZ-UHFFFAOYSA-N 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000008965 mitochondrial swelling Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- DANUORFCFTYTSZ-BIBFWWMMSA-N nigericin Chemical compound C([C@@H]1C[C@H]([C@H]([C@]2([C@@H](C[C@](C)(O2)C2O[C@@](C)(CC2)C2[C@H](CC(O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C)O1)C)OC)[C@H]1CC[C@H](C)C([C@@H](C)C(O)=O)O1 DANUORFCFTYTSZ-BIBFWWMMSA-N 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000002211 ultraviolet spectrum 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
- 230000002407 ATP formation Effects 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 238000002714 localization assay Methods 0.000 description 1
- 230000007762 localization of cell Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000025608 mitochondrion localization Effects 0.000 description 1
- 230000005287 neuromuscular process controlling balance Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 210000001519 tissue Anatomy 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
- C07D417/06—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 linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—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
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
技术领域
本发明涉及一种线粒体靶向的pH和粘度双通道检测荧光探针及其制备方法和用途,以实现在溶液中和细胞中线粒体靶向的pH和粘度双通道检测荧光成像,具有选择性专一、检测高效、生物相容性良好的优点。
背景技术
线粒体是真核细胞母系遗传的细胞质细胞器,是重要的生物能合成工厂。线粒体在细胞新陈代谢,器官、组织以及整个生命体内包括Ca2+平衡的调控、清除多余或者破损的细胞器和触发细胞死亡等方面都起着至关重要的作用。因此,细胞的存活需要维持正常的线粒体水平。线粒体缺陷或功能障碍与一些心血管和神经性疾病等有关。而且线粒体的微环境与其功能关系紧密,尤其是pH值和粘度直接影响其生化过程。例如线粒体表现出一种略碱性基质,穿过内膜的质子动力能驱动ATP合成、离子及代谢物吸收到基质中去。由此可见,线粒体动态变化和微环境的检测对线粒体生物学及相关疾病的研究具有重要意义。
到目前为止,已报道的pH和粘度测定方法包括核磁共振法(NMR)、吸收光谱法、表面增强拉曼散射法(SERS)、水凝胶等。而基于pH荧光探针的荧光成像方法因能排除其他信号分子的干扰,对pH具有特异性响应、灵敏度高和实时性等优点而备受关注。细胞器粘度是一种重要的微环境参数,它通过影响活细胞内生物分子和化学信号的相互作用和传递而发挥生物学功能。
近年来,荧光成像的方法已经被广泛应用于监测生物过程,但对细胞线粒体特异性成像还是非常少,特别是兼具粘度和pH双重检测功能,因此开发细胞pH和粘度双通道检测荧光探针是非常迫切和重要的。
发明内容
本发明旨在提供一种线粒体靶向的pH和粘度双通道检测荧光探针及其制备方法和用途,所要解决的技术问题是通过分子设计得到一种可以特异性靶向线粒体,且可以分别无干扰响应pH和粘度的有机小分子结构,可通过共聚焦荧光成像实时监测活细胞线粒体内pH和粘度的变化,具有选择性高、灵敏度高、光稳定性好等优点,以实现细胞线粒体的荧光成像。细胞毒性测试表明本发明pH和粘度检测荧光探针的细胞相容性良好。
本发明线粒体靶向的pH和粘度双通道检测荧光探针,简记为CMPV,以香豆素为母体,其结构式如下:
本发明线粒体靶向的pH和粘度双通道检测荧光探针的制备方法,包括如下步骤:
步骤1:将3g(18.5mmol)7-羟基香豆素和5mL乙酸酐溶解在25mL二氯甲烷中,25℃活化半小时再加入0.1mL哌啶继续反应1小时;反应结束后,加冰水洗涤,抽滤得到中间体1,为白色固体,3.02g,产率80%。
步骤2:在冰水浴条件下,在圆底烧瓶中加入磁子,20mL三氟乙酸(TFA)和3g(21.4mmol)六亚甲基四胺(HMTA),保持冰水浴搅拌半小时,室温下继续反应1小时,将2g(9.8mmol)中间体1溶解在5mL TFA中然后缓慢加入体系中,80℃回流(rf)反应48h;反应结束后,柱色谱法提纯得淡黄色固体中间体2(洗脱液为石油醚:乙酸乙酯=8:1,v/v),1.21g,产率65%。
步骤3:1.00g(5.2mmol)中间体2,1.5g(5.2mmol)苯并噻唑碘甲烷鎓化物,10mL无水乙醇作为溶剂,再滴加3滴哌啶,90℃回流反应14小时;反应结束后用丙酮洗,过滤得到灰绿色色固体目标产物CMPV。
本发明线粒体靶向的pH和粘度双通道检测荧光探针CMPV的合成过程如下:
本发明线粒体靶向的pH和粘度双通道检测荧光探针的用途,是用于制备特异性靶向线粒体的荧光探针,所述荧光探针能够分别无干扰地响应pH和粘度,从而通过共聚焦荧光成像实时监测活细胞线粒体内pH和粘度的变化。具体检测方法如下:
将本发明CMPV溶于DMSO中制得2mM的母液,各取15μL母液于3mL不同pH的PBS溶剂中,获得10μM CMPV在不同测试液中的荧光和紫外谱图。随着pH的降低,CMPV的吸光度在370nm升高500nm降低,荧光在451nm不断降低。以上结果为表明CMPV在溶液可有效检测pH,可以作为pH检测器。使用不同比例的甘油和蒸馏水配制不同粘度体系,加入15μL探针母液配制成10μM粘度测试体系,荧光在522nm不断升高,结果表明CMPV在溶液中可有效检测粘度。使用线粒体的商业染料MTDR与CMPV在HeLa细胞中进行共定位研究。结果表明CMPV与MTDR的荧光图像重叠良好,并且CMPV与MTDR的Pearson共定位系数计算为0.92。以上结果表明,CMPV可以很好地定位于活细胞的细胞线粒体。探究CMPV在细胞中微环境检测的能力,对细胞使用制霉菌素处理(线粒体肿胀剂)的样品进行处理,制霉菌素处理的细胞随着时间处理增加的情况下荧光逐渐增强。用10mM尼日利亚菌素和高钾离子缓冲溶液均质化细胞内pH,当均质化后不同pH的细胞内加入探针CMPV,共聚焦显微镜下探针的蓝色荧光随着细胞内的pH降低蓝色荧光随之减弱。结果表明,在细胞内CMPV可以检测细胞内粘度和pH。
本发明线粒体靶向的pH和粘度双通道检测探针,在溶液中和细胞中都可有效检测pH和粘度。细胞毒性测试表明CMPV的细胞相溶性良好,双光子共聚焦荧光显微成像实验表明CMPV可以有效定位细胞线粒体(定位系数为0.92),适用于细胞线粒体荧光成像。
附图说明
图1(a)CMPV(10μM)在不同pH的PBS缓冲溶液中的紫外吸收滴定光谱;(b)CMPV(10μM)在不同pH的PBS缓冲溶液中的荧光滴定光谱。
图2(a)CMPV(10μM)在不同pH的PBS缓冲溶液中的非线性拟合;(b)CMPV(10μM)在不同pH的PBS缓冲溶液中的线性拟合。
图3(a)CMPV(10μM)在不同水和甘油体系中的荧光发射光谱;(b)CMPV(10μM)在不同水和甘油体系中的荧光强度与粘度的线性关系。
图4是不同浓度(0μM、5μM、10μM、15μM、20μM)CMPV作用下的HeLa细胞存活率图。
图5是10μMCMPV和1μM商用细胞线粒体荧光探针(MTDR)同时共染HeLa细胞的细胞线粒体共聚焦荧光成像图。探究CMPV的细胞线粒体靶向能力。
图6是10μMCMPV在均质化细胞内pH的共聚焦荧光成像图。探究CMPV的细胞内pH检测能力。
图7是10μMCMPV和5μM制霉菌素共聚焦荧光成像图。探究CMPV在细胞中粘度检测的能力。
具体实施方式
下面通过具体的实施例对本发明技术方案做进一步说明。
实施例1:CMPV的合成
1、将3g(18.5mmol)7-羟基香豆素和5mL乙酸酐溶解在25mL二氯甲烷中,25℃活化半小时再加入0.1mL哌啶继续反应1小时;反应结束后,加冰水洗涤,抽滤得到中间体1,为白色固体,3.02g,产率80%。
2、在冰水浴条件下,在圆底烧瓶中加入磁子,20mL三氟乙酸(TFA)和3g(21.4mmol)六亚甲基四胺(HMTA),保持冰水浴搅拌半小时,室温下继续反应1小时,将2g(9.8mmol)中间体1溶解在5mL TFA中然后缓慢加入体系中,80℃回流(rf)反应48h;反应结束后,柱色谱法提纯得淡黄色固体中间体2(洗脱液为石油醚:乙酸乙酯=8:1,v/v)得淡黄色固体中间体2,1.21g,产率65%。
3、1.00g(5.2mmol)中间体2,1.5g(5.2mmol)苯并噻唑碘甲烷鎓化物,10mL无水乙醇作为溶剂,再滴加3滴哌啶,90℃回流反应14小时;反应结束后用丙酮洗,过滤得到灰绿色色固体目标产物CMPV。
1H NMR(400MHz,DMSO-d6,ppm)δ12.29(s,1H),8.41(d,J=8.4Hz,1H),8.30-8.27(m,3H),8.03(d,J=9.5Hz,1H),7.93-7.85(m,1H),7.80(t,J=7.7Hz,1H),7.73(d,J=8.7Hz,1H),7.00(d,J=8.7Hz,1H),6.36(d,J=9.5Hz,1H),4.26(s,3H).13C NMR(151MHz,DMSO-d6,ppm)δ173.25,163.25,159.96,154.84,145.47,142.66,137.33,133.75,130.03,128.93,128.24,124.80,117.38,116.62,114.19,112.21,111.68,109.16,36.68.
实施例2:CMPV在不同pH溶剂中的光谱测试
为了探究CMPV在溶剂中检测不同pH和粘度的紫外和荧光变化,将本发明CMPV溶于DMSO中制得2mM的母液,取15μL母液于3mL不同pH的PBS溶剂中,获得10μM CMPV在不同测试液中的紫外和荧光谱图(图1)。随着pH的降低,CMPV的吸光度在370nm升高500nm降低,荧光在451nm不断降低。以上结果为表明CMPV在溶液可有效检测pH,可以作为pH检测器。将pH与荧光值进行线性拟合图2发现探针在pH在6.5~8.5的荧光值和pH有很好的线性关系(R2=0.99)。
实施例3:CMPV在不同粘度溶剂中的光谱测试
将本发明CMPV溶于DMSO中制得2mM的母液,使用不同比例的甘油和蒸馏水配制不同粘度体系溶剂,加入15μL探针母液配制成10μM粘度测试体系,测试探针在不同粘度条件下荧光光谱。随着体系粘度的增大荧光最大发射在519nm不断升高,结果表明CMPV在溶液中可有效检测粘度。
实施例4:细胞毒性测试
我们用MTT(5-二甲基噻唑-2-基-2,5-二苯基四唑溴化物)方法进行了细胞毒性实验。CMPV在活HeLa细胞中加入各种浓度(0μM,5.0μM,10.0μM,15.0μM,20.0μM),孵育24小时后测试,结果如图5所示,以上显示CMPV的生物毒性较低,可以进行生物成像应用。
实施例5:细胞定位测试
为了研究CMPV的细胞线粒体定位性能,这里使用细胞线粒体的商业染料(MTDR)与CMPV在HeLa细胞中进行共定位研究。结果表明CMPV的蓝色通道(λem=420-460nm,λex=405nm)和MTDR(λem=650-670nm,λex=633nm)的荧光图像重叠良好,并且CMPV与MTDR的Pearson共定位系数计算为0.92(图5)。这些结果表明,CMPV可以很好地定位于活细胞的细胞线粒体中。
实施例6:CMPV在细胞线粒体上的pH检测实验
为了探究CMPV在细胞线粒体上的pH的检测能力,对探针CMPV与通过尼日利亚菌素和高钾离子缓冲溶液均质化细胞内pH的HeLa细胞共同孵育如图6,与HeLa细胞孵育30min后,CMPV通道Ⅰ的蓝色荧光(ChannelⅠ:λem=440±20nm.λex=405nm)随着细胞内的pH降低一同降低。这表示CMPV的适合监测细胞内的pH检测。
实施例7:CMPV在细胞中粘度检测的能力
制霉菌素(Nystatin)能够引起细胞线粒体肿胀,是一种公认的细胞线粒体增稠剂。为了探究CMPV在细胞中产生粘度检测的能力,制霉菌素处理的Hela细胞样品与探针共同孵育如图7。随着制霉菌素处理时间增加,细胞内绿色荧光明显增强(ChannelⅠ:λem=520±20nm.λex=458nm)。结果表明,在细胞内CMPV在制霉菌素处理的Hela细胞能很好地检测细胞线粒体粘度增加。
Claims (4)
2.一种权利要求1所述的线粒体靶向的pH和粘度双通道检测荧光探针的制备方法,其特征在于包括如下步骤:
步骤1:将7-羟基香豆素和乙酸酐溶解在二氯甲烷中,25℃活化半小时再加入哌啶继续反应1小时;反应结束后,加冰水洗涤,抽滤得到中间体1,为白色固体;
步骤2:在冰水浴条件下,在圆底烧瓶中加入磁子、三氟乙酸和六亚甲基四胺,保持冰水浴搅拌半小时,室温下继续反应1小时,将中间体1溶解在TFA中然后缓慢加入体系,80℃回流反应;反应结束后,柱色谱法提纯得淡黄色固体中间体2;
步骤3:将中间体2和苯并噻唑碘甲烷鎓化物加入溶剂无水乙醇中,再滴加哌啶,90℃回流反应;反应结束后用丙酮洗,过滤得到灰绿色色固体目标产物CMPV。
3.根据权利要求2所述的制备方法,其特征在于:
步骤2中,通过柱色谱法纯化粗产物时的洗脱液为石油醚:乙酸乙酯=8:1,v/v。
4.一种权利要求1所述的双通道检测荧光探针的用途,其特征在于:
所述双通道检测荧光探针用于制备特异性靶向线粒体的荧光探针,所述荧光探针能够分别无干扰地响应pH和粘度。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110979379.5A CN113582985B (zh) | 2021-08-25 | 2021-08-25 | 一种线粒体靶向的pH和粘度双通道检测荧光探针及其制备方法和用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110979379.5A CN113582985B (zh) | 2021-08-25 | 2021-08-25 | 一种线粒体靶向的pH和粘度双通道检测荧光探针及其制备方法和用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113582985A true CN113582985A (zh) | 2021-11-02 |
CN113582985B CN113582985B (zh) | 2022-09-13 |
Family
ID=78239521
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110979379.5A Active CN113582985B (zh) | 2021-08-25 | 2021-08-25 | 一种线粒体靶向的pH和粘度双通道检测荧光探针及其制备方法和用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113582985B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115650897A (zh) * | 2022-10-25 | 2023-01-31 | 广西师范大学 | 一种同时检测Cys和线粒体粘度的荧光探针及其制备方法和应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110078714A (zh) * | 2019-05-16 | 2019-08-02 | 济南大学 | 一种定位线粒体的双光子粘度探针及其制备方法和应用 |
-
2021
- 2021-08-25 CN CN202110979379.5A patent/CN113582985B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110078714A (zh) * | 2019-05-16 | 2019-08-02 | 济南大学 | 一种定位线粒体的双光子粘度探针及其制备方法和应用 |
Non-Patent Citations (3)
Title |
---|
CHA, SUNYOUNG; 等: "Negative solvatochromism of merocyanine dyes: Application as water content probes for organic solvents", 《SENSORS AND ACTUATORS, B: CHEMICAL》 * |
PENG, MENG-JIAO; 等: "A Hybrid Coumarin-Thiazole Fluorescent Sensor for Selective Detection of Bisulfite Anions in Vivo and in Real Samples", 《CHEMISTRY - AN ASIAN JOURNAL》 * |
陈 俊,等: "一种线粒体靶向香豆素基pH荧光探针合成及性能", 《发光学报》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115650897A (zh) * | 2022-10-25 | 2023-01-31 | 广西师范大学 | 一种同时检测Cys和线粒体粘度的荧光探针及其制备方法和应用 |
CN115650897B (zh) * | 2022-10-25 | 2024-03-15 | 广西师范大学 | 一种同时检测Cys和线粒体粘度的荧光探针及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN113582985B (zh) | 2022-09-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Liu et al. | An AIE+ ESIPT ratiometric fluorescent probe for monitoring sulfur dioxide with distinct ratiometric fluorescence signals in mammalian cells, mouse embryonic fibroblast and zebrafish | |
CN111253935B (zh) | 一种双通道检测极性和粘度的双光子荧光探针及其制备方法和用途 | |
CN108003869B (zh) | 一种高灵敏检测次氯酸根的荧光探针及其合成方法与应用 | |
CN111349070B (zh) | 一种用于检测生物细胞粘度的近红外荧光分子探针及其制备方法和应用 | |
CN108398409B (zh) | 一种荧光比率检测次氯酸根的方法 | |
Zhang et al. | A simple pyrazoline-based fluorescent probe for Zn2+ in aqueous solution and imaging in living neuron cells | |
CN112409238B (zh) | 苯并吲哚类化合物与制备方法及其检测半胱氨酸中的应用 | |
CN113563229B (zh) | 基于异佛尔酮-肉桂醛的粘度荧光探针的制备和应用 | |
CN111892923B (zh) | 一种基于二腈乙烯基的双光子荧光粘度探针及其制备方法和用途 | |
Liu et al. | Rhodamine based turn-on fluorescent sensor for Hg2+ and its application of microfluidic system and bioimaging | |
CN107286151B (zh) | 一种基于咔唑的双光子荧光探针及其制备方法和用途 | |
CN113717545A (zh) | 一种具有高亮度、高光稳定性的深红荧光染料及其合成方法 | |
CN114805262B (zh) | 一种粘度和极性响应型平台荧光探针、硫化氢检测荧光探针及其合成工艺与应用 | |
CN113582985B (zh) | 一种线粒体靶向的pH和粘度双通道检测荧光探针及其制备方法和用途 | |
CN113848197B (zh) | 一种荧光探针的用途 | |
Wu et al. | A near-infrared fluorescent probe of dicyanoisophorone derivatives for selective detection and fluorescence cellular imaging of Palladium | |
CN103382189B (zh) | 一类菁类化合物、其制备方法及应用 | |
CN109320537A (zh) | 一种用于面粉和活体内过氧苯甲酰检测的可溶性双光子荧光探针及其制备方法和应用 | |
CN106608862A (zh) | 一种长波长检测肼的荧光探针及其合成方法与应用 | |
CN113956274A (zh) | 一类对癫痫疾病中黏度和过氧化亚硝酸盐变化双响应的荧光探针设计和合成方法 | |
CN111349071A (zh) | 一种用于检测onoo-的氧杂蒽衍生物及其合成方法和应用 | |
KR102359333B1 (ko) | 디페닐보란 유도체를 포함하는 피루베이트 검출 센서, 이를 포함하는 피루베이트 검출용 형광 이미징 센서, 및 이를 이용한 피루베이트 검출 방법 | |
CN115141145A (zh) | 一种检测溶酶体次溴酸荧光探针、制备方法与应用 | |
CN110487761B (zh) | 检测汞离子的荧光探针及其制备方法与使用方法 | |
CN111087362B (zh) | 一种高选择性检测甲醛的荧光探针及其合成方法与应用 |
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 |