CN110467621B - 一种次氯酸离子小分子荧光探针及其制备方法与应用 - Google Patents
一种次氯酸离子小分子荧光探针及其制备方法与应用 Download PDFInfo
- Publication number
- CN110467621B CN110467621B CN201910813749.0A CN201910813749A CN110467621B CN 110467621 B CN110467621 B CN 110467621B CN 201910813749 A CN201910813749 A CN 201910813749A CN 110467621 B CN110467621 B CN 110467621B
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- small molecule
- clo
- hypochlorous acid
- molecule fluorescent
- 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.)
- Active
Links
- -1 Hypochlorous acid ion small-molecule Chemical class 0.000 title claims abstract description 29
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 claims abstract description 10
- 150000003384 small molecules Chemical class 0.000 claims description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000010521 absorption reaction Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 150000002500 ions Chemical class 0.000 claims description 7
- 239000012472 biological sample Substances 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 claims description 3
- 201000011510 cancer Diseases 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 230000005284 excitation Effects 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000002835 absorbance Methods 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 abstract description 11
- 241000252212 Danio rerio Species 0.000 abstract description 8
- 241000894006 Bacteria Species 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 55
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 239000003068 molecular probe Substances 0.000 description 11
- 241000588724 Escherichia coli Species 0.000 description 8
- 230000000694 effects Effects 0.000 description 5
- 230000002194 synthesizing effect Effects 0.000 description 5
- 238000003775 Density Functional Theory Methods 0.000 description 4
- 150000001450 anions Chemical class 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 229910001914 chlorine tetroxide Inorganic materials 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 238000001506 fluorescence spectroscopy Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Chemical compound [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000005281 excited state Effects 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000005311 nuclear magnetism Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000283070 Equus zebra Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005443 coulometric titration Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 238000004770 highest occupied molecular orbital Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004768 lowest unoccupied molecular orbital Methods 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004776 molecular orbital Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000007903 penetration ability Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004313 potentiometry Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- ODHXBMXNKOYIBV-UHFFFAOYSA-N triphenylamine Chemical compound C1=CC=CC=C1N(C=1C=CC=CC=1)C1=CC=CC=C1 ODHXBMXNKOYIBV-UHFFFAOYSA-N 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
- C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种次氯酸离子小分子荧光探针及其制备方法与应用,三苯胺肟和螺吡喃醛结构通过氮氮键连接的母体结构,具有很强共轭π电子,其中螺吡喃环容易开环,结构中螺吡喃环开环后能发射很强的荧光,可以实现荧光技术精确检测次氯酸,并且可以检测活细胞、细菌和斑马鱼体内的内源性的次氯酸,因此在次氯酸检测方面具有良好的应用前景,同时,本发明的合成方法简单、操作方便,不需要苛刻的条件。
Description
技术领域
本发明涉及荧光成像分子探针领域,尤其是涉及一种利用荧光成像技术检测ClO-离子的探针,具体涉及一种次氯酸离子分子探针及制备方法和应用。
背景技术
次氯酸是一种不稳定的弱酸,常用于消毒剂和漂白剂的主要成分。而且,在生物体内具有重要的生理功能,如免疫系统中可以充当抗菌抗病毒等重要功能。此外,次氯酸在维持生物体内环境中起着重要角色,如平衡体内氧化还原过程。但是,次氯酸在生物体内的量一旦超过机体所需的正常范围,便会对机体内环境造成一定损害,例如,过量的次氯酸也可能对一下磷脂,蛋白以及核酸物质产生严重损伤。因此,开发高灵敏度和高选择性的小分子探针来检测次氯酸仍然是一个艰巨的挑战。
目前,用于检测次氯酸的方法很多,例如比色法,电位分析法,化学发光发以及库仑滴定发等。但是,基于荧光探针的次氯酸检测技术正在成为一种重要研究方法。近几年报道了很多小分子探针对次氯酸选择性识别,尽管如此多的次氯酸识别探针被报道,但是目前对于荧光和可视化双功能识别的探针报道甚少,而且该类探针对次氯酸识别能力和选择性都不是很理想,鉴于此,本研究发明一种高选择性和高灵敏度的双功能次氯酸识别荧光探针。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一种高选择性和高灵敏度的次氯酸识别荧光探针。
为实现上述目的,本发明提供一种次氯酸离子小分子荧光探针,所述分子探针分子式为C43H42N4O,其结构式为:
本发明还提供上述小分子荧光探针的制备方法:将分子式为C19H17N3的三苯胺肟和分子式为C24H27NO2的螺吡喃醛溶解在乙醇溶液中,并在室温下搅拌反应4-24h,反应得到黄色固体并用乙醇洗涤干燥得到目标产物;
进一步的,原料的摩尔比为:
1~2份三苯胺肟;
1~2份螺吡喃醛。
本发明还提供上述小分子荧光探针在制备检测、识别环境中或生物样品中次氯酸检查试剂盒中的应用。
作为本发明的一种应用范围,所述小分子荧光探针在制备利用荧光成像检测正常细胞和癌细胞中外源性的ClO-离子的试剂盒中应用。
作为本发明的一种应用范围,所述小分子荧光探针在制备细胞体外ClO-离子检测试剂盒中的应用。
作为本发明上述应用的检测方式,通过紫外分光光度法,在200nm~650nm的波长范围内测定含有ClO-的样品溶液的吸光度;在最大吸收波长530nm下识别环境中或生物样品中ClO-离子。
作为本发明的一种应用方式,通过荧光分光光度法,以408nm为激发波长,在400nm到700nm的波长范围内测定含有CIO-离子样品溶液的荧光强度;在最大发射波长605nm下识别环境中或生物样品中的ClO-离子。
本发明具有如下优点:通过本发明所述制备方法合成小分子探针,以三苯胺肟和螺吡喃醛为基础,使用紫外和荧光光谱法检测次氯酸。三苯胺肟和螺吡喃醛通过氮氮键连接的母体结构,具有很强共轭π电子,三苯胺和螺吡喃醛通过氮氮键连接的母体结构中螺吡喃环容易开环,结构中螺吡喃环开环后能发射很强的荧光。小分子探针在次氯酸的存在下结构中螺吡喃环发生开环,使得其荧光从无到有,紫外吸收峰红移。可以实现荧光技术精确检测次氯酸,并且可以检测活细胞、细菌和斑马鱼体内的内源性的次氯酸。因此在次氯酸检测方面具有良好的应用前景。同时,本发明的合成方法简单、操作方便,不需要苛刻的条件。
附图说明
图1为实施例1中合成小分子探针的路线;
图2为实施例1中合成小分子探针的核磁氢谱;
图3为实施例1中合成小分子探针的质谱;
图4为实施例1中合成小分子探针对次氯酸识别的激发态分子内质子转移(ESIPT)机制;
图5为实施例2中小分子探针对次氯酸识别的紫外和荧光光谱;
图6为实施例4中验证小分子探针对次氯酸识别的密度泛函理论计算;
图7为实施例5中小分子探针在人胚肺细胞(MRC-5)中检测内源性次氯酸的生物成像;
图8为实施例6中小分子探针在大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)中检测内源性次氯酸的生物成像;
图9为实施例7中小分子探针在斑马鱼体内检测内源性次氯酸的生物成像;
图10(a)SPTPA(2.0μM)对各种物质(3.0eq.)以及ClO-在DMSO/H2O溶液体系中的发射光谱;(b)SPTPA添加分析物在605nm处的荧光强度变化,包括F-,Cl-,NO2 -,ClO4 -,HCO3 -,H2PO4 -,SO4 2-,S2O3 2-,CO3 -,H2O2和ClO-。
图11(a)用ClO-(3.0eq.)处理之前和之后SPTPA的时间依赖性荧光;(b)pH对SPTPA(2.0μM)的影响及其对ClO-的识别能力。数据表示在605nm处的荧光强度,并且在25℃下添加ClO-后3min获得光谱,条件:λex=400nm,λem=605nm。
具体实施方式
下面将结合实施例和效果例对本发明做进一步的详述,而非限制本发明的范围。
实施例1合成小分子探针
将三苯胺肟(0.238g,0.83mmol)和螺吡喃醛(0.300g,0.83mmol)溶解在30mL乙醇溶液中,其中三苯胺肟以及螺吡喃醛由安耐吉化学定制,结构式如图1中所示,并在室温下搅拌16小时,得到淡黄色沉淀;将上述淡黄色沉淀过滤,用乙醇反复洗涤,干燥得到黄色固体粉末0.183g,简写记为SPTPA,产率为35%,通过核磁和质谱可以确定该产物即为目标小分子探针。合成小分子探针的路线为图1,图1表示合成小分子荧光探针的路线图,其中EtOH为乙醇,RT为室温。
核磁表征为图2,质谱表征为图3以及小分子荧光探针对次氯酸识别机制模型图为图4。
探针质谱:
HR-MS(ESI)m/z[M+1]+:Calcd for C43H43N4O,631.3431,found,631.3425.
探针核磁氢谱:
1H NMR(600MHz,DMSO-d6)δin ppm:8.80(s,1H),8.49(s,1H),7.75(s,1H),7.62-7.47(m,2H),7.20(br s,6H),7.11-6.87(m,10H),6.76(br s,3H),2.33(s,3H),1.12(s,9H),1.10(s,3H),1.04(s,3H);
实施例2小分子探针对次氯酸识别的紫外和荧光光谱
制备1mL小分子探针(10μM和2.0μM)的DMSO/H2O(v/v=5∶1)溶液。同浓度的ClO-溶液滴加到探针溶液中,
如图5(a)所示,在探针溶液中加入ClO-后,在298nm和408nm处的吸收带逐渐减少,350nm~600nm处有一个新的吸收峰,并在530nm处出现最大吸收峰,其吸光强度随ClO-浓度增加并逐渐增加,最终两者化学计量比为1∶3。在荧光滴定实验中,制备1mL小分子探针(2.0μM)的DMSO/H2O(v/v=5∶1)溶液。同浓度的ClO-溶液滴加到探针溶液中,以408nm为激发波长测量探针从400nm到700nm的荧光值,实验结果见图5(b)。可观察到探针的荧光强度随ClO-浓度增加而增强,当两者浓度比例为1∶3时,荧光强度达到饱和。
实施例3验证小分子探针对次氯酸选择性和竞争性。
制备5.0mL分子探针(5.0×10-6mol/L)的DMSO/H2O(v/v=1∶1)溶液。通过将相应的盐溶于去离子水制备各种阴离子溶液[F-,Cl-,NO2 -,ClO4 -,HCO3 -,H2PO4 -,SO4 2-,S2O3 2-,CO3 -,H2O2,ClO-](1.0×10-3mol/L)。随后,将同等当量的阴离子溶液加入到探针溶液中。通过荧光光谱进行检测,实验结果见图10(a)。取荧光最大吸收波长进行对比,如图10(b)所示,离子包括F-,Cl-,NO2 -,ClO4 -,HCO3 -,H2PO4 -,SO4 2-,S2O3 2-,CO3 -,H2O2,ClO-。除次氯酸离子外,这些阴离子对探针的荧光都没有产生明显变化。在加入次氯酸离子后,小分子探针在605nm处的荧光强度完全淬灭,λex=408nm。而且在探针结合次氯酸离子后,再加入其它阴离子,其它阴离子对体系的荧光强度也不产生干扰,可以判定探针和次氯酸离子具有很强的结合能力。
同时,检测了探针对次氯酸离子响应的时间动力学研究和不同pH值条件下的荧光强度变化,实验结果见图11(a、b)可以看出小分子探针对次氯酸离子有很快的响应,且SPTPA结合次氯酸离子荧光增强并达到值的时间在60s以内。在pH为2-12之间,探针以及探针与次氯酸离子的复合物荧光都能够保持好现有的荧光强度。
实施例4小分子探针对ClO-识别的密度泛函理论研究
为了进一步了解探针的分子结构,荧光和吸收光谱的传感机理,进行了有无ClO-情况下探究了小分子探针的理论计算,以密度泛函理论深入了解它们的电子跃迁(DFT).如图6所示,提出了SPTPA和ClO-反应前后的分子轨道图,并且优化的结构显示SPTPA可以与ClO-反应使C-O形成-OH。这与上述光谱实验的结果一致。此外,SPTPA基态的电子云主要位于分子结构的=N-N=结构中,当转移并分散到整个共轭体系和周围原子激发时,表明中等激发态分子内质子转移(ESIPT)在分子中的作用。此外,闭环和开环结构的能级从HOMO(-0.150/-0.195eV)到LUMO(-0.058/-0.111eV)的水平为0.092eV/0.084eV,SPTPA和其他轨道也是如此。
实施例5小分子探针的在癌细胞中成像效果
为了探索小分子探针对于活细胞中亚细胞标记和保护ClO-的潜在生物学可用性,在MRC-5细胞系中进行细胞生物成像。为了证明探针检测内源性ClO-细胞的能力,我们进行了阴性和阳性对照的成像分析作为有力的证据。当细胞与游离小分子探针一起温育时,观察到可见荧光信号,图7所示。之后,将这些预处理细胞与探针一起温育20分钟,加入ClO-(6.0μM)立即观察到红色通道中的显着荧光强度。这些结果清楚地表明SPTPA探针满足监测活细胞中内源ClO-的要求。
实施例6小分子探针的在正常细胞中成像效果
为了进一步探索其应用,使用探针通过使用荧光成像的生物成像来感测大肠杆菌和金黄色葡萄球菌中的ClO-。图8所示,单独用2.0μM探针染色的大肠杆菌和金黄色葡萄球菌在大肠杆菌的红色通道中几乎没有荧光,在金黄色葡萄球菌中没有荧光。随着ClO-的加入,红色通道中的荧光变得更强大肠杆菌和金黄色葡萄球菌,大肠杆菌和金黄色葡萄球菌的“棒状”和“球状”形状可以从荧光模式中清晰地看到。分别在缩放区域。这些结果证明探针可用于追踪大肠杆菌和金黄色葡萄球菌中的ClO-,这将是研究细菌中ClO-生物转化的潜在工具。
实施例7小分子探针的在斑马鱼中成像效果
在正常的代谢过程中,生物体的器官可以自发地产生各种ROS。到目前为止,大多数研究报告证明荧光成像的可能性是通过预先喂入/注射或将ROS诱导的化学物质添加到生物体中而引入额外的ROS。然而,荧光探针在正常生物中对自发ROS的生物成像仍然面临探针的灵敏度和稳定性及其通过各种生物屏障的能力的问题。在活体动物的数量上产生了许多生物过程,导致ROS(ClO-)在那里自我产生。因此,为了研究探针对生物体内源性和外源性ClO-的潜在生物学应用,5日龄斑马鱼是一种流行的脊椎动物模型,它们被选为我们的研究模型系统。图9所示,斑马鱼在胚胎培养基中培养,并与游离探针(2.0μM)孵育20分钟,以确保探针渗透到斑马鱼的整个组织中,在腹部显示出弱而可见的红色荧光。此外,斑马鱼用探针预处理,用PBS洗涤三次,分别与ClO-(6.0μM)一起孵育,在斑马鱼的头部和腹部显示出显着的红色荧光。因此,这些结果表明探针具有高组织穿透能力,并且可以实现斑马鱼中ClO-的可视化
本发明具有如下优点:通过本发明所述制备方法合成小分子探针,还可以实现紫外和荧光光谱法精确传感ClO-,并且可以快速、准确的检测细胞、细菌和斑马鱼体内中内源性的ClO-。因此在ClO-检测方面具有良好的应用前景。同时,本发明的合成方法简单、操作方便,不需要苛刻的条件。
以上仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理前提下的若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
3.根据权利要求2所述的一种次氯酸离子小分子荧光探针的制备方法,其特征在于:原料的摩尔比为:
1~2份三苯胺肟;
1~2份螺吡喃醛。
4.根据权利要求1所述的次氯酸离子小分子荧光探针的应用,其特征在于,在制备检测、识别环境中或生物样品中次氯酸检查试剂盒中的应用。
5.根据权利要求4所述的次氯酸离子小分子荧光探针的应用,其特征在于,所述小分子荧光探针在制备利用荧光成像检测正常细胞和癌细胞中外源性的ClO-离子的试剂盒中应用。
6.根据权利要求1所述的次氯酸离子小分子荧光探针的应用,其特征在于,所述小分子荧光探针在制备细胞体外ClO-离子检测试剂盒中的应用。
7.根据权利要求4所述的次氯酸离子小分子荧光探针的应用,其特征在于,在制备通过紫外分光光度法,在200nm~650nm的波长范围内测定含有ClO-的样品溶液的吸光度;在最大吸收波长530nm下识别环境中或生物样品中ClO-离子的试剂盒中的应用。
8.根据权利要求4所述的次氯酸离子小分子荧光探针的应用,其特征在于,在制备通过荧光分光光度法,以408nm为激发波长,在400nm到700nm的波长范围内测定含有ClO-离子样品溶液的荧光强度;在最大发射波长605nm下识别环境中或生物样品中的ClO-离子的试剂盒中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910813749.0A CN110467621B (zh) | 2019-08-30 | 2019-08-30 | 一种次氯酸离子小分子荧光探针及其制备方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910813749.0A CN110467621B (zh) | 2019-08-30 | 2019-08-30 | 一种次氯酸离子小分子荧光探针及其制备方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110467621A CN110467621A (zh) | 2019-11-19 |
CN110467621B true CN110467621B (zh) | 2020-08-25 |
Family
ID=68514133
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910813749.0A Active CN110467621B (zh) | 2019-08-30 | 2019-08-30 | 一种次氯酸离子小分子荧光探针及其制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110467621B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112679529B (zh) * | 2020-03-25 | 2022-02-11 | 常州大学 | 一种识别Cr3+、Fe3+和Al3+离子的螺吡喃比色探针及其制备方法 |
CN111892552A (zh) * | 2020-07-20 | 2020-11-06 | 山西大学 | 一种三苯胺衍生物及其制备方法和在对硫化氢的双通道荧光检测中的应用 |
CN113686844B (zh) * | 2021-08-04 | 2023-11-21 | 齐鲁工业大学 | 三苯胺衍生物在检测过氧化氢和/或次氯酸根中的应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009141236A1 (en) * | 2008-05-20 | 2009-11-26 | Basf Se | Spiropyrans having fluorophoric groups |
-
2019
- 2019-08-30 CN CN201910813749.0A patent/CN110467621B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009141236A1 (en) * | 2008-05-20 | 2009-11-26 | Basf Se | Spiropyrans having fluorophoric groups |
Non-Patent Citations (4)
Title |
---|
Photochromic Spiropyran Dendrimers: ‘Click’ Syntheses, Characterization, and Optical Properties;Qiong Zhang,等;《Macromol. Rapid Commun.》;20081231;第29卷(第3期);第193-201页 * |
Synthesis of 5"-Functionalized Indolinospiropyrans with Vinylene Unit as Linker;NIU, Chengshan,等;《Chinese Journal of Chemistry》;20091231;第27卷(第10期);第2001-2006页 * |
次氯酸荧光探针的研究进展;王延宝,赵宝祥;《有机化学》;20160318;第36卷;第1539-1554页 * |
识别次氯酸的荧光探针;李海东,等;《化学进展》;20170105;第29卷(第1期);第17-35页 * |
Also Published As
Publication number | Publication date |
---|---|
CN110467621A (zh) | 2019-11-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110467621B (zh) | 一种次氯酸离子小分子荧光探针及其制备方法与应用 | |
Chen et al. | Pillararene-based fluorescent chemosensors: recent advances and perspectives | |
Müller et al. | Red‐to NIR‐Emitting, BODIPY‐Based, K+‐Selective Fluoroionophores and Sensing Materials | |
Fan et al. | Energy transfer cassettes based on organic fluorophores: construction and applications in ratiometric sensing | |
Guo et al. | Recent progress in the development of near-infrared fluorescent probes for bioimaging applications | |
US9315465B2 (en) | Photostable AIE luminogens for specific mitochondrial imaging and its method of manufacturing thereof | |
Gao et al. | A self-assembled fluorescent organic nanoprobe and its application for sulfite detection in food samples and living systems | |
CN107922834B (zh) | 用具有aie特性的荧光光稳定线粒体特异生物探针实时监测线粒体自噬过程 | |
Chen et al. | Energy transfer-based biodetection using optical nanomaterials | |
CN110563685B (zh) | 一种以3-羟基黄酮为荧光团的小分子荧光探针及其制备方法与应用 | |
Novakova et al. | Azaphthalocyanines: red fluorescent probes for cations | |
CN107602600B (zh) | 氨基取代氮杂氟硼二吡咯近红外pH荧光探针及其制法和用途 | |
He et al. | The ICT-based fluorescence and colorimetric dual sensing of endogenous hypochlorite in living cells, bacteria, and zebrafish | |
Srivastava et al. | Protein assisted fluorescence enhancement of a dansyl containing fluorescent reagent: Detection of Hg+ ion in aqueous medium | |
CN111116534B (zh) | 一种小分子探针cmsh以及制备方法及应用 | |
CN109705111B (zh) | 一种汞离子检测探针及其制备方法和应用 | |
Zhan et al. | Small molecule-based bioluminescence and chemiluminescence probes for sensing and imaging of reactive species | |
Cen et al. | A double-cavity nor-seco-cucurbit [10] uril-based fluorescent probe for detection of ClO-and its application in cell imaging | |
CN106496217A (zh) | 一种新型检测h2s荧光分子探针的制备方法和应用 | |
Nair et al. | Naked eye instant reversible sensing of Cu2+ and its in situ imaging in live brine shrimp Artemia | |
Ambikapathi et al. | Bioimaging of peroxynitrite in MCF-7 cells by a new fluorescent probe rhodamine B phenyl hydrazide | |
Huang et al. | Nanomolar detection of adenosine triphosphate (ATP) using a nanostructured fluorescent chemosensing ensemble | |
CN110818646B (zh) | 基于聚集诱导发光小分子荧光探针及其制备方法及应用 | |
Ganesan et al. | Function-oriented synthesis of fluorescent chemosensor for selective detection of Al3+ in neat aqueous solution: Paperstrip detection & DNA bioimaging | |
Adegoke et al. | Conjugation of mono-substituted phthalocyanine derivatives to CdSe@ ZnS quantum dots and their applications as fluorescent-based sensors |
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 |