CN106674284A - 一种通过光调控精确检测线粒体内铜离子的荧光探针及其制备方法和应用 - Google Patents
一种通过光调控精确检测线粒体内铜离子的荧光探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN106674284A CN106674284A CN201611217246.XA CN201611217246A CN106674284A CN 106674284 A CN106674284 A CN 106674284A CN 201611217246 A CN201611217246 A CN 201611217246A CN 106674284 A CN106674284 A CN 106674284A
- Authority
- CN
- China
- Prior art keywords
- fluorescence probe
- fluorescein
- preparation
- light
- copper ion
- 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.)
- Pending
Links
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 229910001431 copper ion Inorganic materials 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- 239000007850 fluorescent dye Substances 0.000 title abstract 7
- 239000000523 sample Substances 0.000 claims abstract description 54
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 22
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical class O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 46
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 claims description 34
- 238000001514 detection method Methods 0.000 claims description 20
- 238000000746 purification Methods 0.000 claims description 20
- 238000010898 silica gel chromatography Methods 0.000 claims description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 18
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 16
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 16
- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 230000001105 regulatory effect Effects 0.000 claims description 10
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 230000001276 controlling effect Effects 0.000 claims description 8
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 8
- -1 fluorescein derivative compound Chemical class 0.000 claims description 7
- HXBMIQJOSHZCFX-UHFFFAOYSA-N 1-(bromomethyl)-2-nitrobenzene Chemical class [O-][N+](=O)C1=CC=CC=C1CBr HXBMIQJOSHZCFX-UHFFFAOYSA-N 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- 238000000967 suction filtration Methods 0.000 claims description 6
- 230000033228 biological regulation Effects 0.000 claims description 3
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 238000000034 method Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 12
- 210000000805 cytoplasm Anatomy 0.000 abstract description 5
- 230000035699 permeability Effects 0.000 abstract description 3
- 238000010226 confocal imaging Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 abstract description 2
- 230000002900 effect on cell Effects 0.000 abstract 1
- 230000000214 effect on organisms Effects 0.000 abstract 1
- 230000008685 targeting Effects 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 239000010949 copper Substances 0.000 description 9
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 8
- 238000005286 illumination Methods 0.000 description 7
- 238000002189 fluorescence spectrum Methods 0.000 description 6
- 229910021645 metal ion Inorganic materials 0.000 description 5
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-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
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 229960001866 silicon dioxide Drugs 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000011095 buffer preparation Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 0 CC(c(cccc1)c1N1OCC1)Oc1ccc(C(c2ccccc2C2=O)(c(ccc(OCCCC[P+](c3ccccc3)(c3ccccc3)C3=CCCC=C3)c3)c3O3)N2N)*3c1 Chemical compound CC(c(cccc1)c1N1OCC1)Oc1ccc(C(c2ccccc2C2=O)(c(ccc(OCCCC[P+](c3ccccc3)(c3ccccc3)C3=CCCC=C3)c3)c3O3)N2N)*3c1 0.000 description 1
- GOUJZNFZGIZBHQ-UHFFFAOYSA-N CC(c1ccccc1[N+]([O-])=O)Oc1ccc(C(c2ccccc2C2=O)(c(c(O3)c4)ccc4OCCCCBr)N2N)c3c1 Chemical compound CC(c1ccccc1[N+]([O-])=O)Oc1ccc(C(c2ccccc2C2=O)(c(c(O3)c4)ccc4OCCCCBr)N2N)c3c1 GOUJZNFZGIZBHQ-UHFFFAOYSA-N 0.000 description 1
- 102100030497 Cytochrome c Human genes 0.000 description 1
- 108010075031 Cytochromes c Proteins 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009740 ion physiology Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 230000008811 mitochondrial respiratory chain Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
- C07F9/65615—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing a spiro condensed ring system of the formula where at least one of the atoms X or Y is a hetero atom, e.g. S
-
- 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
- G01N21/6486—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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Materials Engineering (AREA)
- Biomedical Technology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
本发明涉及一种通过光调控精确检测线粒体内铜离子的荧光探针及其制备方法和应用，属于有机荧光探针领域。所述的荧光探针为Mito‑Cu2+荧光探针，Mito‑Cu2+荧光探针的结构式如(Ⅰ)所示。本发明荧光探针能够精确检测线粒体内的铜离子而避免细胞质内的铜离子的干扰，另外，该探针具有较好的线粒体靶向性、化学稳定性、生物兼容性和选择性等特点。激光共聚焦成像实验表明该探针具有较好的细胞通透性，对细胞和生物体无毒副作用。
Description
技术领域
本发明涉及一种通过光调控精确检测线粒体内铜离子的荧光探针及其制备方法和应用，属于有机荧光探针领域。
背景技术
在线粒体中，铜离子是一个重要的微量元素,是一系列重要生理酶中不可分割的一部分,如运输和催化功能的酶。当线粒体内的铜离子失衡会破坏线粒体呼吸链，会表现出活性氧簇化合物含量升高、线粒体膜电位下降、脂质过氧化、细胞色素c的释放等一系列细胞凋亡的征兆。铜离子和蛋白质的相互作用也被视为神经变性疾病的信号，如帕金森和阿尔茨海默病。对线粒体内Cu2+的精确检测，有助于我们进一步了解铜离子在神经退行性疾病中的起到的作用，有助于增进我们对线粒体内铜离子生理过程的了解，为神经疾病病理的研究提供一个线粒体内铜离子可视化工具。
线粒体在生命中的重要角色，促使它一直作为科学研究中的重中之重。在线粒体的研究中，多以荧光探针主，一个理想的荧光探针是无数个科学家的梦想，至今已经报道出来了很多荧光探针，如检测活性氧簇、活性氮簇、金属离子，生物小分子等的荧光探针，但是这些荧光探针都存在一个致命的缺点就是在经过细胞质靶向线粒体的过程中不能避免来自细胞质内的被检测物的干扰，造成研究结果缺乏说服力。为了解决这一存在已久的问题，急需研发一种可以定位线粒体又能避免细胞质内被检测物的荧光探针。
发明内容
为了解决以上的问题，本发明提供一种可定位活细胞中线粒体，并通过光调控精确检测线粒体内铜离子的荧光探针及其制备方法和应用。
本发明采用的技术方案是：一种通过光调控精确检测线粒体内铜离子的荧光探针，所述的荧光探针为Mito-Cu2+荧光探针，Mito-Cu2+荧光探针的结构式如(Ⅰ)所示：
本发明采用的另一技术方案是：所述的通过光调控精确检测线粒体内的铜离子荧光探针的制备方法，所述的Mito-Cu2+荧光探针的制备方法包括如下步骤：将荧光素衍生化合物和三苯基膦依次加入到甲苯中，加热回流8-16小时，冷却抽滤，除去溶剂，经硅胶柱层析提纯得到目标产物Mito-Cu2+荧光探针，反应式如下：
优选的，所述荧光素衍生化合物和三苯基膦的摩尔比为1:1.2-2。
优选的，所述的荧光素衍生物的制备方法如下：
(1)将荧光素溶解在甲醇中，滴加的水合肼，回流8-14小时，除去溶剂，经硅胶柱层析提纯得到荧光素酰肼；
(2)将荧光素酰肼和2-硝基苄溴溶到DMF中，加入碳酸铯常温6-13个小时，水洗萃取，经硅胶柱层析提纯得到光保护荧光素酰肼；
(3)将光保护荧光素酰肼和1，4-二溴丁烷溶到DMF中，加入碳酸铯和碘化钾常温反应10-24个小时，水洗萃取，经硅胶柱层析提纯得到荧光素衍生物。
优选的，所述步骤(1)中荧光素与水合肼的质量比为1:4.2-5。
优选的，所述步骤(2)中荧光素酰肼、2-硝基苄溴和碳酸铯的摩尔比为1:1-1.5:4。
优选的，所述步骤(3)中光保护荧光素酰肼、1，4-二溴丁烷和碳酸铯的摩尔比为1:1.2-1.5:4。
本发明采用的另一技术方案是：一种通过光调控精确检测线粒体内铜离子荧光探针的应用，所述的荧光探针可应用于生理体系中精确检测线粒体内的铜离子。
有益效果：
本发明的荧光探针具有较好的线粒体靶向性、化学稳定性、生物兼容性和铜离子选择性。激光共聚焦成像实验表明该探针具有较好的细胞通透性，对细胞和生物体无毒副作用。
本发明的荧光探针在可应用于细胞体系中通过光调控精确检测线粒体内的铜离子而避免来自细胞质中铜离子的干扰的应用。
本发明的精确测线粒体内铜离子的荧光探针可用于活细胞线粒体铜离子的精确检测，主要用的活细胞为Hela细胞株、MCF-7细胞株或者RAW264.7细胞株。
附图说明
图1是实施例1制备的Mito-Cu2+(5uM)UV光照5分钟后对不同Cu(NO3)2浓度Hepes缓冲溶液(0.2uM-20uM)的荧光光谱图。
图2是实施例1制备的Mito-Cu2+(5uM)UV光照5分钟后对20uM Cu(NO3)2浓度Hepes缓冲溶液不同响应时间的荧光光谱图。
图3是实施例1制备的Mito-Cu2+(5uM)UV光照5分钟后对20当量不同金属离子浓度Hepes缓冲溶液的荧光光谱图。
图4是实施例1制备的Mito-Cu2+(5uM)在细胞线粒体中的共聚焦显微成像。
具体实施方式
实施例1
通过光调控精确测线粒体内铜离子的荧光探针的制备方法如下：
(1)荧光素酰肼的制备：将1g荧光素溶解在50mL甲醇中，滴加4mL的水合肼，回流10小时，除去溶剂，经硅胶柱层析提纯得到荧光素酰肼；
(2)光保护荧光素酰肼的制备：将108mg荧光素酰肼和82mg 2-硝基苄溴溶到3mLDMF中，加入405mg碳酸铯常温6个小时，水洗萃取，经硅胶柱层析提纯得到光保护荧光素酰肼；
(3)光保护荧光素衍生物的制备：将光保护荧光素酰肼253mg和1，4-二溴丁烷131.7mg溶到4mL DMF中，加入碳酸铯664.4mg和碘化钾催化量常温反应12个小时，水洗萃取，经硅胶柱层析提纯得到荧光素衍生物。1H NMR(300MHz,CDCl3):δ8.02(d,J＝4.8Hz,1H),7.92(d,J＝1.5Hz,1H),7.75(m,1H),7.58(t,J＝4.5Hz,1H),7.42(m,3H),7.00(t,J＝3.9Hz,1H),6.69(s,1H),6.62(d,J＝9.9Hz,1H),6.51(d,J＝6.9Hz,5H),6.03(t,J＝3.3Hz,1H),3.98(t,J＝3.45Hz,2H),3.66(s,2H),3.46(t,J＝3.75Hz,2H),2.03(m,2H),1.92(m,2H),1.69(d,J＝3.9Hz,3H).13C NMR(75MHz,CDCl3):δ166.36,160.00,158.37,153.14,150.92,147.47,138.70,134.27,132.92,129.76,128.75,128.57,128.49,128.24,127.52,124.88,123.81,123.30,122.92,112.70,112.47,112.13,111.44,110.60,103.15,101.68,71.84,67.24,65.30,33.32,29.46,27.82,23.63.
(4)Mito-Cu2+荧光探针的制备：将摩尔比1:1.2的荧光素衍生化合物和三苯基膦依次加入到甲苯中，加热回流12个小时，冷却抽滤，除去溶剂，经硅胶柱层析提纯得到目标产物Mito-Cu2+荧光探针。1H NMR(300MHz,DMSO-d6):δ8.05(d,J＝4.8Hz,1H),7.74(m,17H),7.55(m,3H),7.48(s,1H),6.95(d,J＝3Hz,1H),6.41-6.50(s,1H),6.59(m,2H)6.53(m,3H),5.59(d,J＝3.9Hz,1H),4.42(bs,2H),4.06(t,J＝3.45Hz,2H),3.69(t,J＝6.75Hz,2H),1.91(m,2H),1.72(m,2H),1.65(d,J＝3.9Hz,3H).13C NMR(75MHz,DMSO-d6):δ165.49,159.03,157.46,152.13,151.00,147.39,136.87,134.85,134.18,133.56,133.43,132.64,130.24,130.08,129.14,128.57,128.26,127.82,127.28,124.52,123.25,122.43,118.97,117.83,112.29,111.87,111.37,102.31,101.30,71.23,66.24,64.30,28.77,22.90,19.33,18.33.IT-TOF-MS:m/z[M+H]+calcd:812.29,found:812.28.
实施例2
通过光调控精确测线粒体内铜离子的荧光探针的制备方法如下：
(1)荧光素酰肼的制备：将0.8g荧光素溶解在50mL甲醇中，滴加3.7mL的水合肼，回流12小时，除去溶剂，经硅胶柱层析提纯得到荧光素酰肼；
(2)光保护荧光素酰肼的制备：将158mg荧光素酰肼和125mg 2-硝基苄溴溶到4mLDMF中，加入593.6mg碳酸铯常温10个小时，水洗萃取，经硅胶柱层析提纯得到光保护荧光素酰肼；
(3)光保护荧光素衍生物的制备：将光保护荧光素酰肼100mg和1，4-二溴丁烷57mg溶到2mL DMF中，加入碳酸铯267mg和碘化钾催化量常温反应16个小时，水洗萃取，经硅胶柱层析提纯得到荧光素衍生物
(4)Mito-Cu2+荧光探针的制备：将摩尔比1:1.6的荧光素衍生化合物和三苯基膦依次加入到甲苯中，加热回流14个小时，冷却抽滤，除去溶剂，经硅胶柱层析提纯得到目标产物Mito-Cu2+荧光探针。
实施例3
通过光调控精确测线粒体内铜离子的荧光探针制备方法如下：
(1)荧光素酰肼的制备：将1g荧光素溶解在50mL甲醇中，滴加4.5mL的水合肼，回流14小时，除去溶剂，经硅胶柱层析提纯得到荧光素酰肼；
(2)光保护荧光素酰肼的制备：将200mg荧光素酰肼和185.3mg 2-硝基苄溴溶到4mL DMF中，加入751.4mg碳酸铯常温12个小时，水洗萃取，经硅胶柱层析提纯得到光保护荧光素酰肼；
(3)光保护荧光素衍生物的制备：将光保护荧光素酰肼123mg和1，4-二溴丁烷76mg溶到3mL DMF中，加入碳酸铯323mg和碘化钾催化量常温反应20个小时，水洗萃取，经硅胶柱层析提纯得到荧光素衍生物
(4)Mito-Cu2+荧光探针的制备：将摩尔比1:1.75的荧光素衍生化合物和三苯基膦依次加入到甲苯中，加热回流15个小时，冷却抽滤，除去溶剂，经硅胶柱层析提纯得到目标产物Mito-Cu2+荧光探针。
实施例4
通过光调控精确测线粒体内铜离子的荧光探针制备方法如下：
(1)荧光素酰肼的制备：将1g荧光素溶解在50mL甲醇中，滴加4.8mL的水合肼，回流14小时，除去溶剂，经硅胶柱层析提纯得到荧光素酰肼；
(2)光保护荧光素酰肼的制备：将120mg荧光素酰肼和119mg 2-硝基苄溴溶到4mLDMF中，加入450mg碳酸铯常温13个小时，水洗萃取，经硅胶柱层析提纯得到光保护荧光素酰肼；
(3)光保护荧光素衍生物的制备：将光保护荧光素酰肼85mg和1，4-二溴丁烷55.3mg溶到2mL DMF中，加入碳酸铯223mg和碘化钾催化量常温反应20个小时，水洗萃取，经硅胶柱层析提纯得到荧光素衍生物
(4)Mito-Cu2+荧光探针的制备：将摩尔比1:2的荧光素衍生化合物和三苯基膦依次加入到甲苯中，加热回流16个小时，冷却抽滤，除去溶剂，经硅胶柱层析提纯得到目标产物Mito-Cu2+荧光探针。
实施例5
通过光调控精确测线粒体内铜离子的荧光探针对铜离子的响应检测
1、Mito-Cu2+对不同铜离子浓度的荧光响应(图1)：
首先配制浓度25mM的Hepes缓冲溶液(pH＝7.35),用缓冲溶液配制5uMMito-Cu2+的铜离子探针，UV光照5分钟后,加入0.2-20当量的铜离子，用荧光分光光度法测试，并绘制Mito-Cu2+的铜离子探针对不同当量铜离子的荧光光谱图。
2、Mito-Cu2+对20当量铜离子不同响应时间的荧光响应(图2)：
用缓冲溶液配制5uM Mito-Cu2+的铜离子探针，UV光照5分钟后,加入20当量的铜离子，用荧光分光光度法分别测试响应5分钟、10分钟、20分钟、30分钟、40分钟、50分钟、60分钟的荧光强度，并绘制Mito-Cu2+对20当量铜离子不同响应时间的荧光光谱图。
3、Mito-Cu2+对20当量不同金属离子的荧光响应(图3)：
用缓冲溶液配制10uM Mito-Cu2+的铜离子探针，UV光照5分钟后,加入20当量的不同金属离子，用荧光分光光度法分别测试，并绘制Mito-Cu2+的铜离子探针对不同金属离子的荧光光谱图。
4、Mito-Cu2+在细胞线粒体中的共聚焦显微成像(图4)：
向含有Hela细胞的培养皿中加Mito-Cu2+DMSO溶液，与细胞培养液混合均匀后，使Mito-Cu2+在培养液中的浓度为5uM，染色1个小时后，用pH＝7.35的缓冲溶液冲洗3次，将该培养皿在共聚焦显微镜下成像，取出培养皿，UV光照5分钟后，静置1个小时，再次将该培养皿在共聚焦显微镜下成像。
实验结果表明，Mito-Cu2+具有良好的细胞通透性、很好线粒体靶向性。证明了通过光调控的方法控制探针和铜离子的反应是非常成功的，实现了精确检测线粒体内的铜离子而不受细胞质内铜离子的干扰。
本发明的不局限于上述实施例所述的具体技术方案，凡采用等同替换形成的技术方案均为本发明要求的保护范围。
Claims (8)
1.一种通过光调控精确检测线粒体内铜离子的荧光探针，其特征在于，所述的荧光探针为Mito-Cu2+荧光探针，Mito-Cu2+荧光探针的结构式如(Ⅰ)所示：
2.一种如权利要求1所述的通过光调控精确检测线粒体内的铜离子荧光探针的制备方法，其特征在于，所述的Mito-Cu2+荧光探针的制备方法包括如下步骤：将荧光素衍生化合物和三苯基膦依次加入到甲苯中，加热回流8-16小时，冷却抽滤，除去溶剂，经硅胶柱层析提纯得到目标产物Mito-Cu2+荧光探针，反应式如下：
3.根据权利要求2所述的通过光调控精确检测线粒体内铜离子荧光探针的制备方法，其特征在于，所述荧光素衍生化合物和三苯基膦的摩尔比为1:1.2-2。
4.根据权利要求2所述的通过光调控精确检测线粒体内铜离子荧光探针的制备方法，其特征在于，所述的荧光素衍生物的制备方法如下：
(1)将荧光素溶解在甲醇中，滴加的水合肼，回流8-14小时，除去溶剂，经硅胶柱层析提纯得到荧光素酰肼；
(2)将荧光素酰肼和2-硝基苄溴溶到DMF中，加入碳酸铯常温6-13个小时，水洗萃取，经硅胶柱层析提纯得到光保护荧光素酰肼；
(3)将光保护荧光素酰肼和1，4-二溴丁烷溶到DMF中，加入碳酸铯和碘化钾常温反应10-24个小时，水洗萃取，经硅胶柱层析提纯得到荧光素衍生物。
5.根据权利要求4所述的通过光调控精确检测线粒体内铜离子荧光探针的制备方法，其特征在于：所述步骤(1)中荧光素与水合肼的质量比为1:4.2-5。
6.根据权利要求4所述的通过光调控精确检测线粒体内的铜离子荧光探针的制备方法，其特征在于：所述步骤(2)中荧光素酰肼、2-硝基苄溴和碳酸铯的摩尔比为1:1-1.5:4。
7.根据权利要求4所述的通过光调控精确检测线粒体内铜离子荧光探针的制备方法，其特征在于：所述步骤(3)中光保护荧光素酰肼、1，4-二溴丁烷和碳酸铯的摩尔比为1:1.2-1.5:4。
8.一种如权利要求1所述的通过光调控精确检测线粒体内铜离子荧光探针的应用，所述的荧光探针可应用于生理体系中精确检测线粒体内的铜离子。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611217246.XA CN106674284A (zh) | 2016-12-26 | 2016-12-26 | 一种通过光调控精确检测线粒体内铜离子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 |
|---|---|---|---|
| CN201611217246.XA CN106674284A (zh) | 2016-12-26 | 2016-12-26 | 一种通过光调控精确检测线粒体内铜离子的荧光探针及其制备方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106674284A true CN106674284A (zh) | 2017-05-17 |
Family
ID=58870545
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611217246.XA Pending CN106674284A (zh) | 2016-12-26 | 2016-12-26 | 一种通过光调控精确检测线粒体内铜离子的荧光探针及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106674284A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108358966A (zh) * | 2018-03-05 | 2018-08-03 | 南京工业大学 | 一种新型靶向-增强线粒体功能药物Mito-VB3及其制备方法和应用 |
| CN112341472A (zh) * | 2020-11-20 | 2021-02-09 | 济南大学 | 一种酪氨酸酶激活的双淬灭诊疗前药及其制备 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100234379A1 (en) * | 2005-07-28 | 2010-09-16 | Bahr Ben A | Dual modulation of endocannabinoid transport and fatty-acid amide hydrolase for treatment of excitotoxicity |
-
2016
- 2016-12-26 CN CN201611217246.XA patent/CN106674284A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100234379A1 (en) * | 2005-07-28 | 2010-09-16 | Bahr Ben A | Dual modulation of endocannabinoid transport and fatty-acid amide hydrolase for treatment of excitotoxicity |
Non-Patent Citations (3)
| Title |
|---|
| XULIN XIA等: "The synthesis and properties of novel, functional azobenzene based metal complexes", 《DYES AND PIGMENTS》 * |
| YUAN LIN等: "Photocontrollable Analyte-Responsive Fluorescent Probes: A Photocaged Copper-Responsive Fluorescence Turn-On Probe", 《CHEM. EUR. J.》 * |
| 姜娜等: "线粒体荧光探针最新研究进展", 《化工学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108358966A (zh) * | 2018-03-05 | 2018-08-03 | 南京工业大学 | 一种新型靶向-增强线粒体功能药物Mito-VB3及其制备方法和应用 |
| CN108358966B (zh) * | 2018-03-05 | 2020-04-28 | 南京工业大学 | 一种靶向-增强线粒体功能药物Mito-VB3及其制备方法和应用 |
| CN112341472A (zh) * | 2020-11-20 | 2021-02-09 | 济南大学 | 一种酪氨酸酶激活的双淬灭诊疗前药及其制备 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Preparation of a Nile Red–Pd-based fluorescent CO probe and its imaging applications in vitro and in vivo | |
| Lu et al. | A simple two-output near-infrared fluorescent probe for hydrazine detection in living cells and mice | |
| Li et al. | A benzothiazole-based fluorescent probe for efficient detection and discrimination of Zn2+ and Cd2+, using cysteine as an auxiliary reagent | |
| Wang et al. | A novel DCM-NBD conjugate fluorescent probe for discrimination of Cys/Hcy from GSH and its bioimaging applications in living cells and animals | |
| CN104220438B (zh) | 用于检测过氧亚硝酸盐的二芳基胺‑基荧光探针 | |
| CN106279278B (zh) | 一种具有线粒体靶向和双光子性质的硫化氢分子荧光探针及其制备方法和应用 | |
| US20090219526A1 (en) | Reagents for biomolecular labeling, detection and quantification employing raman spectroscopy | |
| CN100410261C (zh) | 季胺基团修饰水溶性手性席夫碱金属配合物及其合成方法 | |
| EP2778161B1 (en) | Two-photon fluorescent probe using naphthalene as matrix and preparation method and use thereof | |
| CN106518855B (zh) | 一种以半川菁及黄酮醇为荧光团的二氧化硫衍生物比例荧光探针及其应用 | |
| CN106046059B (zh) | 一种具有线粒体靶向功能的磷光铱配合物探针及其制备和应用 | |
| Lan et al. | A new dicyanoisophorone-based ratiometric and colorimetric near-infrared fluorescent probe for specifically detecting hypochlorite and its bioimaging on a model of acute inflammation | |
| CN106220663A (zh) | 一种过氧化氢荧光探针化合物的制备与应用 | |
| CN106967102A (zh) | 一种基于罗丹明衍生物的过氧化氢增强型荧光探针 | |
| CN109266331A (zh) | 一种基于半花菁结构测次氯酸根离子的近红外荧光探针、其制备方法及应用 | |
| CN106634968B (zh) | 一种线粒体靶向的粘度荧光探针及其制备方法和应用 | |
| CN104177341A (zh) | 一种检测二价铜离子的化合物及其制备与应用 | |
| Zhou et al. | Fluorescence turn-on detection of fluoride using HPQ-silyl ether reactive probes and its in vivo application | |
| CN109897625A (zh) | 选择性检测半胱氨酸荧光探针及其合成方法和应用 | |
| CN106674284A (zh) | 一种通过光调控精确检测线粒体内铜离子的荧光探针及其制备方法和应用 | |
| García et al. | Design of fluorescent coumarin-hydroxamic acid derivatives as inhibitors of HDACs: Synthesis, anti-proliferative evaluation and docking studies | |
| Cui et al. | A rhodamine B-based turn on fluorescent probe for selective recognition of mercury (II) ions | |
| CN109851622B (zh) | 一种靶向溶酶体的次氯酸根荧光探针 | |
| CN108003173A (zh) | 一种特异性识别水合肼的荧光探针 | |
| CN105906619B (zh) | 一种双光子荧光探针及其制备方法和用途 |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170517 |