CN105601658B - 一种能够区分生物硫醇的荧光探针的制备与应用 - Google Patents
一种能够区分生物硫醇的荧光探针的制备与应用 Download PDFInfo
- Publication number
- CN105601658B CN105601658B CN201610027345.5A CN201610027345A CN105601658B CN 105601658 B CN105601658 B CN 105601658B CN 201610027345 A CN201610027345 A CN 201610027345A CN 105601658 B CN105601658 B CN 105601658B
- Authority
- CN
- China
- Prior art keywords
- fluorescence
- fluorescence probe
- cysteine
- homocysteine
- glutathione
- 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.)
- Expired - Fee Related
Links
- 239000000523 sample Substances 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 5
- 125000003396 thiol group Chemical class [H]S* 0.000 title 1
- 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 claims abstract description 60
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 claims abstract description 33
- 235000018417 cysteine Nutrition 0.000 claims abstract description 32
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims abstract description 31
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229960003180 glutathione Drugs 0.000 claims abstract description 30
- 108010024636 Glutathione Proteins 0.000 claims abstract description 28
- 150000003573 thiols Chemical class 0.000 claims abstract description 9
- 238000004458 analytical method Methods 0.000 claims abstract description 5
- 239000012472 biological sample Substances 0.000 claims abstract 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 13
- 230000004044 response Effects 0.000 claims description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- IGHBXJSNZCFXNK-UHFFFAOYSA-N 4-chloro-7-nitrobenzofurazan Chemical compound [O-][N+](=O)C1=CC=C(Cl)C2=NON=C12 IGHBXJSNZCFXNK-UHFFFAOYSA-N 0.000 claims description 3
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims 1
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Substances C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 abstract description 10
- 150000001413 amino acids Chemical class 0.000 abstract description 10
- 238000001514 detection method Methods 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 26
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 25
- 230000000694 effects Effects 0.000 description 12
- 238000000034 method Methods 0.000 description 9
- 238000002189 fluorescence spectrum Methods 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-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
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 150000001945 cysteines Chemical class 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- UXAMZEYKWGPDBI-UHFFFAOYSA-N C(CCCCCCCCCCCCCCC)Br(C)(C)C Chemical class C(CCCCCCCCCCCCCCC)Br(C)(C)C UXAMZEYKWGPDBI-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 238000006845 Michael addition reaction Methods 0.000 description 1
- 208000029549 Muscle injury Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 206010041349 Somnolence Diseases 0.000 description 1
- 229930185324 Trichochrome Natural products 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 229940077388 benzenesulfonate Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 201000010251 cutis laxa Diseases 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 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
- 230000009257 reactivity Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- 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
-
- 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
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1048—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with oxygen
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明公开了一种能够区分生物硫醇的新型化合物,具体涉及一种新型荧光探针的制备方法及其应用,属于化学分析检测技术领域。其分子结构式如下:该荧光探针用于环境或生物样品中的半胱氨酸、同型半胱氨酸和谷胱甘肽的荧光传感分析,通过和探针分子作用后输出信号的差异,能够很好地区分半胱氨酸/同型半胱氨酸和谷胱甘肽,具有选择性好,抗干扰能力强,激发和发射波长处于近红外区等优点,可以灵敏快速地从多种氨基酸中区分出半胱氨酸、同型半胱氨酸和谷胱甘肽,具有很好的应用前景。
Description
技术领域
本发明涉及的是化学分析检测技术领域,具体涉及一种能够区分生物硫醇的荧光探针的制备方法以及该荧光探针在实验环境和细胞环境检测生物硫醇方面的应用。
背景技术
氨基酸是构成蛋白质的基本物质,并且与生物的生命活动有着密切的联系。半胱氨酸(Cysteine,Cys)、同型半胱氨酸(Homocysteine,Hcy)和谷胱甘肽 (Glutataione,GSH)是生物体内常见的生物硫醇,在维持生物的正常生理活动中起着重要的作用。医学研究表明不正常的生理浓度可能引起很多疾病,比如半胱氨酸生理浓度的异常,会引起毛发色素脱色、水肿、嗜睡、肌肉损伤、皮肤松弛过敏、免疫能力下降等,同型半胱氨酸在临床上的应用主要作为心血管疾病,尤其是冠状动脉粥硬化和心肌梗塞的危险指标,它的浓度升高程度与疾病的危险性成正比,谷胱甘肽的浓度异常也可能会引起阿尔兹海默氏症、心血管疾病、癌症的发生。它们在生物体内含量变化可以作为这些疾病诊断的依据。因此,在生理条件下高选择性、高灵敏性地检测小分子生物硫醇是非常重要的,当前已引起广泛的关注及深入研究。目前已经应用的技术包括高效液相色谱法、毛细管电泳法、电化学检测、光学分析和质谱鉴定,这些方法仅可以在体外监测半胱氨酸、同型半胱氨酸和谷胱甘肽。荧光分子探针不仅灵敏度高选择性好,而且能够在活细胞中检测分析物,所以研究者们开始关注将荧光分子探针的这项技术应用于对体外或者活细胞内的半胱氨酸、同型半胱氨酸和谷胱甘肽进行监测或者细胞荧光成像。目前已经报导了多种基于化学反应的该类荧光探针,例如Michael加成和亲核取代反应。在这些方法中,在荧光分子内引入荧光淬灭剂2,4-二硝基-苯磺酸盐、2,4-二硝基苯磺酰胺基团或者丙烯酰基团,导致荧光猝灭,在半胱氨酸、同型半胱氨酸和谷胱甘肽诱导下发生的裂解,荧光得以恢复,这是一种特别有效的方法。由于这三种氨基酸都含有巯基(-SH)并且在结构和反应活性上相差较小,所以这类荧光探针很难将谷胱甘肽和半胱氨酸/同型半胱氨酸区分开,因此研发能够输出不同响应信号、水溶性好、斯托克斯位移大、发射波长在近红外区的该类荧光探针是很有必要的。
发明内容
本发明目的之一是提供一种合成简单、反应条件温和、成本较低的荧光探针合成方法;目的之二是提供一种灵敏度高、选择性好,抗干扰能力强,发射波长在近红外区,能够对体外或者活细胞内的半胱氨酸、同型半胱氨酸和谷胱甘肽进行监测或者细胞荧光成像的荧光探针。
本发明解决问题采取的技术方案为,一种检测半胱氨酸、同型半胱氨酸和谷胱甘肽的“关-开”型荧光探针,其分子结构式如下:合成路线如下:
具体合成方法如下:(1)称取原料1(0.04mmol,0.0201g),4-氯-7-硝基苯并-2-氧杂-1,3-二唑 (NBD-Cl)(0.04mmol,0.0081g)和无水碳酸钾(0.15mmol,0.0213g)置于5mL单颈圆底烧瓶,加入2mL无水丙酮,氩气保护,室温搅拌4小时。待反应结束,除去丙酮,剩余物用柱层析进一步纯化(二氯甲烷:石油醚=1:1,v/v)得到黑色固体。产量:0.0242g。收率:94%。
本发明的荧光探针使用方法如下,将探针分子溶解在含有10mM十六烷基三甲基溴化铵(CTAB)、pH为7.4的PBS缓冲溶液中,室温下进行测试。并且对低浓度的半胱氨酸、同型半胱氨酸和谷胱甘肽可以进行定量检测,具体实施方法在实施实例中详细介绍。
本发明的荧光探针的作用机理如下,探针分子与半胱氨酸或同型半胱氨酸作用后,NBD部分从探针分子上离去,并伴随重排现象,探针分子的荧光由无到有,并有双荧光信号输出,探针分子与谷胱甘肽作用后,NBD部分从探针分子上离去,探针分子的荧光由无到有,从而实现了对半胱氨酸、同型半胱氨酸和谷胱甘肽的检测过程。探针分子与半胱氨酸和同型半胱氨酸的响应过程:(半胱氨酸:n=1;同型半胱氨酸:n=2)。探针分子与谷胱甘肽的响应过程:
本发明的荧光探针与半胱氨酸或同型半胱氨酸作用后荧光发射峰在 545nm和705nm处,与谷胱甘肽作用后的荧光发射峰仅出现在705nm处。
本发明所述的探针分子合成简单,成本较低,对半胱氨酸、同型半胱氨酸和谷胱甘肽的选择性好、抗干扰能力强、响应速度快,激发和发射波长处于近红外区,并能通过输出信号不同特异性区分半胱氨酸/同型半胱氨酸和谷胱甘肽,使得该荧光探针在生物化学,环境科学等领域具有实际的应用价值。
附图说明
图1为本发明荧光探针的选择性,荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mMCTAB,pH=7.4)中,在650nm激发下与不同种类氨基酸作用后的荧光光谱,横坐标为波长,纵坐标为荧光强度。
图2为本发明荧光探针的选择性,荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mMCTAB,pH=7.4)中,在476nm激发下与不同种类氨基酸作用后的荧光光谱,横坐标为波长,纵坐标为荧光强度。
图3为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与不同浓度半胱氨酸作用后的荧光光谱变化,横坐标为波长,纵坐标为荧光强度。
图4为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与不同浓度同型半胱氨酸作用后的荧光光谱变化,横坐标为波长,纵坐标为荧光强度。
图5为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与不同浓度谷胱甘肽作用后的荧光光谱变化,横坐标为波长,纵坐标为荧光强度。
图6为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与半胱氨酸浓度的线性关系,横坐标为浓度,纵坐标为荧光强度。
图7为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与同型半胱氨酸浓度的线性关系,横坐标为浓度,纵坐标为荧光强度。
图8为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与谷胱甘肽的线性关系,横坐标为浓度,纵坐标为荧光强度。
图9为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,476nm激发下与不同浓度半胱氨酸作用后的荧光光谱变化,横坐标为波长,纵坐标为荧光强度。
图10为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,476nm激发下与不同浓度同型半胱氨酸作用后的荧光光谱变化,横坐标为波长,纵坐标为荧光强度。
图11为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,476nm激发下与半胱氨酸浓度的线性关系,横坐标为浓度,纵坐标为荧光强度。
图12为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,476nm激发下与同型半胱氨酸浓度的线性关系,横坐标为浓度,纵坐标为荧光强度。
图13为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与10倍当量半胱氨酸的时间扫描,横坐标为时间,纵坐标为荧光强度。
图14为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与10倍当量同型半胱氨酸的时间扫描,横坐标为时间,纵坐标为荧光强度。
图15为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,650nm激发下与10倍当量谷胱甘肽的时间扫描,横坐标为时间,纵坐标为荧光强度。
图16为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,476nm激发下与10倍当量半胱氨酸的时间扫描,横坐标为时间,纵坐标为荧光强度。
图17为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,476nm激发下与10倍当量同型半胱氨酸的时间扫描,横坐标为时间,纵坐标为荧光强度。
图18为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,476nm激发下与10倍当量谷胱甘肽的时间扫描,横坐标为时间,纵坐标为荧光强度。
图19为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,半胱氨酸抗干扰测试,横坐标为半胱氨酸和不同种类其他氨基酸混合类别,纵坐标为荧光比值。
图20为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,同型半胱氨酸抗干扰测试,横坐标为同型半胱氨酸和不同种类其他氨基酸混合类别,纵坐标为荧光比值。
图21为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mMCTAB,pH=7.4)中,谷胱甘肽抗干扰测试,横坐标为谷胱甘肽和不同种类其他氨基酸混合类别,纵坐标为荧光比值。
图22为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,与半胱氨酸作用前后的荧光强度,横坐标为pH,纵坐标为荧光强度。
图23为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,与同型半胱氨酸作用前后的荧光强度,横坐标为pH,纵坐标为荧光强度。
图24为本发明的荧光探针(5.0×10-6mol/L)在PBS缓冲溶液(10mM CTAB,pH=7.4)中,与谷胱甘肽作用前后的荧光强度,横坐标为pH,纵坐标为荧光强度。
具体实施实例
实施例1:探针分子的合成
称取原料1(0.04mmol,0.0201g),4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)(0.04 mmol,0.0081g)和无水碳酸钾(0.15mmol,0.0213g)置于5mL单颈圆底烧瓶,加入2mL无水丙酮,氩气保护,室温搅拌4小时。待反应结束,除去丙酮,剩余物用柱层析进一步纯化(二氯甲烷:石油醚=1:1,v/v)得到黑色固体。产量: 0.0242g。收率:94%。1H NMR(500MHz,CDCl3)δ8.53(d,J=8.3Hz,1H),7.69 -7.63(m,1H),7.53(d,J=8.4Hz,2H),7.41(d,J=8.4Hz,2H),7.28-7.19(m,2H), 6.96(t,J=7.4Hz,1H),6.89(d,J=14.6Hz,1H),6.76(d,J=7.9Hz,1H),6.63(d,J =8.3Hz,1H),6.57(s,1H),5.98(s,1H),5.72(d,J=12.2Hz,1H),3.24(s,3H),2.59 (s,3H),1.65(s,5H),1.58(s,3H),1.50(s,3H).13C NMR(100MHz,CDCl3)δ162.0,156.6,153.7,153.2,150.3,145.2,144.4,144.1,142.3,139.2,137.2,136.8,135.0,133.7,133.5,133.2,131.6,131.3,131.1,130.4,128.4,128.0,121.6,121.3,120.9,119.8,118.4,113.6,108.0,107.0,98.5,46.6,46.4,29.6,29.3,28.7,15.0,14.2.ESI-MS:[M+H]+calcd for 687.2697,found 687.2706.
实施例2:本发明:荧光探针的应用
将探针溶于缓冲溶液(含10mM CTAB的PBS溶液,pH=7.4)中配制成5.0×10-6mol/L的溶液,向溶液中加入氨基酸(Ala,Gln,Glu,His,Ile,Leu,Lys,Phe,Pro,Ser, Thr,Try,Tyr,Val.)没有引起荧光的变化,加人氨基酸(Cys,Hcy,GSH)引起了荧光变化,该荧光探针对半胱氨酸、同型半胱氨酸和谷胱甘肽表现出很高灵敏度并且能够区分半胱氨酸/同型半胱氨酸和谷胱甘肽。当半胱氨酸、同型半胱氨酸和谷胱甘肽与干扰物质(Asn,Ala,Val,Phe,His,Leu,Ser,Ile,Trp,Lys,Arg,Pro,Gly, Met,Tyr,Glu,Thr)共存时,探针不受干扰因素的影响,表现出来很好的抗干扰能力。该探针分子与半胱氨酸、同型半胱氨酸和谷胱甘肽作用后荧光信号明显,即可观察到荧光的变化。探针分子在pH为6至10的范围内都可以对半胱氨酸/ 同型半胱氨酸和谷胱甘肽选择性区分识别,表现出了较好的检测范围。
Claims (3)
1.一种能够区分生物硫醇的荧光探针,其结构为:
2.如权利要求1所述的能够区分生物硫醇的荧光探针的制备方法,其特征在于按以下步骤进行制备:
(a)称取原料14-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)和无水碳酸钾置于单颈圆底烧瓶,加入无水丙酮,氩气保护,室温搅拌4小时;
(b)待反应结束,除去丙酮,剩余物用柱层析进一步纯化,得到黑色固体。
3.根据权利要求1所述的能够区分生物硫醇的荧光探针的非诊断或治疗目的的用途,其特征在于该荧光探针用于半胱氨酸/同型半胱氨酸和谷胱甘肽的区分以及环境或生物样品中半胱氨酸、同型半胱氨酸的荧光检测和分析。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610027345.5A CN105601658B (zh) | 2016-01-15 | 2016-01-15 | 一种能够区分生物硫醇的荧光探针的制备与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610027345.5A CN105601658B (zh) | 2016-01-15 | 2016-01-15 | 一种能够区分生物硫醇的荧光探针的制备与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105601658A CN105601658A (zh) | 2016-05-25 |
CN105601658B true CN105601658B (zh) | 2018-03-30 |
Family
ID=55982077
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610027345.5A Expired - Fee Related CN105601658B (zh) | 2016-01-15 | 2016-01-15 | 一种能够区分生物硫醇的荧光探针的制备与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105601658B (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108358906B (zh) * | 2018-03-14 | 2021-04-02 | 中南大学 | 一种特异性区分不同硫醇的荧光探针 |
CN108982394A (zh) * | 2018-08-07 | 2018-12-11 | 青岛科技大学 | 一种利用opa特异性识别生物硫醇分子的方法 |
CN112630179B (zh) * | 2020-12-09 | 2023-07-21 | 安徽师范大学 | 具有氧化物模拟酶性质的普鲁士蓝量子点及其制备方法及检测l-半胱氨酸的方法 |
CN116840197A (zh) * | 2022-03-23 | 2023-10-03 | 中国科学院苏州生物医学工程技术研究所 | 一种2’-o-甲基转移酶活性检测方法、试剂盒及应用 |
CN116925756B (zh) * | 2023-07-21 | 2024-04-19 | 锦州医科大学 | 一种特异性检测生物体中半胱氨酸的纳米探针及其应用 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102928392A (zh) * | 2012-10-29 | 2013-02-13 | 山西大学 | 一种检测半胱氨酸的方法 |
CN103289681B (zh) * | 2013-06-09 | 2014-12-03 | 南京工业大学 | 谷胱甘肽荧光探针及其制备方法和用途 |
CN104447421B (zh) * | 2014-10-28 | 2016-06-08 | 苏州罗兰生物科技有限公司 | 一种新型半胱氨酸和同型半胱氨酸荧光探针的制备与应用 |
CN104673278A (zh) * | 2015-02-15 | 2015-06-03 | 浙江理工大学 | 一种检测谷胱甘肽的荧光探针及其制备方法与使用方法 |
-
2016
- 2016-01-15 CN CN201610027345.5A patent/CN105601658B/zh not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
CN105601658A (zh) | 2016-05-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105601658B (zh) | 一种能够区分生物硫醇的荧光探针的制备与应用 | |
Guan et al. | A near-infrared fluorescent sensor for selective detection of cysteine and its application in live cell imaging | |
CN108727326B (zh) | 识别半胱氨酸和谷胱甘肽的荧光探针及制备方法与应用 | |
CN106946801B (zh) | 一种特异性识别半胱氨酸的新型荧光探针的制备与应用 | |
Wang et al. | A highly sensitive fluorescent probe for hydrogen sulfide based on dicyanoisophorone and its imaging in living cells | |
CN105524055B (zh) | 一种能够区分半胱氨酸/同型半胱氨酸和谷胱甘肽荧光探针的制备与应用 | |
Gan et al. | A novel fluorescent probe for selective imaging of cellular cysteine with large Stokes shift and high quantum yield | |
CN110698454B (zh) | 一种异佛尔酮类硫化氢荧光探针及其制备方法与应用 | |
CN106929006B (zh) | 一种以萘酰亚胺为母核的识别半胱氨酸和同型半胱氨酸荧光探针及其制备与应用 | |
Xia et al. | A fluorescent turn-on probe for highly selective detection of cysteine and its bioimaging applications in living cells and tissues | |
Zhu et al. | A simple highly specific fluorescent probe for simultaneous discrimination of cysteine/homocysteine and glutathione/hydrogen sulfide in living cells and zebrafish using two separated fluorescence channels under single wavelength excitation | |
Lan et al. | A near-infrared Nile red fluorescent probe for the discrimination of biothiols by dual-channel response and its bioimaging applications in living cells and animals | |
Zhu et al. | A novel highly sensitive fluorescent probe for bioimaging biothiols and its applications in distinguishing cancer cells from normal cells | |
CN106046012A (zh) | 一种新型香豆素类生物硫醇荧光探针及其制备方法 | |
Xia et al. | A novel HPQ-based fluorescent probe for the visualization of carbon monoxide in zebrafish | |
Ge et al. | A novel NIR fluorescence probe with cysteine-activated structure for specific detection of cysteine and its application in vitro and in vivo | |
CN109438319A (zh) | 一种检测亮氨酸氨肽酶的化合物及其制备方法和应用 | |
Wang et al. | A turn-on fluorescent probe via substitution-rearrangement for highly sensitive and discriminative detection of cysteine and its imaging in living cells | |
Zhou et al. | Fluorescent probe for highly selective detection of cysteine in living cells | |
Tan et al. | A simple lysosome-targeted fluorescent probe based on flavonoid for detection of cysteine in living cells | |
Wu et al. | Novel near-infrared frequency up-conversion luminescence probe for monitoring biothiols in vitro and in vivo | |
Zheng et al. | A novel ratiometric fluorescent probe for the detection and imaging of cysteine in living cells | |
Zhu et al. | A simple long-wavelength fluorescent probe for simultaneous discrimination of cysteine/homocysteine and glutathione/hydrogen sulfide with two separated fluorescence emission channels by single wavelength excitation | |
Yan et al. | Simultaneous discrimination of Cys/Hcy and GSH with simple fluorescent probe under a single-wavelength excitation and its application in living cells, tumor tissues, and zebrafish | |
CN110498786A (zh) | 一种检测半胱氨酸/同型半胱氨酸和谷胱甘肽的新型比值型荧光探针 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into 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: 20180330 Termination date: 20190115 |
|
CF01 | Termination of patent right due to non-payment of annual fee |