CN108329301A - 一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途 - Google Patents
一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途 Download PDFInfo
- Publication number
- CN108329301A CN108329301A CN201810256444.XA CN201810256444A CN108329301A CN 108329301 A CN108329301 A CN 108329301A CN 201810256444 A CN201810256444 A CN 201810256444A CN 108329301 A CN108329301 A CN 108329301A
- Authority
- CN
- China
- Prior art keywords
- photon
- autophagy
- probe
- cell
- fluorescence probe
- 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
- 230000004900 autophagic degradation Effects 0.000 title claims abstract description 37
- 238000012544 monitoring process Methods 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000007850 fluorescent dye Substances 0.000 title abstract description 12
- 238000005259 measurement Methods 0.000 title abstract 3
- 210000004027 cell Anatomy 0.000 claims abstract description 64
- 239000000523 sample Substances 0.000 claims abstract description 53
- 210000003712 lysosome Anatomy 0.000 claims abstract description 18
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- 230000001868 lysosomic effect Effects 0.000 claims description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- 239000012452 mother liquor Substances 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- UJOBWOGCFQCDNV-UHFFFAOYSA-N 9H-carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 claims description 4
- 229940126062 Compound A Drugs 0.000 claims description 4
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 claims description 4
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 3
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 3
- 238000010792 warming Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- HYZJCKYKOHLVJF-UHFFFAOYSA-N 1H-benzimidazole Chemical compound C1=CC=C2NC=NC2=C1 HYZJCKYKOHLVJF-UHFFFAOYSA-N 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 230000002132 lysosomal effect Effects 0.000 claims description 2
- 230000004044 response Effects 0.000 claims description 2
- 238000001228 spectrum Methods 0.000 claims description 2
- AXQNJCVTWOBBNH-UHFFFAOYSA-N 2-methoxyethynylbenzene Chemical group COC#CC1=CC=CC=C1 AXQNJCVTWOBBNH-UHFFFAOYSA-N 0.000 claims 1
- 239000000284 extract Substances 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 6
- 231100000263 cytotoxicity test Toxicity 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 230000035699 permeability Effects 0.000 abstract description 2
- 230000008045 co-localization Effects 0.000 abstract 1
- 230000002900 effect on cell Effects 0.000 abstract 1
- 238000000799 fluorescence microscopy Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 8
- 230000005284 excitation Effects 0.000 description 8
- 238000003384 imaging method Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000004083 survival effect Effects 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
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 108010037721 cytase Proteins 0.000 description 3
- 230000005283 ground state Effects 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 210000003463 organelle Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- PFTCBBOADJQVTP-UHFFFAOYSA-N acetic acid;boric acid;phosphoric acid Chemical compound CC(O)=O.OB(O)O.OP(O)(O)=O PFTCBBOADJQVTP-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 235000003642 hunger Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000037351 starvation Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- KBIAVTUACPKPFJ-UHFFFAOYSA-N 1-ethynyl-4-methoxybenzene Chemical class COC1=CC=C(C#C)C=C1 KBIAVTUACPKPFJ-UHFFFAOYSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- UWSONZCNXUSTKW-UHFFFAOYSA-N 4,5-Dimethylthiazole Chemical compound CC=1N=CSC=1C UWSONZCNXUSTKW-UHFFFAOYSA-N 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004957 autophagosome Anatomy 0.000 description 1
- 125000003785 benzimidazolyl group Chemical group N1=C(NC2=C1C=CC=C2)* 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000005061 intracellular organelle Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical group 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
-
- 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
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途，其中监测细胞自噬的双光子pH比率计量荧光探针的结构如下：本发明荧光探针能够对pH有专一性的荧光信号响应。本发明通过细胞共定位实验确信其能够专一性靶向细胞溶酶体，细胞毒性测试表明本发明对于细胞几乎没有什么毒副作用，双光子共聚焦荧光显微成像实验表明本发明对于MCF‑7细胞的渗透性良好，同时，经计算本发明荧光探针的pKa为3.88，非常适合细胞溶酶体pH变化范围的监测，能够通过检测细胞溶酶体pH的变化实时监测细胞自噬过程进行的情况。
Description
技术领域
本发明涉及一种双光子比率型荧光探针，具体地说是一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途。
背景技术
自噬是细胞在受到破坏性刺激时吞噬自身细胞质蛋白或细胞器并内陷成囊泡进而与溶酶体融合形成自噬溶酶体，降解其所包裹的内容物的过程。细胞自噬是细胞成分降解和回收利用的基础，在细胞中起着“清道夫”的作用，作为细胞内细胞器和其它结构自然代谢的正常途径当组织细胞受到各种理化因素伤害时，自噬溶酶体大量增加，对细胞的损伤起一种保护作用。传统的自噬监测包括透射电子显微镜(TEM)，Western印迹(Atg8/LC3)和GFP-Atg8/LC3荧光显微镜。然而这些监测手段都有其局限性，如扫描电镜和蛋白免疫印迹，它们并不能监测活细胞里的自噬状态，同时，这些方法监测的结果可视化效果并不理想，因此，一种能够形象而又简易的监测自噬的方法对细胞自噬过程的研究十分必要。
溶酶体是真核细胞中一种单层膜囊状结构的细胞器，内含多种水解酶，在细胞中主要作用是分解从外界进入到细胞内的物质及消化细胞自身的局部细胞质或细胞器，当细胞衰老时，其溶酶体破裂，释放出水解酶，消化整个细胞而使其死亡。由于细胞微环境(如极性、pH、粘度)在溶酶体和自噬体中非常不同，所以当细胞发生自噬时两者融合形成的自噬溶酶体内的微环境势必发生变化，因此，我们可以通过检测该过程中细胞微环境的变化来监测细胞自噬发生的情况。
pH是细胞中生化反应平衡的重要影响因素，作为生物体系微环境中的一个重要性质，影响着细胞正常生命活动机制的运行以及生物分子的顺利转换。不正常的细胞pH变化与生物体系中多种疾病及生理过程有紧密的联系。
双光子荧光探针作为一种测试工具，当测试物质性质或所处环境发生变化时荧光信号就会发生相应改变，其灵敏度高，操作方便，稳定性高，光漂白性小，细胞穿透性强在细胞层面定性分析时展现出优越的性能。因此，可以选择双光子荧光探针通过检测细胞溶酶体内pH的变化来监测细胞自噬过程。
发明内容
本发明旨在提供一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途。本发明通过分子设计遴选出一种合适的荧光探针结构，以实现双光子成像定性检测细胞溶酶体pH变化。本发明荧光探针专一性强，灵敏度高，细胞毒性实验表明本发明荧光探针对细胞几乎没有毒副作用。
本发明监测细胞自噬的双光子pH比率计量荧光探针(Lyso-MPBC)，简称为荧光探针，是以咔唑为母体，4-甲氧基苯乙炔为供电子基，溶酶体定位基团为吗啉，苯并咪唑为pH的响应基团，其结构式如下：
本发明监测细胞自噬的双光子pH比率计量荧光探针的制备方法，包括如下步骤：
将化合物A1g、邻苯二胺0.46g、对甲苯磺酸0.0082g以及N,N-二甲基甲酰胺30mL加入到三口烧瓶中，在油浴锅中升温至120℃并通氩气保护反应12h；反应结束后将液体旋干，用二氯甲烷和水萃取，收集上层有机相并旋干得到粗产物；将所得粗产物制样用柱层析色谱柱分离，得到目标产物0.7g(1.28mmoL)，收率60％。
所述化合物A的结构式为：
柱层析色谱柱分离时所用洗脱液为二氯甲烷和甲醇按体积比100:1混合得到。
本发明监测细胞自噬的双光子pH比率计量荧光探针Lyso-MPCB的合成过程如下：
本发明监测细胞自噬的双光子pH比率计量荧光探针Lyso-MPCB的用途，是在检测细胞中溶酶体pH时作为检测试剂使用。本发明荧光探针(Lyso-MPCB)可以通过检测溶酶体pH变化来监测细胞自噬过程。
将本发明荧光探针溶于DMSO中制得1mM的母液，取100μL的该母液于10mL容量瓶中，再用不同pH值的待测溶液(磷酸-醋酸-硼酸溶液体系，通过加入1mM盐酸或氢氧化钠溶液调至不同pH值)定容，配制成10μM。荧光探针单光子和双光子的激发波长分别为370nm和760nm，检测390-700nm波长范围内的荧光光谱变化，可以看到随着缓冲溶液的pH从碱性9.60降到酸性3.20，荧光最大发射峰从410nm红移至475nm，共65nm，且荧光强度红移增强。
本发明荧光探针的作用机理是荧光探针分子中的苯并咪唑基团的三级氮含有一对孤电子对，在测试体系pH值较小和较大时荧光探针分子会以不同的形式存在，当探针分子受光激发发射荧光时电子由基态跃迁到激发态所需要的激发能不同，得到的荧光光谱也有很大变化。在低pH值体系中孤对电子与体系中的质子结合形成一种有机盐结构，此时，荧光探针分子的吸电子能力增强，推拉电子的性能增强，基态电子跃迁到激发态所需要的激发能减小，荧光发射光谱发生紅移荧光强度增加；在高pH值体系中孤电子对无法与质子结合，整个荧光探针分子的共轭性较差，推拉电子的能力减弱，基态电子跃迁到激发态所需要的激发能增强，荧光强度减小。
本发明荧光探针结构简单，合成方便，操作简便，反应灵敏。以荧光强弱和颜色的变化来检测微环境pH的变化，并且对细胞毒性很小，能够用于检测细胞溶酶体内的pH变化，进而监测细胞自噬过程。
本发明通过细胞共定位实验确信其能够专一性靶向细胞溶酶体，细胞毒性测试表明本发明对于细胞几乎没有什么毒副作用，双光子共聚焦荧光显微成像实验表明本发明对于MCF-7细胞的渗透性良好，同时，经计算本发明荧光探针的pKa为3.88，非常适合细胞溶酶体pH变化范围的监测，能够通过检测细胞溶酶体pH的变化实时监测细胞自噬过程进行的情况。
附图说明
图1是本发明荧光探针Lyso-MPCB(10μM)在不同pH值的缓冲溶液中的紫外吸收光谱。
图2是本发明荧光探针Lyso-MPCB(10μM)在不同pH值的缓冲溶液中的荧光发射光谱图，每条线都是在加入探针分子后立马进行的测试。
图3是本发明荧光探针Lyso-MPCB在用1mM盐酸和氢氧化钠溶液来回调节的测试溶液中，当pH＝4.2和pH＝7.2时分别测定荧光发射谱图，再计算荧光发射波长为475nm和410nm下荧光强度比值(I475nm/I410nm)的循环图。
图4是本发明荧光探针Lyso-MPCB(0.1mM)在不同pH值的缓冲溶液中不同的波长激发下双光子吸收截面值。
图5是本发明荧光探针Lyso-MPCB在MCF-7细胞培养24h后的细胞存活率。
图6是本发明荧光探针Lyso-MPCB在MCF-7细胞中的溶酶体定位成像照片。探针Lyso-MPCB(10μM)加入到MCF-7细胞中培养30分钟，然后再向其中加入溶酶体染料LysoTracker Red FM(0.5μM)，继续培养10分钟。其中图(a)绿色通道(460-490nm)，λex＝760nm；(b)红色通道(580-620nm)，λex＝559nm；(c)是(a)和(b)通道的叠加图；(d)是(a)和(b)通道对应荧光强度的散点图分析。
图7是用本发明荧光探针Lyso-MPCB分子(10μM)培养MCF-7细胞0.5小时后饥饿诱导自噬4小时后的荧光变化情况。其中，图(a1-2)蓝色通道(390-420nm)；(b1-2)绿色通道(460-490nm)；(c1-2)是明场；(d1)是(a1)、(b1)和(c1)通道的叠加图；(d2)是(a2)、(b2)和(c2)通道的叠加图。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步说明。
实施例1：荧光探针分子Lyso-MPCB的合成
将化合物A1g、邻苯二胺0.46g、对甲苯磺酸0.0082g以及N,N-二甲基甲酰胺30mL加入到三口烧瓶中，在油浴锅中升温至120℃并通氩气保护反应12h；反应结束后将液体旋干，用二氯甲烷和水萃取，收集上层有机相并旋干得到粗产物；将所得粗产物制样用柱层析色谱柱分离，得到目标产物得到目标产物0.7g(1.28mmoL)，收率60％。所述化合物A的结构式为：
1H NMR(400MHz,DMSO-d6)δ12.84(s,1H),9.04(s,1H),8.41(s,1H),8.33(d,J＝8.6Hz,1H),7.84(d,J＝8.7Hz,1H),7.73(d,J＝8.5Hz,1H),7.65(d,J＝8.5Hz,2H),7.55(t,J＝11.5Hz,3H),7.20(dd,J＝5.5,2.6Hz,2H),7.02(d,J＝8.3Hz,2H),4.49(t,J＝7.0Hz,2H),3.82(s,3H),3.53(s,4H),2.30(s,6H),1.89-1.79(m,2H),1.53-1.47(m,2H).13C NMR(101MHz,DMSO-d6)δ160.23,153.36,142.25,141.14,133.69,130.29,125.90,124.60,123.28,122.87,122.62,120.14,115.89,115.39,114.33,111.23,111.09,90.22,88.74,67.07,58.31,56.25,54.12,43.37,30.00,27.13.
实施例2：荧光探针分子的荧光测试及双光子测试
将本发明荧光探针Lyso-MPCB溶于DMSO中制得1mM的母液，取100μL的该母液于10mL容量瓶中，再用不同pH值的缓冲溶液(磷酸-醋酸-硼酸溶液体系，通过加入1mM盐酸或氢氧化钠溶液调至不同pH值)定容，配制成10μM。荧光探针单光子和双光子的激发波长分别为370nm和760nm，检测390-700nm波长范围内的荧光光谱变化。并通过不同pH值对应的I475nm/I410nm计算出探针的pKa值(3.88)。
荧光探针Lyso-MPCB在用1mM盐酸和氢氧化钠溶液来回调节的测试溶液中，当pH＝4.2和pH＝7.2时分别测定荧光发射谱图，再计算荧光发射波长为475nm和410nm下荧光强度比值(I475nm/I410nm)的循环图(图3)，循环次数为6次。
利用双光子测试技术，测试荧光探针(Lyso-MPCB)在pH＝3.2时的双光子吸收截面，从图4可以看出，荧光探针分子在pH＝3.2时的最大双光子吸收截面为335GM，双光子激发波长在760nm。
实施例3：细胞毒性测试
MTT(3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐)实验是根据已报道的文章，做一些细胞毒性测试。分别在同一批细胞中加入0，5，10，15，20μM的荧光探针，此条件是在37℃、含5％CO2的细胞培养箱中孵育24小时，根据细胞存活度的公式：细胞存活率％＝OD570(样品)/OD570(对照组)×100，可算得细胞存活率(图5)。从图5中我们可以看出，浓度为20μM时，细胞存活率还有90％左右，说明了本发明荧光探针对细胞无毒性作用，因此可以用来检测细胞中的黏度。
实施例4：细胞定位测试
MCF-7细胞由DEME(invitrogen)培养液培养，成像前一天，MCF-7细胞放于激光共聚焦皿中，成像时MCF-7细胞和10μM的荧光探针Lyso-MPCB的DMSO溶液于37℃、含5％CO2的细胞培养箱中孵育0.5小时，用中性的PBS缓冲溶液洗涤3次后，再往培养皿中加入0.5μM商品化溶酶体染色剂LysoTracker Red FM溶液继续孵育0.5小时，用中性的PBS缓冲溶液洗涤3次。用双光子荧光共聚焦成像，设置绿色通道tracker1，激发波长为760nm，发射波段为460-490nm，这个通道用来接受探针分子Lyso-MPCB发射的荧光。设置红色通道tracker2，激发波长为559nm，发射波长为580-620nm，这个通道用来接收商品化溶酶体染色剂LysoTracker Red FM发射的荧光(图6)。
实施例5：细胞自噬监测
MCF-7细胞由DEME(invitrogen)培养液培养，成像前一天，MCF-7细胞放于平底表面皿中，成像时MCF-7细胞和10μM的荧光探针Lyso-MPCB的DMSO溶液于37℃、含5％CO2的细胞培养箱中孵育0.5小时，用中性的PBS缓冲溶液或培养液充分洗涤后，将培养基换成HBSS(诱导细胞发生自噬过程的饥饿培养基)。然后，在开始时(0h)以及处理一段时间(4h)后，用双光子荧光共聚焦成像，得图7。其中，图(a1-2)蓝色通道(390-420nm)；(b1-2)绿色通道(460-490nm)；(c1-2)是明场；(d1)是(a1)、(b1)和(c1)通道的叠加图；(d2)是(a2)、(b2)和(c2)通道的叠加图。
Claims (7)
1.一种监测细胞自噬的双光子pH比率计量荧光探针，其特征在于：是以咔唑为母体，4-甲氧基苯乙炔为供电子基，溶酶体定位基团为吗啉，苯并咪唑为pH的响应基团。
2.根据权利要求1所述的荧光探针，其特征在于其结构式如下：
3.一种权利要求1或2所述的监测细胞自噬的双光子pH比率计量荧光探针的制备方法，其特征在于包括如下步骤：
将化合物A 1g、邻苯二胺0.46g、对甲苯磺酸0.0082g以及N,N-二甲基甲酰胺30mL加入到三口烧瓶中，升温至120℃并通氩气保护反应12h；反应结束后将液体旋干，用二氯甲烷和水萃取，收集上层有机相并旋干得到粗产物；将所得粗产物制样用柱层析色谱柱分离，得到目标产物；
所述化合物A的结构式为：
4.根据权利要求3所述的制备方法，其特征在于：
柱层析色谱柱分离时所用洗脱液为二氯甲烷和甲醇按体积比100:1混合得到。
5.一种权利要求1或2所述的监测细胞自噬的双光子pH比率计量荧光探针的用途，其特征在于：在检测细胞中自噬溶酶体pH变化时作为检测试剂应用。
6.一种权利要求1或2所述的监测细胞自噬的双光子pH比率计量荧光探针的应用，其特征在于：所述荧光探针作为检测试剂通过检测细胞溶酶体pH变化来监测细胞自噬进行的程度。
7.根据权利要求6所述的应用，其特征在于包括如下步骤：
将所述荧光探针溶于DMSO中制得1mM的母液，取100μL的该母液于10mL容量瓶中，再用待测溶液定容，通过检测通道一：390～420nm以及通道二：460～490nm波长范围内的荧光光谱峰值比率计量变化I475nm/I410nm实现定量检测细胞自噬溶酶体的pH变化，从而达到监测细胞自噬过程的目的。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810256444.XA CN108329301B (zh) | 2018-03-27 | 2018-03-27 | 一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810256444.XA CN108329301B (zh) | 2018-03-27 | 2018-03-27 | 一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108329301A true CN108329301A (zh) | 2018-07-27 |
| CN108329301B CN108329301B (zh) | 2020-10-02 |
Family
ID=62931610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810256444.XA Active CN108329301B (zh) | 2018-03-27 | 2018-03-27 | 一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108329301B (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004246A (zh) * | 2019-12-13 | 2020-04-14 | 山西大学 | 监测线粒体自噬的罗丹明类pH荧光探针及制备和应用 |
| CN111100627A (zh) * | 2019-12-20 | 2020-05-05 | 中国科学院化学研究所 | 荧光探针及其应用 |
| CN113429335A (zh) * | 2021-06-25 | 2021-09-24 | 安徽大学 | 一种溶酶体靶向的双响应双光子荧光探针及其制备方法和用途 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496239A (zh) * | 2016-10-19 | 2017-03-15 | 中南大学 | 一种新型溶酶体内pH比率荧光探针的制备与应用 |
| CN106833625A (zh) * | 2017-03-14 | 2017-06-13 | 山西大学 | 一种双光子溶酶体pH荧光探针及其制备方法和应用 |
-
2018
- 2018-03-27 CN CN201810256444.XA patent/CN108329301B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496239A (zh) * | 2016-10-19 | 2017-03-15 | 中南大学 | 一种新型溶酶体内pH比率荧光探针的制备与应用 |
| CN106833625A (zh) * | 2017-03-14 | 2017-06-13 | 山西大学 | 一种双光子溶酶体pH荧光探针及其制备方法和应用 |
Non-Patent Citations (1)
| Title |
|---|
| XIN ZHANG, ET AL.: "Rational Design of d-PeT Phenylethynylated-Carbazole Monoboronic Acid Fluorescent Sensors for the Selective Detection of α-Hydroxyl Carboxylic Acids and Monosaccharides", 《J. AM. CHEM. SOC.》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004246A (zh) * | 2019-12-13 | 2020-04-14 | 山西大学 | 监测线粒体自噬的罗丹明类pH荧光探针及制备和应用 |
| CN111004246B (zh) * | 2019-12-13 | 2021-03-30 | 山西大学 | 监测线粒体自噬的罗丹明类pH荧光探针及制备和应用 |
| CN111100627A (zh) * | 2019-12-20 | 2020-05-05 | 中国科学院化学研究所 | 荧光探针及其应用 |
| CN113429335A (zh) * | 2021-06-25 | 2021-09-24 | 安徽大学 | 一种溶酶体靶向的双响应双光子荧光探针及其制备方法和用途 |
| CN113429335B (zh) * | 2021-06-25 | 2023-05-16 | 安徽大学 | 一种溶酶体靶向的双响应双光子荧光探针及其制备方法和用途 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108329301B (zh) | 2020-10-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | A ratiometric fluorescence probe for lysosomal polarity | |
| Chen et al. | A NIR fluorescent probe for detection of viscosity and lysosome imaging in live cells | |
| Liu et al. | A sensitive and selective fluorescent probe for the detection of hydrogen peroxide with a red emission and a large Stokes shift | |
| Hou et al. | A colorimetric and ratiometric fluorescent probe for cyanide sensing in aqueous media and live cells | |
| CN107849441A (zh) | 用于癌症的可视化和治疗的aie发光体 | |
| Zhu et al. | A red-emitting fluorescent probe for mitochondria-target microviscosity in living cells and blood viscosity detection in hyperglycemia mice | |
| Ye et al. | Highly photostable, lysosome-targeted BODIPYs with green to near-infrared emission for lysosome imaging in living cells | |
| CN104610959B (zh) | 一种检测硫化氢的荧光探针及其制备方法与使用方法 | |
| CN103382313B (zh) | 一种萘酰亚胺荧光染料及其制备和应用 | |
| CN105017196B (zh) | 一种检测肼的近红外比率荧光探针及其应用 | |
| CN108329301A (zh) | 一种监测细胞自噬的双光子pH比率计量荧光探针及其制备方法和用途 | |
| Niu et al. | A ratiometric emission NIR-fluorescent probe for sensing and imaging pH changes in live cells | |
| CN105733563B (zh) | 一种基于香豆素的双光子溶酶体极性探针、其制备方法及其用途 | |
| CN105523960B (zh) | 一种新型pH响应双发射波长荧光分子探针的合成方法及其在生物成像中的应用 | |
| Zhi et al. | A novel carbazolyl GFP chromophore analogue: synthesis strategy and acidic pH-activatable lysosomal probe for tracing endogenous viscosity changes | |
| Tong et al. | A ratiometric fluorescent pH probe based on keto–enol tautomerization for imaging of living cells in extreme acidity | |
| CN105802608A (zh) | 一种荧光探针的制备方法及其在铝离子检测中的应用 | |
| CN108069908A (zh) | 荧光探针及其应用 | |
| CN110003173B (zh) | 一种基于咔唑的双光子极性荧光探针及其制备方法和用途 | |
| CN103820104B (zh) | 一类以尼罗蓝为母体的近红外荧光探针、其制法及应用 | |
| CN106543226B (zh) | 一种定位线粒体的atp荧光探针的制备及应用 | |
| Jin et al. | Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring | |
| KR101233679B1 (ko) | 서로 다른 형광색의 이광자 형광 프로브를 이용하여 나트륨/칼슘 활성을 동시에 이미지화하는 방법 및 이광자 형광 프로브의 제조방법 | |
| CN106634968A (zh) | 一种线粒体靶向的粘度荧光探针及其制备方法和应用 | |
| CN106892870B (zh) | 一种溶酶体靶向的双光子黏度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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |