CN117603203A - 一种比率荧光探针及其制备方法和应用 - Google Patents
一种比率荧光探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN117603203A CN117603203A CN202410069665.1A CN202410069665A CN117603203A CN 117603203 A CN117603203 A CN 117603203A CN 202410069665 A CN202410069665 A CN 202410069665A CN 117603203 A CN117603203 A CN 117603203A
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- hclo
- probe
- detection
- ratio
- 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
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title abstract description 8
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims abstract description 74
- 238000001514 detection method Methods 0.000 claims abstract description 25
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 210000004027 cell Anatomy 0.000 claims abstract description 19
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims abstract description 9
- -1 10-hexyl-phenothiazine-3-acetaldehyde Chemical compound 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 9
- 241000252212 Danio rerio Species 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- VZRIFNLQJXPRJI-UHFFFAOYSA-M 2,3-dimethyl-1,3-benzothiazol-3-ium;iodide Chemical compound [I-].C1=CC=C2[N+](C)=C(C)SC2=C1 VZRIFNLQJXPRJI-UHFFFAOYSA-M 0.000 claims description 5
- 210000002540 macrophage Anatomy 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 230000025608 mitochondrion localization Effects 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 19
- 210000003470 mitochondria Anatomy 0.000 abstract description 12
- 230000008685 targeting Effects 0.000 abstract description 9
- 238000001727 in vivo Methods 0.000 abstract description 7
- WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 abstract description 4
- 230000002438 mitochondrial effect Effects 0.000 abstract description 4
- 229950000688 phenothiazine Drugs 0.000 abstract description 4
- 238000011895 specific detection Methods 0.000 abstract description 2
- 238000006000 Knoevenagel condensation reaction Methods 0.000 abstract 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 239000002158 endotoxin Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 229920006008 lipopolysaccharide Polymers 0.000 description 7
- 239000003642 reactive oxygen metabolite Substances 0.000 description 7
- 230000008045 co-localization Effects 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 238000002073 fluorescence micrograph Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000003896 Myeloperoxidases Human genes 0.000 description 2
- 108090000235 Myeloperoxidases Proteins 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000005311 nuclear magnetism Effects 0.000 description 2
- 239000012038 nucleophile Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000011897 real-time detection Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 102000008015 Hemeproteins Human genes 0.000 description 1
- 108010089792 Hemeproteins Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 229910052798 chalcogen Inorganic materials 0.000 description 1
- 150000001787 chalcogens Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000030544 mitochondrion distribution Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 125000003544 oxime group Chemical group 0.000 description 1
- NWVVVBRKAWDGAB-UHFFFAOYSA-N p-methoxyphenol Chemical compound COC1=CC=C(O)C=C1 NWVVVBRKAWDGAB-UHFFFAOYSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 229910052714 tellurium Inorganic materials 0.000 description 1
- PORWMNRCUJJQNO-UHFFFAOYSA-N tellurium atom Chemical compound [Te] PORWMNRCUJJQNO-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- 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"
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Optics & Photonics (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- General Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明属于探针检测技术领域,为解决现有线粒体内次氯酸检测的比率荧光探针存在工作区间小和检测极限较小的问题,提出一种比率荧光探针,并提出了具体的准备方法及应用。本发明的荧光探针,以10‑己基‑吩噻嗪‑3‑乙醛、2,3‑二甲基苯并[d]噻唑‑3‑鎓碘化物、KOH和DMSO为原料,采用Knoevenagel缩合反应制备得到荧光探针。本发明合成了一种基于吩噻嗪的比率荧光探针,用于HClO的特异性检测。具有良好的选择性、生物相容性和低检测限性。此外,还具有良好的线粒体靶向性,能够在活细胞和体内中快速检测出内源性HClO,这使得它在生命科学中具有广泛的应用潜力。
Description
技术领域
本发明属于探针检测技术领域,具体涉及一种比率荧光探针及其制备方法和应用。
背景技术
次氯酸(HClO)是最重要的活性氧(ROS)之一,参与着许多生理过程,在生命活动中起着至关重要的作用。内源性HClO可在髓过氧化物酶(MPO) 的催化作用下,由过氧化氢和氯化物反应生成。
HClO具有很强的氧化性,可与多种蛋白质侧链和肽键发生反应,在杀菌免疫中起重要作用。另一方面,过量的HClO会使含有硫醇、硫醚、血红素蛋白和氨基的生物分子过氧化,从而引起细胞和组织损伤,进而引发各种心血管疾病、神经变性、关节炎和癌症。因此,开发可在体内实时检测的分析方法,对于了解HClO的生理功能及其在相关疾病的研究中具有重要意义。
目前虽然已有比色法、电化学法、化学发光法等分析方法来实现HClO的体外检测,但上述方法难以满足体内实时检测次氯酸的要求。另一方面,HClO的半衰期短,在体内的扩散距离小,体内的H2O2、ONOO-、O2 -等强氧化剂会对HClO的检测产生干扰。这些因素使得内源性HClO的特异性检测变得困难。
近年来,荧光探针因其特异性强、效率高、灵敏度高、无损检测等优点被广泛应用于HClO检测。荧光探针的设计主要基于HClO对反应基团的选择性氧化。常用的反应基团主要有硫族(硫、硒、碲元素)、碳碳双键、对甲氧基苯酚/胺及其衍生物、羟胺或肟基、肼等。HClO容易氧化上述基团,将反应基团转化为相关的氧化产物,从而改变这些富电子基团在荧光团上的电荷转移(CT)或光致电子转移(PET)效应,进而引起探针的荧光信号变化。
虽然基于上述反应基团的荧光探针极大地丰富了HClO的检测手段,但需要指出的是,大多数探针仍然存在抗干扰能力差等缺点。
许多识别HClO的荧光探针迅速发展起来,其中一些可以识别活细胞中的HClO。然而,线粒体被认为是主要的能量来源,也是产生ROS的主要来源。比率荧光探针可以减少由探针浓度、环境温度、环境pH等外部因素变化引起的测量偏差。因此,将线粒体靶向和比率荧光结合在一个分子探针中,将会在分析和检测领域带来突破。
现有申请号为“201711118691.5”《一种应用于线粒体内次氯酸检测的比率荧光探针的制备及应用》,通过制备探针实现对次氯酸的检测,但是该方案仍然存在以下问题:1.该探针工作区间小,为0.5-3.5μM,不能满足检测的需求;2.该探针的检测极限较小,同样不能满足检测需求。
发明内容
基于以上存在的问题,本发明设计了一种比率荧光探针。实验证明具有良好的线粒体靶向性,并成功用于活细胞和斑马鱼中外源性和内源性HClO的检测,检测极限为21nM。
为了实现上述目的,本发明所采用的技术方案如下:
一种比率荧光探针,结构式如下:
。
一种比率荧光探针的制备方法,包括:
步骤1:将10-己基-吩噻嗪-3-乙醛、2,3-二甲基苯并[d]噻唑-3-鎓碘化物、KOH和DMSO在室温下搅拌并放置12h;
步骤2:将步骤1的粗产物从乙醇中过滤并重结晶,得到深红色固体。
优选的,所述10-己基-吩噻嗪-3-乙醛的摩尔量为0.64 mmol,2,3-二甲基苯并[d]噻唑-3-鎓碘化物的摩尔量为1.28 mmol。
优选的,所述KOH以质量分数计的浓度为50%。
优选的,所述KOH体积为5mL,DMSO的体积为10mL。
本发明制备的比率荧光探针作为细胞中线粒体的靶向定位用途、还可以用作巨噬细胞和斑马鱼体内HClO的检测。
一种HClO检测装置,包含有比率荧光探针制备方法。
与现有技术相比,本发明的有益效果是:
本发明设计并合成了一种用于线粒体中外源性和内源性HClO成像的比率荧光探针。通过激光共聚焦荧光图像证实了对线粒体具有良好的靶向能力,共定位系数为0.99,能够对线粒体和斑马鱼中存在的HClO做出高选择性和高灵敏度的反应(检测限为21 nM)。因此,可以看出,本发明制备的比率荧光探针具有生物相容性、靶向性和比率成像能力等优势,在肿瘤临床诊断方面具有重大的潜力。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
在附图中:
附图1 :(A)加入HClO (0 ~ 20 μM)后 (10 μM)的吸收光谱;(B) 对HClO的吸收比;(C) 吸收比与HClO(0 ~ 10 μM)浓度增加的校准曲线;(D)加入HClO (0 ~ 20 μM)后(10 μM)的荧光光谱;(E) 对HClO的发射强度比;(F) 发射强度比与HClO(0-10 μM)浓度增加的校准曲线;(G) (10 μM)对各种活性氧(H2O2、ONOO-、•OH、Cl -、Br -、SO4 2-)和生理亲核试剂(GSH和Cys)的荧光响应;(H)竞争实验;(J)紫外灯下对HClO荧光响应的颜色。
附图2为 对HClO的感应机理。
附图3为RAW264.7细胞共定位实验。(A) RAW264.7细胞用10 μM (λex=405 nm, λem=450 ~ 600 nm)培养;(B) 用Mito Tracker Deep Red (λex=644 nm, λem=650 ~ 570 nm)培养;(C) A与B的合并图像;(D) 亮场图像,标尺:20 μm。
附图4 为RAW 264.7细胞中HClO的荧光图像。(a) 探针(10 μM)染色30 min;(b) 5μM HClO;(c) 50 μM HClO;(d) LPS (1 μg/mL)预处理1 h,处理30 min,蓝色通道:405 ~440 nm,绿色通道:460 ~ 600 nm。标尺:10 μm。
附图5 为RAW 264.7细胞中HClO的荧光图像。(a) 探针(30 μM)染色30 min;(b)15 μM HClO;(c) 100 μM HClO;(d) LPS (5 μg/mL)预处理1 h,预处理30 min,蓝色通道:405 ~ 440 nm,绿色通道:460 ~ 600 nm。标尺:10 μm。
附图6为本发明的荧光探针反应机理图。
附图7为本发明的制备方法流程图。
具体实施方式
以下结合附图1至图7对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
一种比率荧光探针,结构式如下:
。
实施例:
一种比率荧光探针的制备方法,包括:
步骤1:将10-己基-吩噻嗪-3-乙醛(0.2g,0.64 mmol)、2,3-二甲基苯并[d]噻唑-3-鎓碘化物(0.21g,1.28 mmol)、KOH 5mL和DMSO 10 mL在室温下搅拌过夜;所述KOH以质量分数计的浓度为50%。
步骤2:将步骤1的粗产物从乙醇中过滤并重结晶,得到深红色固体(0.27 g,产率73%)。
本实施例制备的比率荧光探针应用于细胞中线粒体的靶向定位。
本实施例制备的比率荧光探针应用于巨噬细胞中HClO的检测。
本实施例制备的比率荧光探针应用于斑马鱼体内的HClO的检测。
实验:
1.结构表征
通常,如果在吩噻嗪的氮原子上附着一个烷基取代基,则吩噻嗪中的硫原子可以被HClO氧化成亚砜,利用这一原理制备了基于吩噻嗪的HClO探针比率荧光探针。通过质谱得到比率荧光探针的质核比为467.1786;其氢谱核磁为1H NMR (400 MHz, DMSO) δ 8.39(d, J = 7.5 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.10 (s, 1H), 7.92 (m, 2H),7.84 (m, 2H), 7.77 (m, 1H), 7.24 (m, 1H), 7.20 – 7.13 (m, 2H), 7.11 (d, J =8.3 Hz, 1H), 7.02 (m, 1H), 3.98 (m, 2H), 1.75 – 1.64 (m, 2H), 1.41 (s, 3H),1.25 (m, 6H), 0.83 (m, 3H);其碳谱核磁为13C NMR (100 MHz, DMSO) δ 171.65,148.35, 147.49, 142.84, 141.98, 131.24, 129.23, 128.32, 128.13, 127.98,127.54, 127.39, 127.24, 124.10, 123.67, 123.55, 122.20, 116.58, 116.52,115.69, 111.21, 46.89, 36.17, 30.72, 26.06, 25.61, 22.00, 13.79。
2.对HClO的光学响应
为了研究探针对HClO的光学响应行为,进行了如图1所示的滴定实验。
从吸收光谱(图1A)可以看出,加入HClO后,探针在262 nm处的吸收峰逐渐减小,在314 nm处的吸收峰逐渐增大。在0 ~ 10 μM的浓度范围内,两个波长的吸光度比呈良好的线性关系(y=0.21+0.027x, R2=0.99)(图1C)。从荧光光谱可以看出,495 nm处的荧光强度逐渐降低,385 nm处的荧光强度逐渐增加(图1D)。在2-10 μM的HClO浓度范围内,385 nm和495nm处信号的荧光强度比(F385/F495)增强了近1160倍,线性关系良好(y=-3.18+1.4x, R2=0.98)(图1F),对HClO的检测限低至21 nM (LOD=3σ/Slope) 23,证明了其生物系统中的应用潜力。
在紫外灯下对HClO的荧光响应颜色如图1I所示。
为了研究探针比率荧光探针对HClO的选择性,测试探针在体内存在多种潜在干扰物质时的荧光响应。(如图1G,其中1-9依次为H2O2, ONOO-,•OH, 1O2, Cl-,Br-,SO4 2-、GSH和Cys),(如图1H,其中1为HClO,2-10依次为H2O2, ONOO-,•OH, 1O2, Cl-,Br-,SO4 2-、GSH和Cys),可以看出在HClO存在的情况下,荧光强度比(F385/F495)发生了显著变化,而其他活性氧(H2O2, ONOO-,•OH, 1O2, Cl-,Br-,SO4 2-)和生理亲核试剂如GSH和Cys等诱导的荧光强度比(F385/F495)变化则不明显。
为了测试探针对HClO识别的竞争能力,分别用荧光光谱对干扰物进行了竞争实验。当HClO与干扰物共存时,荧光强度比(F385/F495)保持不变。结果表明,探针对HClO具有较强的抗竞争能力,这也表明探针在生物系统中具有良好的应用前景。
3.对外源性和内源性HClO的荧光成像
线粒体是细胞代谢的重要介质,是活性氧(ROS)的产生者和靶点,而次氯酸(HClO)一种是典型的ROS,因此对线粒体的检测具有重要意义。
用激光共聚焦显微镜对比率荧光探针细胞器靶向能力进行验证。比率荧光探针与线粒体一起获得的共定位结果如图3(A - D)所示。可以清楚地看到比率荧光探针具有优越的线粒体靶向能力,比率荧光探针的共定位系数为0.99。因此,探针比率荧光探针在生物系统成像中具有很大的应用潜力。
图 3 RAW264.7细胞共定位实验。(A) RAW264.7细胞用10 μM (λex=405 nm, λem=450 ~ 600 nm)培养;(B) 用Mito Tracker Deep Red (λex=644 nm, λem=650 ~ 570 nm)培养;(C) A与B的合并图像;(D) 亮场图像。标尺:20 μm。
如图4所示,比率荧光探针能够在RAW264.7细胞中对外源性HClO进行荧光检测。用10 μM 比率荧光探针与RAW264.7细胞培养后,分别用5 μM和50 μM HClO处理RAW264.7细胞2 h。正如预期的那样,蓝色通道的明亮荧光逐渐增强,而绿色通道的荧光随之减弱。比值信号(Fblue/Fgreen)的增强幅度分别为0.027 ~ 2.04和7.71。
随后,在RAW264.7巨噬细胞中进一步评估检测内源性HClO的潜力,RAW264.7巨噬细胞在脂多糖(LPS)的生理刺激下可以产生高水平的ROS。细胞先用1 μg/mL LPS处理4 h,再用10 μM 比率荧光探针培养0.5 h。如图4所示,蓝色通道荧光显著增强,绿色通道荧光略有减弱,而比值信号(Fblue/Fgreen)的增强范围在0.027 ~ 1.80之间。
图 4 RAW 264.7细胞中HClO的荧光图像。(a) 比率荧光探针探针(10 μM)染色30min;(b) 5 μM HClO;(c) 50 μM HClO;(d) LPS (1 μg/mL)预处理1 h,比率荧光探针处理30 min,蓝色通道:405 ~ 440 nm,绿色通道:460 ~ 600 nm。标尺:10 μm。
比率荧光探针在细胞中外源性和内源性HClO的出色比率成像,促使我们探索比率荧光探针在体内检测HClO的能力。准备斑马鱼(3-4天),分别用比率荧光探针和HClO刺激。如图5所示,与比率荧光探针 (30 μM)培养1 h后,在绿色通道中观察到明亮的荧光,而在蓝色通道中没有荧光。用15 μM HClO和100 μM HClO刺激斑马鱼2 h后,绿色通道的荧光明显减弱,蓝色通道的荧光明显增强。在使用5 μg/mL LPS处理4 h,30 μM 比率荧光探针培养0.5 h后。其结果如图5所示,蓝色通道的荧光明显增强,而绿色通道的荧光略有减弱证明了比率荧光探针在生物成像应用中的潜力。
综上所述,本发明设计并合成了一种用于线粒体中外源性和内源性HClO成像的比率荧光探针。通过激光共聚焦荧光图像证实了比率荧光探针对线粒体具有良好的靶向能力,共定位系数为0.99,能够对线粒体和斑马鱼中存在的HClO做出高选择性和高灵敏度的反应(检测限为21 nM)。比率荧光探针具有生物相容性、靶向性和比率成像能力等优势,在肿瘤临床诊断方面具有重大的潜力。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (8)
1.一种比率荧光探针,其特征在于,结构式如下:
。
2.一种如权利要求1所述的比率荧光探针的制备方法,其特征在于,包括:
步骤1:将10-己基-吩噻嗪-3-乙醛、2,3-二甲基苯并[d]噻唑-3-鎓碘化物、KOH和DMSO在室温下搅拌并放置12h;
步骤2:将步骤1的粗产物从乙醇中过滤并重结晶,得到深红色固体,所述深红色固体为比率荧光探针。
3.根据权利要求2所述的一种比率荧光探针的制备方法,其特征在于,所述10-己基-吩噻嗪-3-乙醛的摩尔量为0.64mmol,2,3-二甲基苯并[d]噻唑-3-鎓碘化物的摩尔量为1.28mmol。
4.根据权利要求3所述的一种比率荧光探针的制备方法,其特征在于,所述KOH以质量分数计的浓度为50%。
5.根据权利要求4所述的一种比率荧光探针的制备方法,其特征在于,所述KOH体积为5mL,DMSO的体积为10mL。
6.如权利要求1所述的比率荧光探针作为细胞中线粒体的靶向定位用途。
7.如权利要求1所述的比率荧光探针用作巨噬细胞中HClO的检测。
8.如权利要求1所述的比率荧光探针用作斑马鱼体内的HClO的检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410069665.1A CN117603203B (zh) | 2024-01-18 | 2024-01-18 | 一种比率荧光探针及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410069665.1A CN117603203B (zh) | 2024-01-18 | 2024-01-18 | 一种比率荧光探针及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117603203A true CN117603203A (zh) | 2024-02-27 |
CN117603203B CN117603203B (zh) | 2024-04-12 |
Family
ID=89946541
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202410069665.1A Active CN117603203B (zh) | 2024-01-18 | 2024-01-18 | 一种比率荧光探针及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117603203B (zh) |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106518860A (zh) * | 2016-11-07 | 2017-03-22 | 山东大学 | 一种基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针及其应用 |
CN106905310A (zh) * | 2017-03-09 | 2017-06-30 | 济南大学 | 一种检测次氯酸的荧光探针及其制备方法和应用 |
CN108285449A (zh) * | 2017-10-24 | 2018-07-17 | 泰山医学院 | 一种由噻唑修饰的吡啶并[1,2-a]苯并咪唑可以检测次氯酸根离子的荧光探针及应用 |
CN109266331A (zh) * | 2018-12-03 | 2019-01-25 | 许昌学院 | 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 |
CN109781678A (zh) * | 2017-11-14 | 2019-05-21 | 泰山医学院 | 一种应用于线粒体内次氯酸检测的比率荧光探针的制备及应用 |
CN111154288A (zh) * | 2020-01-03 | 2020-05-15 | 华南理工大学 | 检测次氯酸根离子的吩噻嗪类染料及其制备方法与应用 |
CN113135948A (zh) * | 2021-03-12 | 2021-07-20 | 徐州医科大学 | 一种用于检测ClO-/ONOO-的比率荧光探针及其制备方法和应用 |
CN117342803A (zh) * | 2023-08-22 | 2024-01-05 | 湖南科技大学 | 一种次氯酸光电化学传感器的制备方法及其在检测次氯酸中的应用 |
-
2024
- 2024-01-18 CN CN202410069665.1A patent/CN117603203B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106518860A (zh) * | 2016-11-07 | 2017-03-22 | 山东大学 | 一种基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针及其应用 |
CN106905310A (zh) * | 2017-03-09 | 2017-06-30 | 济南大学 | 一种检测次氯酸的荧光探针及其制备方法和应用 |
CN108285449A (zh) * | 2017-10-24 | 2018-07-17 | 泰山医学院 | 一种由噻唑修饰的吡啶并[1,2-a]苯并咪唑可以检测次氯酸根离子的荧光探针及应用 |
CN109781678A (zh) * | 2017-11-14 | 2019-05-21 | 泰山医学院 | 一种应用于线粒体内次氯酸检测的比率荧光探针的制备及应用 |
CN109266331A (zh) * | 2018-12-03 | 2019-01-25 | 许昌学院 | 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 |
CN111154288A (zh) * | 2020-01-03 | 2020-05-15 | 华南理工大学 | 检测次氯酸根离子的吩噻嗪类染料及其制备方法与应用 |
CN113135948A (zh) * | 2021-03-12 | 2021-07-20 | 徐州医科大学 | 一种用于检测ClO-/ONOO-的比率荧光探针及其制备方法和应用 |
CN117342803A (zh) * | 2023-08-22 | 2024-01-05 | 湖南科技大学 | 一种次氯酸光电化学传感器的制备方法及其在检测次氯酸中的应用 |
Non-Patent Citations (2)
Title |
---|
CHENG-LU ZHANG: "A Dual Functional Fluorescent Probe Based on Phenothiazine for Detecting Hg2+ and ClO- and its Applications", JOURNAL OF FLUORESCENCE, 7 December 2023 (2023-12-07), pages 1 - 14 * |
LEI WANG: "Deep-Red AIE-Active Fluorophore for Hypochlorite Detection and Bioimaging in Live Cells", INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH, vol. 57, no. 23, 25 May 2018 (2018-05-25), pages 7735 - 7741 * |
Also Published As
Publication number | Publication date |
---|---|
CN117603203B (zh) | 2024-04-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Guo et al. | Fluorescence chemosensors for hydrogen sulfide detection in biological systems | |
WO2021103700A1 (zh) | 一种可响应硝基还原酶的乏氧探针化合物及其制备与应用 | |
Tang et al. | A mitochondria-targetable far-red emissive fluorescence probe for highly selective detection of cysteine with a large Stokes shift | |
CN109336815B (zh) | 一种检测细胞内质网内次氯酸的双光子荧光探针 | |
CN112745303B (zh) | 一种乏氧荧光探针及其应用 | |
CN113801105B (zh) | 线粒体靶向的过氧亚硝酸根/亚硫酸氢根双响应荧光探针 | |
Yan et al. | A near-infrared and mitochondria-targeted fluorescence probe for ratiometric monitoring of sulfur dioxide derivatives in living cells | |
Zhong et al. | Aggregation-induced fluorescence probe for hypochlorite imaging in mitochondria of living cells and zebrafish | |
CN109608474B (zh) | 一种检测酪氨酸酶的化合物及其制备方法和应用 | |
Li et al. | A bioluminescent probe for imaging endogenous hydrogen polysulfides in live cells and a murine model of bacterial infection | |
Han et al. | Photostable ratiometric two-photon fluorescent probe for visualizing hydrogen polysulfide in mitochondria and its application | |
Yuan et al. | Luminescence of coelenterazine derivatives with C-8 extended electronic conjugation | |
Xin et al. | A thiocarbonate-caged fluorescent probe for specific visualization of peroxynitrite in living cells and zebrafish | |
CN109651249A (zh) | 一种检测细胞内质网半胱氨酸的荧光探针及其合成和应用 | |
Shen et al. | Employing an ICT-ESIPT strategy for ratiometric tracking of HClO based on sulfide oxidation reaction | |
CN108752373B (zh) | 一种基于苯硼酯识别过氧化氢的荧光探针 | |
Liu et al. | A pH-switchable azo-based fluorescence reporter for lysosome-confined visualization of hypoxia status | |
CN117603203B (zh) | 一种比率荧光探针及其制备方法和应用 | |
CN111548790A (zh) | 一种近红外比率型荧光探针及其合成方法与应用 | |
CN107011891B (zh) | 一种Cu+荧光探针及其制备方法和应用 | |
CN112457360B (zh) | 一种肝靶向的过氧亚硝酸根荧光探针及制备方法和应用 | |
CN110746339B (zh) | 一种吡咯双腙衍生物荧光探针及其制备方法和应用 | |
CN113200975A (zh) | 基于靛红衍生物专一响应onoo-的水溶性荧光探针、制备方法及应用 | |
CN107915705B (zh) | 一种二硫苏糖醇荧光探针 | |
CN116925025B (zh) | 一种快速检测硝基还原酶的近红外荧光探针及其制备和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |