CN111825718A - 基于喹啉-氧杂蒽的碱性磷酸酶荧光探针的制备和应用 - Google Patents
基于喹啉-氧杂蒽的碱性磷酸酶荧光探针的制备和应用 Download PDFInfo
- Publication number
- CN111825718A CN111825718A CN202010709048.5A CN202010709048A CN111825718A CN 111825718 A CN111825718 A CN 111825718A CN 202010709048 A CN202010709048 A CN 202010709048A CN 111825718 A CN111825718 A CN 111825718A
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- alp
- probe
- alkaline phosphatase
- 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.)
- Granted
Links
- 102000002260 Alkaline Phosphatase Human genes 0.000 title claims abstract description 85
- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims abstract description 85
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 70
- NXOWJSWURYSBHA-UHFFFAOYSA-N N1=CC=CC2=CC=CC=C12.C1=CC=CC=2OC3=CC=CC=C3CC12 Chemical compound N1=CC=CC2=CC=CC=C12.C1=CC=CC=2OC3=CC=CC=C3CC12 NXOWJSWURYSBHA-UHFFFAOYSA-N 0.000 title abstract description 8
- 238000002360 preparation method Methods 0.000 title abstract description 7
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 3
- 239000012043 crude product Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012265 solid product Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 29
- 230000004044 response Effects 0.000 abstract description 11
- 238000001514 detection method Methods 0.000 abstract description 10
- 239000001018 xanthene dye Substances 0.000 abstract description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 229910052760 oxygen Inorganic materials 0.000 abstract description 3
- 239000001301 oxygen Substances 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 2
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 abstract 1
- 150000002500 ions Chemical class 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 125000003396 thiol group Chemical class [H]S* 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 238000002189 fluorescence spectrum Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 208000020084 Bone disease Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- IVIJMWMIZGPBFU-UHFFFAOYSA-N C1=CC=CC=2OC3=CC=CC=C3CC12.N1=CC=CC2=CC=C3C(=C12)C=CC=C3 Chemical compound C1=CC=CC=2OC3=CC=CC=C3CC12.N1=CC=CC2=CC=C3C(=C12)C=CC=C3 IVIJMWMIZGPBFU-UHFFFAOYSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229940027991 antiseptic and disinfectant quinoline derivative Drugs 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 150000003248 quinolines Chemical class 0.000 description 1
- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 229910000166 zirconium phosphate Inorganic materials 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
-
- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
技术领域
本发明属于荧光探针技术领域,具体涉及基于喹啉-氧杂蒽染料的碱性磷酸酶荧光探针的制备和应用。
背景技术
碱性磷酸酶(ALP)作为一种能够将蛋白质和非蛋白质底物去磷酸化的水解酶,广泛存在于哺乳动物的肝脏、肾脏、肠道、骨骼和胎盘在内的许多组织中(K.Ooi,K.Shiraki,Y.Morishita,J.Clin.Lab.Anal.2007,21,133;J.N.Fernandez,A.B.Kidney,Vet.Clin.Pathol.2007,36,223-233.)。ALP作为目前公认的生物标志物,参与许多重要的生理和病理过程(J.E.Coleman.Annu.Rev.Biophys.Biomol.Struct.1992,21,441-483;J.Stebbing,L.C.Lit,H.Zhang,R.S.Darrington,O.Melaiu,B.Rudraraju,G.Giamas,Oncogene.2014,33,939-953.)。同时,ALP的活性与细胞的分化和生存能力有关,而且已经证实ALP水平异常与许多疾病的发生和发展密切相关,例如:乳腺癌,前列腺癌,心脏病,骨病,糖尿病等(R.H.Christenson,Clin.Biochem.1997,30,573-593;M.Syakalima,M.Takiguchi,J.Yasuda,Y.Mortal,A.Hashimoto,Vet.Q.1998,20,18-22;P.H.Lange,J.L.Millan,T.Stigbrand,R.L.Vessella,E.Ruoslahti,W.H.Fishman,Cancer.Res.1982,42,3244-3247;A.C.Hellberg,F.D.Nat.Rev.Cancer.2006,6,307-320;K.Ooi,K.Shiraki,Y.Morishita,T.Nobori,J.Clin.Lab.Anal.2007,21,133-139.)。因此,开发一种方便可靠的实时检测生物体中ALP活性的方法显得非常重要。
ALP的传统检测方法包括电化学法、比色法和色谱法,这些方法通常不仅需要复杂的操作,而且无法实现体内检测。荧光方法因其具有操作简便、灵敏度高、选择性好、实时和无创性等诸多优点而备受关注(G.Deng,S.Li,Z.Sun,W.Li,L.Zhou,J.Zhang,P.Gong,L.Cai,Theranostics.2018,8,4116-4128;G.Hong,S.Diao,J Chang,A.L.Antaris,C.Chen,B.Zhang,S.Zhao,D.N.Atochin,P.L.Huang,K.I.Andreasson,C.J.Kuo,H.Dai,Nat.Photonics.2014,8,723-730)。到目前为止,已经开发了一些检测ALP的荧光探针,用于实时监测细胞或者活体内的ALP活性(X.F.Hou,Q.X.Yu,F.Zeng,H.J.Ye,S.J.Wu,J.Mater.Chem.B.2015,3,1042-1048;J.Liang,R.T.K.Kwok,H.Shi,B.Z.Tang,B.Liu,ACSAppl.Mater.Interfaces.2013,5,8784-8789;H.Zhang,C.Xu,J.Liu,X.Li,L.Guo,X.Li,Chem.Commun.2015,51,7031-7034;X.Gu,G.Zhang,Z.Wang,W.Liu,L.Xiao,D.Zhang,Analyst.2013,138,2427-2431.)。但是,由于这些探针发射波长短,没有达到近红外范围。使得探针容易受到自身背景荧光的干扰,从而阻碍了它们在生物系统中的应用。因此,设计一种具有近红外发射的荧光探针是至关重要的。
喹啉-氧杂蒽作为一种新型的荧光染料,具有水溶性好、灵敏度高等优点。特别是,由于其染料具有近红外发射,因此具有较深的组织穿透深度,不易受到生物自体荧光的干扰,对生物成像更有利。研究发现,利用喹啉衍生物的荧光探针已经成功应用于检测了一些目标物,如:亚硫酸盐、Zn2+等(L.Tan,W.Y.Lin,S.S.Zhu,L.Yuan,K.B.Zheng,Org.Biomol.Chem.2014,12,4637-4643;Z.Q.Mao,L.Hu,X.H.Dong,C.Zhong,B.F.Liu,Z.H.Liu,Anal.Chem.2014,86,6548-6554.)。但是,到现在为止,还没有基于喹啉-氧杂蒽染料作为荧光探针来检测ALP。因此,设计和合成一种基于喹啉-氧杂蒽染料的荧光探针来检测ALP是非常有必要的。
发明内容
根据所提出的要求,本发明人对此进行了深入研究,在付出了大量创造性劳动后,提供了一种基于苯喹啉-氧杂蒽染料的碱性磷酸酶近红外荧光探针。
本发明的技术方案是,一种碱性磷酸酶近红外荧光探针,其结构式如下:
一种碱性磷酸酶近红外荧光探针的制备方法。步骤如下:
在N2保护下,将0.5当量的QX-OH,2.0~3.0当量的三氯氧磷,2.0~3.0当量的吡啶分别加入到25mL圆底烧瓶中,然后加入5~8mL的CH2Cl2将其溶解。反应在室温下搅拌4小时后,将所得混合物倒入冰中并继续搅拌过夜。反应完成后,减压除去溶剂,所得粗产物用展开剂CH2Cl2/CH3OH=2∶1进行柱层析纯化,得到深蓝色固体产物QX-P,即为所述的荧光探针。
本发明的有益效果是,一种基于喹啉-氧杂蒽染料的碱性磷酸酶近红外荧光探针的良好的光谱响应性能。首先,研究该探针的荧光光谱性质。探针本身在770nm处没有明显的近红外发射峰;当探针中加入ALP后,在770nm处出现了明显的近红外发射峰。并且随着ALP浓度的增大,探针的近红外荧光强度不断增强。当加入1.0U/mL的ALP时,荧光强度大约增强6倍,因此可以很好的检测ALP。该探针的检测范围从0.05U/mL到1.0U/mL,检测限为0.017U/mL,这说明该探针可以高灵敏的检测ALP。接着,研究了探针的紫外吸收光谱。在没有加入ALP时,探针在568nm处有吸收带;加入ALP后,568nm处的吸收峰逐渐降低,在717nm附近出现新的吸收峰。然后,研究探针的选择性,考察了探针与各种金属离子(Na+,Ca2+,Mg2+)和阴离子(Cl-,Br-,OH-),活性氧(ClO-,O2 -,H2O2),生物硫醇(Cys,Hcy,GSH),氨基酸(Tyr,Pro,Phe,Lue),生物酶(AChE,GGT,PDE,trypsin)以及检测物(ALP)的荧光响应情况。结果发现,只有ALP能引起荧光光谱的改变,其他检测物对探针的荧光光谱没有明显的影响。最后,研究了pH值对荧光探针测定ALP的影响,当pH值在7.0到9.0之间时,不影响荧光探针对ALP的测定。此外,该荧光探针响应比较迅速,响应时间在10分钟以内。
一种碱性磷酸酶近红外荧光探针的应用。在对照组细胞中观察不到明显的荧光,当细胞中加入荧光探针后,可以明显地观察到较强的荧光,这说明细胞中的ALP含量较高。而用钒酸钠(Na3VO4)处理抑制细胞内ALP的产生,发现细胞内的荧光明显减弱。这些结果说明荧光探针能检测到细胞内产生的ALP,这为监控人体内和碱性磷酸酶相关病变提供一种可靠的手段。
附图说明
图1为荧光探针的合成路线。
图2为荧光探针与不同浓度的ALP作用后的荧光光谱图。
横坐标为波长,纵坐标为荧光强度。荧光探针的浓度为10μM,ALP的浓度分别为:0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0U/mL。发射波长为770nm,对应的激发波长为717nm。
图3为荧光探针对不同ALP浓度的荧光线性响应图。
图4为荧光探针与ALP作用后的紫外可见吸收光谱图。
横坐标为波长,纵坐标为吸光度。荧光探针的浓度为10μM,ALP浓度为1.0U/mL。
图5为荧光探针的选择性图。
荧光探针的浓度为10μM,ALP浓度为1.0U/mL,其它分析物浓度均为2eq。
图6为pH对荧光探针的影响图。
图7为荧光探针与ALP作用后荧光强度随时间变化的关系曲线图。
图8为细胞毒性实验图。横坐标为荧光探针的浓度,纵坐标为细胞的存活率。
图9荧光探针与ALP作用的细胞成像图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不限于此。
实施例1:
荧光探针的合成
合成路线如图1。在N2保护下,将QX-OH(190mg,0.5mmol),三氯氧磷(230mg,1.5mmol),吡啶(119mg,1.5mmol)分别加入到25mL圆底烧瓶中,然后加入6mL的CH2Cl2将其溶解。反应在室温下搅拌4小时后,将所得混合物倒入冰中并继续搅拌过夜。反应完成后,减压除去溶剂,所得粗产物用柱层析纯化(CH2Cl2/CH3OH=2∶1),得到深蓝色固体产物QX-P(150mg,产率65%),即为所述的荧光探针。1H NMR(400MHz,DMSO)δ8.66(d,J=9.4Hz,1H),8.56(t,J=10.8Hz,2H),8.34(d,J=9.3Hz,1H),8.22(d,J=7.7Hz,1H),8.07–7.97(m,1H),7.80–7.75(m,1H),7.25(d,J=8.4Hz,1H),7.07(s,1H),6.92(s,1H),6.71–6.66(m,2H),4.93–4.88(m,2H),2.64(t,J=6.2Hz,4H),1.84–1.79(m,2H),1.52(t,J=6.9Hz,3H).13CNMR(100MHz,DMSO)δ160.8,156.8,154.3,141.6,138.7,134.6,130.0,129.6,128.6,128.1,127.3,126.2,121.0,118.5,114.3,113.3,112.3,110.8,102.8,45.7,29.1,24.6,20.8,13.7.MS(TOF):462.2.
实施例2:
荧光探针和ALP溶液配制
探针溶液的制备:称取一定量探针溶解在二甲基亚砜中,配成1×10-4M的备用溶液。将1.0mL探针的备用溶液加入到10mL的容量瓶中,用Tris-HCl缓冲溶液定容后,得到浓度为1.0×10-5mol/L的荧光探针溶液。将ALP分别配制为以下浓度(0,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0U/mL)。
实施例3:
荧光探针与ALP作用的荧光光谱的测定
图2为荧光探针与ALP作用的荧光光谱,荧光探针的浓度为10μM,ALP的浓度依次为0,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0U/mL。实验所用激发波长为717nm,发射波长范围为730~900nm。狭缝宽度为10.0nm/10.0nm,所用的荧光测定仪器为日立F4600荧光分光光度计。从图2可以看出,加入ALP之前,由于磷酸根的淬灭作用,探针本身几乎没有发射峰;随着ALP的加入,在770nm处发射峰大幅度的增强,并且随着ALP浓度的增大,探针的荧光强度不断增强。图3为探针对不同ALP浓度的线性响应图。荧光强度跟ALP的浓度呈现线性关系,线性范围是0.05U/mL~1.0U/mL,检测限是0.017U/mL。这说明该探针可以高灵敏的检测ALP。
实施例4:
荧光探针与ALP作用的紫外可见吸收光谱的测定
图4为荧光探针与ALP作用后的紫外可见吸收光谱图,荧光探针的浓度为10μM,ALP的加入量为1.0U/mL。紫外可见吸收光谱测定用的仪器为安捷伦Cary60紫外可见分光光度计。从图4中可以看出,探针本身在568nm处有吸收带;加入ALP之后,568nm处的吸收峰逐渐降低,在717nm附近出现新的强吸收峰。
实施例5:
荧光探针对ALP测定的选择性
图5为荧光探针对ALP测定的选择性图。考察在浓度为10μM的荧光探针中加入ALP(1.0U/mL)及各种金属离子(Na+,Ca2+,Mg2+)和阴离子(Cl-,Br-,OH-),活性氧(ClO-,O2 -,H2O2),生物硫醇(Cys,Hcy,GSH),氨基酸(Tyr,Pro,Phe,Lue),生物酶(AChE,GGT,PDE,trypsin)的荧光响应情况。从图5中可以看出,只有ALP能引起荧光光谱的明显增强,其他检测物对探针的荧光光谱没有明显的影响。这些结果表明,荧光探针对ALP有良好的选择性。
实施例6:
溶液pH值对荧光探针测定ALP的荧光性质的影响
考察pH值对荧光探针测定ALP的荧光光谱的影响,其结果如图6。我们研究的pH范围为2.0~10.0,荧光探针的浓度为10μM,ALP的浓度为1.0U/mL。从图中可以看出,荧光探针随着pH的变化,荧光强度基本不变,说明pH对探针本身没有很大的影响。然而,加入ALP之后,在pH在7.0~9.0范围内,荧光强度比值显著增强。综上所述,当pH值在7.0到9.0之间时,不影响荧光探针对ALP的测定,是比较合适的pH值范围,这非常有利于该探针用于实际样品中ALP的测定。
实施例7:
荧光探针与ALP作用的响应时间的测定
我们研究了荧光探针对ALP的响应时间,其结果如图7。从图中可以看出,该探针对ALP的响应时间为10min,这能够满足在实际样品中进行实时监测的要求。从图7我们还可以看出,荧光强度达到最大值后,在之后的时间里,荧光强度不再发生变化,这表明此荧光探针光稳定性较好。
实施例8:
荧光探针在活细胞中的应用
首先,我们做了细胞毒性试验,如图8所示。当加入0~30μM探针,结肠癌细胞HCT116的存活率在90%以上。这可以说明,该荧光探针毒性较小,可应用于检测活细胞内的ALP。然后,我们研究荧光探针在活细胞中的应用,选择结肠癌细胞HCT116进行共聚焦显微成像,结果如图9所示。在对照组细胞中,几乎没有观察到荧光。然后细胞中加入探针,观察到荧光明显增强。当在细胞中加入ALP抑制剂Na3VO4后再加入探针,发现细胞内的荧光几乎消失。这些结果说明该探针可以高灵敏性的检测细胞内的ALP。
Claims (3)
2.根据权利要求1所述的一种碱性磷酸酶近红外荧光探针的制备方法,其特征在于,反应步骤如下:
在N2保护下,将0.5当量的QX-OH,2.0~3.0当量的三氯氧磷,2.0~3.0当量的吡啶分别加入到25mL圆底烧瓶中,然后加入5~8mL的CH2Cl2将其溶解。反应在室温下搅拌4小时后,将所得混合物倒入冰中,并继续搅拌过夜。反应完成后,减压除去溶剂,所得粗产物用展开剂CH2Cl2/CH3OH=2∶1进行柱层析纯化,得到深蓝色固体产物QX-P,即为所述的荧光探针。
3.根据权利要求1所述的一种碱性磷酸酶近红外荧光探针的应用,其特征在于,所述荧光探针应用于活细胞内碱性磷酸酶含量的检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010709048.5A CN111825718B (zh) | 2020-07-21 | 2020-07-21 | 基于喹啉-氧杂蒽的碱性磷酸酶荧光探针的制备和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010709048.5A CN111825718B (zh) | 2020-07-21 | 2020-07-21 | 基于喹啉-氧杂蒽的碱性磷酸酶荧光探针的制备和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111825718A true CN111825718A (zh) | 2020-10-27 |
CN111825718B CN111825718B (zh) | 2022-07-29 |
Family
ID=72924672
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010709048.5A Active CN111825718B (zh) | 2020-07-21 | 2020-07-21 | 基于喹啉-氧杂蒽的碱性磷酸酶荧光探针的制备和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111825718B (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113402505A (zh) * | 2021-05-18 | 2021-09-17 | 华南理工大学 | 一种可吸收近红外光的有机材料及其制备方法与应用 |
CN113429389A (zh) * | 2021-06-29 | 2021-09-24 | 湘潭大学 | 一种检测一氧化碳的固态荧光探针的制备和应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106753341A (zh) * | 2016-12-27 | 2017-05-31 | 湘潭大学 | 一种近红外碱性磷酸酶荧光探针的制备方法和应用 |
CN107312524A (zh) * | 2017-07-19 | 2017-11-03 | 山西大学 | 一种用于检测碱性磷酸酶的荧光探针及其制备方法 |
US20180149596A1 (en) * | 2016-11-30 | 2018-05-31 | Korea Research Institute Of Bioscience And Bioscience And Biotechnology | Near-infrared fluorescent probe for detecting alkaline phosphatase and manufacturing method thereof |
CN109369719A (zh) * | 2018-11-05 | 2019-02-22 | 深圳大学 | 一种用于碱性磷酸酶检测的分子探针及制备方法与应用 |
CN110511245A (zh) * | 2019-09-03 | 2019-11-29 | 天津理工大学 | 一种基于硫代半菁染料的近红外荧光探针SHCy-P及其制备方法和应用 |
-
2020
- 2020-07-21 CN CN202010709048.5A patent/CN111825718B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20180149596A1 (en) * | 2016-11-30 | 2018-05-31 | Korea Research Institute Of Bioscience And Bioscience And Biotechnology | Near-infrared fluorescent probe for detecting alkaline phosphatase and manufacturing method thereof |
CN106753341A (zh) * | 2016-12-27 | 2017-05-31 | 湘潭大学 | 一种近红外碱性磷酸酶荧光探针的制备方法和应用 |
CN107312524A (zh) * | 2017-07-19 | 2017-11-03 | 山西大学 | 一种用于检测碱性磷酸酶的荧光探针及其制备方法 |
CN109369719A (zh) * | 2018-11-05 | 2019-02-22 | 深圳大学 | 一种用于碱性磷酸酶检测的分子探针及制备方法与应用 |
CN110511245A (zh) * | 2019-09-03 | 2019-11-29 | 天津理工大学 | 一种基于硫代半菁染料的近红外荧光探针SHCy-P及其制备方法和应用 |
Non-Patent Citations (1)
Title |
---|
FANGHUI LIANG ET AL.: "A novel near-infrared fluorescent probe for the dynamic monitoring of", 《SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113402505A (zh) * | 2021-05-18 | 2021-09-17 | 华南理工大学 | 一种可吸收近红外光的有机材料及其制备方法与应用 |
CN113429389A (zh) * | 2021-06-29 | 2021-09-24 | 湘潭大学 | 一种检测一氧化碳的固态荧光探针的制备和应用 |
CN113429389B (zh) * | 2021-06-29 | 2022-05-10 | 湘潭大学 | 一种检测一氧化碳的固态荧光探针的制备和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN111825718B (zh) | 2022-07-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110540837B (zh) | 一种过氧化氢近红外荧光探针的制备和应用 | |
Wei et al. | NBD-based colorimetric and fluorescent turn-on probes for hydrogen sulfide | |
Zhao et al. | Transforming the recognition site of 4-hydroxyaniline into 4-methoxyaniline grafted onto a BODIPY core switches the selective detection of peroxynitrite to hypochlorous acid | |
Wu et al. | A novel lipid droplets-targeting ratiometric fluorescence probe for hypochlorous acid in living cells | |
CN111499604B (zh) | 一种溶酶体靶向的Cys近红外荧光探针及其制备方法和应用 | |
Gao et al. | A sensitive ratiometric fluorescent probe for quantitive detection and imaging of alkaline phosphatase in living cells | |
CN111518071B (zh) | 一种半胱氨酸近红外荧光探针的制备和应用 | |
CN109053802B (zh) | 一种比率型近红外荧光探针及其合成方法与应用 | |
CN111825718B (zh) | 基于喹啉-氧杂蒽的碱性磷酸酶荧光探针的制备和应用 | |
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 | |
Liu et al. | Lysosome-targeted near-infrared fluorescent dye and its application in designing of probe for sensitive detection of cysteine in living cells | |
Zhang et al. | Endoplasmic reticulum targeted fluorescent probe for the detection of hydrogen sulfide based on a twist-blockage strategy | |
Han et al. | Rational design of a lysosomal-targeted ratiometric two-photon fluorescent probe for imaging hydrogen polysulfides in live cells | |
Liu et al. | A fluorescein-based fluorescence probe for the fast detection of thiol | |
CN111518083A (zh) | 一种检测一氧化碳的打开型荧光探针的制备和应用 | |
CN113563229A (zh) | 基于异佛尔酮-肉桂醛的粘度荧光探针的制备和应用 | |
Qi et al. | Near-infrared turn-on fluorescent probe for discriminative detection of Cys and application in in vivo imaging | |
Zhai et al. | Development of a ratiometric two-photon fluorescent probe for imaging of hydrogen peroxide in ischemic brain injury | |
Zhou et al. | A novel colorimetric and ratiometric fluorescent probe for monitoring lysosomal HOCl in real time | |
CN114181204B (zh) | 一种检测粘度的近红外荧光探针及其制备和应用 | |
Jun-Ying et al. | A Nile red-based near-infrared fluorescent probe for the detection of superoxide radical anion in living cells | |
CN110669503B (zh) | 一种一氧化碳近红外荧光探针的制备和应用 | |
Guo et al. | Novel dual-site fluorescent probe for monitoring cysteine and sulfite in living cells | |
CN113637048B (zh) | 一种γ-谷氨酰转肽酶的双光子荧光探针及其制备方法和应用 | |
CN114634464A (zh) | 一种用于检测次氯酸的溶酶体靶向近红外荧光探针及其制备方法和应用 |
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 |