CN106753341B - 一种近红外碱性磷酸酶荧光探针的制备方法和应用 - Google Patents
一种近红外碱性磷酸酶荧光探针的制备方法和应用 Download PDFInfo
- Publication number
- CN106753341B CN106753341B CN201611230342.8A CN201611230342A CN106753341B CN 106753341 B CN106753341 B CN 106753341B CN 201611230342 A CN201611230342 A CN 201611230342A CN 106753341 B CN106753341 B CN 106753341B
- Authority
- CN
- China
- Prior art keywords
- probe
- alkaline phosphatase
- fluorescence
- alp
- fluorescence probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000000523 sample Substances 0.000 title claims abstract description 85
- 102000002260 Alkaline Phosphatase Human genes 0.000 title claims abstract description 83
- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims abstract description 83
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 238000001514 detection method Methods 0.000 claims abstract description 14
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims abstract description 12
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 150000001413 amino acids Chemical class 0.000 claims abstract description 5
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 5
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 230000000694 effects Effects 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 10
- 230000003834 intracellular effect Effects 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 230000004044 response Effects 0.000 claims description 8
- 239000003112 inhibitor Substances 0.000 claims description 7
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 5
- 230000035515 penetration Effects 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 150000003573 thiols Chemical class 0.000 claims description 4
- 229910020700 Na3VO4 Inorganic materials 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 238000005292 vacuum distillation Methods 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 239000012216 imaging agent Substances 0.000 claims 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000006911 enzymatic reaction Methods 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 abstract description 10
- 238000003384 imaging method Methods 0.000 abstract description 7
- 230000035479 physiological effects, processes and functions Effects 0.000 abstract description 4
- 238000001727 in vivo Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 208000037273 Pathologic Processes Diseases 0.000 abstract description 2
- 238000013461 design Methods 0.000 abstract description 2
- 238000011835 investigation Methods 0.000 abstract description 2
- 230000009054 pathological process Effects 0.000 abstract description 2
- 244000139794 beach naupaka Species 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 125000003396 thiol group Chemical class [H]S* 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 239000000975 dye Substances 0.000 description 7
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000004847 absorption spectroscopy Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 244000258825 Scaevola taccada Species 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000007541 cellular toxicity Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 101150060184 ACHE gene Proteins 0.000 description 1
- 108700023418 Amidases Proteins 0.000 description 1
- 101100229716 Aspergillus niger gox gene Proteins 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
- 101100392469 Escherichia coli (strain K12) glcD gene Proteins 0.000 description 1
- 101100392473 Escherichia coli (strain K12) glcE gene Proteins 0.000 description 1
- 101100282781 Escherichia coli (strain K12) glcF gene Proteins 0.000 description 1
- 101150082479 GAL gene Proteins 0.000 description 1
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 description 1
- 108010000445 Glycerate dehydrogenase Proteins 0.000 description 1
- 102000004459 Nitroreductase Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 102000005922 amidase Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 108010029645 galactitol 2-dehydrogenase Proteins 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 108020001162 nitroreductase Proteins 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- SPVXKVOXSXTJOY-UHFFFAOYSA-N selane Chemical compound [SeH2] SPVXKVOXSXTJOY-UHFFFAOYSA-N 0.000 description 1
- 229910000058 selane Inorganic materials 0.000 description 1
- 150000003958 selenols Chemical class 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- IHIXIJGXTJIKRB-UHFFFAOYSA-N trisodium vanadate Chemical compound [Na+].[Na+].[Na+].[O-][V]([O-])([O-])=O IHIXIJGXTJIKRB-UHFFFAOYSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- 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
-
- 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
- C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
- C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
- C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
-
- 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
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet 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
- G01N21/6486—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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种近红外碱性磷酸酶荧光探针的制备方法和应用。该荧光探针的结构式为：。本发明以IR‑780、间苯二酚和三氯氧磷为原料，设计合成了一种基于半花菁生物的近红外荧光探针。该探针可用来检测溶液中ALP浓度，表现好的灵敏度，线性范围为0.01‑2.0 U/mL，检测限为0.003 U/mL。该探针表现出很好的选择性，不受其它酶、生物硫醇、氨基酸以及金属离子的影响。更为重要的是，该探针还可应用于生物成像，检测细胞内和组织中的ALP含量，这对于深入研究ALP在生物体内生理和病理过程具有重要意义。
Description
技术领域
本发明属于荧光探针技术领域，具体涉及一种近红外碱性磷酸酶荧光探针的制备方法和应用。
背景技术
碱性磷酸酶(ALP)广泛分布于人体肝脏、骨骼、肠、肾和胎盘等组织中，是经肝脏向胆外排出的一种酶，它在生理过程中发挥着关键的作用(文献1:J.E.Coleman,Annu.Rev.Biophys.Biomol.Struct.,1992,21,441-483)。ALP在生物体内的浓度的变化与某些生命过程正常与否息息相关。研究表明，ALP的水平异常与许多疾病有关，包括乳腺癌，前列腺癌，糖尿病，骨疾病，以及肝功能异常(文献2:J.A.Lorente,H.Valenzuela,J.Morote,A.Gelabert,Eur.J.Nucl.,1999,26,625-632；文献3:D.L.Robertson,G.F.Joyce,Nature,1990,344,467-468；文献4:P.Colombatto,A.Randone,G.Civitico,J.M.Gorin,L.Dolci,N.Medaina,F.Oliveri,G.Verme,G.Marchiaro,R.Pagni,P.Karayiannis,H.C.Thomas,G.Hess,F.Bonino,M.R.Brunetto,J.Viral Hepat.,1996,3,301-306；文献5:M.M.Couttenye,P.C.D'Haese,V.O.Van Hoof,E.Lemoniatou,W.Goodman,G.A.Verpooten,M.E.De Broe,Nephrol.Dial.Transplant.,1996,11,1065-1072)。因此，设计一种有效检测ALP的方法，以便更好地了解其生理和病理功能，是具有重要意义的。
荧光探针以其高选择性、灵敏度，操作简单，并且可应用于生物成像等优点，受到了广泛的关注。近年来，报道了一些用于检测ALP的荧光探针(文献6:J.J.Deng,P.Yu,Y.X.Wang,L.Q.Mao,Anal.Chem.,2015,87,3080-3086；文献7:Y.Li,Y.N.Li,Z.G.Liu,X.G.Su,RSC Advances.,2014,4,42825-42830；文献8:X.G.Gu,G.X.Zhang,Z.Wang,W.W.Liu,L.Xiao,D.Q.Zhang,Analyst,2013,138,2427-2431；文献9：M.Kawaguchi,K.Hanaoka,T.Komatsu,T.Terai,T.Nagano,Bioorg Med Chem Lett.,2011,21,5088-5091；文献10：X.F.Hou,Q.X.Yu,F.Zeng,J.H.Ye,S.Z.Wu,Journal of Materials Chemistry B.,2015,3,1042-1048；文献11：Z.X.Lu,J.S.Wu,W.M.Liu,G.Y.Zhang,P.F.Wang,RSCAdvances.,2016,6,32046-32051)。我们注意到，尽管这些荧光探针实现了对ALP的检测，但他们仍然存在一个共同的问题，这些探针的发射波长都是在可见光区域，这将会影响探针在生物系统中的应用。因此，设计和合成一个近红外探针检测ALP是很有必要的。
近红外染料的荧光发射波长范围在650-900nm，与可见光区的染料相比，具有一些独特的优势，如组织渗透能力强，对生物样品光损伤小，背景干扰少(文献12:L.Yuan,W.Lin,Y.S.Zhao,W.S.Gao,B.Chen,L.W.He,S.S.Zhu,J.Am.Chem.Soc.,2012,134,13510-13523)。到目前为止，一些近红外荧光探针已被设计合成，并成功用于酶，pH，小分子及金属离子的检测(文献13:H.Yu,M.Sun,K.Zhang,H.Zhu,Z.Liu,Y.Zhang,J.Zhao,L.Wu,Z.Zhang,S.Wang,Sens.Actuators.B.,2015,219,294-300；文献14:K.Xu,F.Wang,X.Pan,R.Liu,J.Ma,F.Kong,B.Tang,Chem.Commun.,2013,49,2554-2556；文献15:J.Zhang,B.Yu,L.Ning,X.Zhu,J.Wang,Z.Chen,X.Liu,X.Yao,X.Zhang,H.Zhang,Eur.J.Org.Chem.,2015,2015,1711-1718；文献16Y.Y.Yang,T.Cheng,W.Zhu,Y.Xu,X.Qian,Org.Lett.,2011,13,264-267)。值得我们注意的是，这些近红外荧光探针基本上都是基于花菁染料的。
与传统花菁染料相比，半花菁染料耐光性好，荧光量子产率高，更有利于成像。到目前为止，半花菁染料已被设计合成，并用于检测硒化氢，肼，半胱氨酸，pH，一氧化氮，β-内酰胺酶，硒醇，超氧根离子，硝基还原酶(文献17:F.Kong,L.Ge,X.Pan,K.Xu,X.Liu,B.Tang,Chem.Sci.,2016,7,1051-1056；文献18:J.Zhang,L.Ning,J.Liu,J.Wang,B.Yu,X.Liu,X.Yao,Z.Zhang,H.Zhang,Anal.Chem.,2015,87,9101-9107；文献19:J.Zhang,J.Wang,J.Liu,L.Ning,X.Zhu,B.Yu,X.Liu,X.Yao,H.Zhang,Anal.Chem.,2015,87,4856-4863；文献20:Y.Li,Y.Wang,S.Yang,Y.Zhao,L.Yuan,J.Zheng,R.Yang,Anal.Chem.,2015,87,2495-2503；文献21:A.T.Wrobel,T.C.Johnstone,A.Deliz Liang,S.J.Lippard,Rivera-Fuentes,P.J.Am.Chem.Soc.,2014,136,4697-4705；文献22:L.Li,Z.Li,W.Shi,X.Li,H.Ma,Anal.Chem.,2014,86,6115-6120；文献23:H.Chen,B.Dong,Y.Tang,W.Lin,Chemistry–AEuropean Journal.,2015,21,11696-11700；文献24:J.Zhang,C.Li,R.Zhang,F.Zhang,W.Liu,X.Liu,X.M.Lee,H.Zhang,Chem.Commun.,2016,52,2679-2682；文献25:Z.Li,X.He,Z.Wang,R.Yang,W.Shi,H.Ma,Biosens.Bioelectron.,2015,63,112-116)。但是用于检测ALP的近红外荧光探针几乎没有。因此，设计一个基于半花菁染料的近红外荧光探针用于生物体内ALP的检测是非常重要的。
本文设计并合成了一种基于半花菁生物的近红外荧光探针，此探针与ALP反应后，产生强烈的红色荧光，表现出很高的灵敏度。该荧光探针与ALP作用迅速，并具有高选择性。并且可成功用于活细胞以及组织中的荧光成像。
发明内容
本发明的目的是提供一种近红外ALP的荧光探针，该荧光探针具有长波长(在近红外区)、高灵敏度以及高选择性的检测ALP，并能够应用于活细胞以及组织中的荧光成像。
本发明的技术方案是，一种基于半花菁衍生物的ALP荧光探针，其结构式如下：
所述的一种基于近红外ALP荧光探针的制备方法，步骤如下：
1)在100mL圆底烧瓶中，依次加入间苯二酚和碳酸钾，其摩尔比为1:1，加入20mL乙腈作为溶剂，室温下，磁力搅拌10min后，将溶解在20mL乙腈中的IR-780溶液逐滴加入到反应液中，其中IR-780和间苯二酚的摩尔比为1:2，N2保护下，加热至50℃，继续搅拌约4h，反应完成，用旋转蒸发仪除去溶剂，粗产品以二氯甲烷/甲醇为20:1(体积比)为淋洗剂，柱层析分离提纯，得蓝绿色固体(CyOH)。
2)将CyOH溶解在无水吡啶中，搅拌下逐滴加入三氯氧磷，其中CyOH和三氯氧磷的摩尔比为1:2，室温下搅拌4h以后，往混合溶液中加入冰，继续在室温条件下搅拌24h，反应完成，减压蒸馏除去溶剂，粗产品用二氯甲烷/甲醇为2:1(体积比)的洗脱剂，柱层析分离提纯，得蓝色固体(CyP)。
反应路线如下：
本发明的有益效果是：本发明的近红外荧光探针在ALP存在下荧光发生显著变化，可以用于高灵敏度的检测ALP，该荧光探针的线性范围为0.01-2.0U/mL，检测限为0.003U/mL。同时，该荧光探针对ALP的响应迅速，响应时间在20min以内。并且该近红外荧光探针对ALP表现出很好的选择性，不受其它酶(Gox，trypsin，AchE，GDH，Gal，thrombin)、生物硫醇(Cys，Hcy，ALP)、氨基酸(Pro，Tyr，Asn，Met，Phe，Nleu，Asp，Orn，Ala，Trp，His，Ser，Gly，Val，Lys，Glu，Arg，Heu，Thr)及其它的金属离子(K+，Ca2+，Zn2+，Na+，Mg2+)的影响。
所述的一种近红外碱性磷酸酶荧光探针的应用：将该荧光探针应用于细胞成像中，细胞内的ALP含量较多，因此直接向细胞内加入探针，能检测到强的红色荧光信号。但是，当在加入探针之前加入一定量的ALP抑制剂Na3VO4时，细胞内没有荧光信号。将该荧光探针应用于组织成像中，组织中的ALP含量也较多，因此直接向组织中加入探针，能检测到强的红色荧光信号。但是，当在加入探针之前加入一定量的ALP抑制剂Na3VO4时，组织中没有荧光信号。这些现象表明该近红外荧光探针不仅能检测溶液中的ALP，还可应用于检测细胞内和组织中的ALP含量，这对于深入研究ALP在生物体内生理和病理过程具有重要意义。
附图说明
图1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。荧光探针的浓度均为10μM，ALP浓度分别为：0，0.01，0.25，0.5，0.75，1.0，1.25，1.5，1.75，2.0U/mL。荧光激发波长为670nm。插图为探针对ALP浓度的线性响应图。
图2为荧光探针与ALP的作用机理图。
图3为荧光探针与ALP作用后的紫外可见吸收光谱图。
图4为荧光探针在相同ALP浓度下，荧光强度随时间变化的关系曲线图。
图5为荧光探针的选择性图。F0和F表示探针溶液加入ALP前后的荧光强度。
图6为细胞毒性试验。横坐标为荧光探针的浓度，纵坐标为细胞的存活率。
图7为ALP的细胞成像图。
图8为ALP的组织成像图。
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
CyOH的合成：在100mL的圆底烧瓶中，依次加入间苯二酚(1.0mmol,110mg)和碳酸钾(1.0mmol,138mg)，再加入20mL乙腈作为溶剂。室温下，磁力搅拌10min后，将溶解在20mL乙腈中的IR-780(0.5mmol,305mg)溶液逐滴加入到反应液中。N2保护下，加热至50℃继续搅拌约4h，反应完成，用旋转蒸发仪除去溶剂，粗产品以二氯甲烷/甲醇为20:1(体积比)为淋洗剂，柱层析分离提纯，得蓝绿色固体169mg，产率为82％。1H NMR(400MHz,CDCl3):δ8.02(d,1H,J＝13.2Hz),7.25-7.21(m,2H),7.14(t,1H,J＝7.6Hz),7.06-7.03(m,2H),6.97(s,1H),6.90(d,1H,J＝8.0Hz),6.81(d,1H,J＝8.0Hz),5.56(d,1H,J＝13.2Hz),3.76(t,2H,J＝7.2Hz),2.67(t,2H,J＝6.0Hz),2.59(t,2H,J＝6.0Hz),1.91-1.82(m,4H),1.67(s,6H),1.04(t,3H,J＝7.6Hz).MS(TOF)m/z 412.2.
CyP探针的合成：在100mL的圆底烧瓶中，将CyOH(0.2mmol,82.4g)溶解在25mL无水吡啶中，在室温下，逐滴加入三氯氧磷(0.4mmol,61.3mg)，搅拌4小时以后，向混合溶液中加入冰，继续搅拌24小时，反应完成。减压蒸馏除去溶剂，粗产品用二氯甲烷/甲醇为2:1(体积比)的洗脱剂，柱层析分离提纯，得蓝色固体70.9mg，产率为72％。1H NMR(400MHz,CDCl3):δ8.64(d,1H,J＝15.2Hz),7.55-7.51(m，2H),7.44(t,1H,J＝7.6Hz),7.36-7.33(m,2H),7.27(s,1H),7.20(d,1H,J＝8.0Hz),7.11(d,1H,J＝8.0Hz),6.08(d,1H,J＝13.2Hz),4.16(t,2H,J＝7.6Hz),2.77(t,2H,J＝6.0Hz),2.69(t,2H,J＝6.0Hz),2.00-1.93(m,4H),1.83(s,6H),1.12(t,3H,J＝7.6Hz).13C NMR(100MHz,CDCl3):δ174.4,166.8,163.1,155.5,143.1,142.0,141.2,137.7,132.6,128.8,125.7,123.8,123.7,122.5,118.3,114.7,111.0,103.0,100.1,49.9,46.4,29.7,28.6,24.3,22.7,20.9,11.7.MS(TOF)m/z492.2.Anal.calcd.for C28H31NO5P+(CyP):C,81.52,H,7.33,N,3.40.Found:C,83.56,H,7.45,N,3.47.结果表明，所得产物结构正确。
实施例2：
荧光探针与ALP作用的溶液配制
将一定量的荧光探针溶解在EtOH中，得到浓度为1.0×10-4mol·L-1探针的备用溶液。将1.0mL探针的备用溶液加入到10mL的容量瓶中，用缓冲溶液定容后，得到浓度为1.0×10-5mol·L-1的荧光探针溶液。将ALP分别配制为以下浓度(0,0.01,0.25,0.5,0.75,1.0,1.25,1.5,1.75,2.0U/mL)。
实施例3：
荧光探针与ALP作用的荧光光谱的测定
用pH值为8的缓冲溶液为溶剂测定了荧光探针与ALP作用的荧光光谱，结果如图1。荧光探针的浓度为10μM，ALP的浓度依次为0，0.01，0.25，0.5，0.75，1.0，1.25，1.5，1.75，2.0U/mL，激发波长固定为595nm，发射波长范围为685～850nm，狭缝宽度为10.0nm/10.0nm。从图1可以看出，随着ALP的加入，在703nm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。如图1的插图所示，荧光强度跟ALP的浓度呈现线性关系，线性范围是0.01-2.0U/mL，检测限是0.003U/mL。所用的荧光测定仪器为Perkin Elmer LS 55荧光分光光度计。图2为荧光探针与ALP作用的机理图，从图中可以看出，荧光探针与ALP发生反应后，使得磷酸根离去，生成CyOH，从而导致荧光发生显著变化。
实施例4：
荧光探针与ALP作用的紫外可见吸收光谱性质的测定
图3为荧光探针与ALP作用后的紫外可见吸收光谱图。从图3中可以看出，加入ALP之后，686nm处的吸收峰大幅度增强。紫外可见吸收光谱测定用的仪器为Perkin ElmerLambda 25型紫外可见分光光度计。
实施例5：
荧光探针与ALP作用的响应时间的测定
为了研究荧光探针对ALP的响应时间，我们考察了荧光探针在相同ALP浓度下(2.0U/mL)的荧光光谱的变化情况，其结果如图4。从图中可以看出，该探针对ALP的响应时间不到20min，满足在实际样品中进行实时监测时对响应时间的要求。
实施例6：
荧光探针对ALP测定的选择性
在浓度为10μM的荧光探针溶液中加入酶(ALP，Gox，trypsin，AchE，GDH，Gal，thrombin)、生物硫醇(Cys，Hcy，GSH)、氨基酸(Pro，Tyr，Asn，Met，Phe，Nleu，Asp，Orn，Ala，Trp，His，Ser，Gly，Val，Lys，Glu，Arg，Heu，Thr)及金属离子(K+，Ca2+，Zn2+，Na+，Mg2+)。从图5中可以看出，除ALP外，加入其它的酶，生物硫醇和其它氨基酸，荧光强度都没有明显的改变。F0和F表示探针溶液加入ALP前后的荧光强度。这些现象表现探针CyP对ALP的测定表现出良好的选择性。
实施例7：
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6所示，当加入0～30μM ALP探针，20min之后，细胞的成活率均在97％以上，因此可以说明，该荧光探针可应用于检测活细胞内的ALP，并且毒性较小。
一般情况下，细胞内的ALP含量非常丰富，因此直接向细胞内加入探针，也能检测到强的红色荧光信号。但是，当在加入探针之前加入一定量的ALP抑制剂Na3VO4时，细胞内没有荧光信号。(图7)。因此可以说明，该探针可高选择性的检测细胞内的ALP。
实施案例8：
荧光探针在组织中的应用
近红外的探针具有背景低，穿透性强的优点。如图8所示，(a)图是在小鼠肝冷冻切片中先加入10μM探针CyP的荧光成像图。(b)图是在小鼠肝冷冻切片中先加入ALP抑制剂Na3VO4，20min后再加入10μM探针CyP的荧光成像图。从图可以看出，直接向组织中加入探针，能检测到强的红色荧光信号。但是，当在加入探针之前加入一定量的ALP抑制剂Na3VO4时，组织中没有荧光信号。探针CyP的穿透深度范围为40-140μm，由此我们能得出探针CyP的组织穿透能力比较强。
Claims (5)
1.一种近红外碱性磷酸酶荧光探针，该探针被命名为CyP，其结构式如下：
。
2.根据权利要求1所述的一种近红外碱性磷酸酶荧光探针的制备方法，其特征在于它的具体制备步骤为：
1）在100 mL圆底烧瓶中，依次加入间苯二酚和碳酸钾，其摩尔比为1:1，加入20 mL乙腈作为溶剂，室温下，磁力搅拌10 min后，将溶解在20 mL乙腈中的IR-780溶液逐滴加入到反应液中，其中IR-780和间苯二酚的摩尔比为1:2，N2保护下，加热至50 ℃，继续搅拌4 h，反应完成，用旋转蒸发仪除去溶剂，粗产品以体积比为20:1的二氯甲烷/甲醇为淋洗剂，柱层析分离提纯，得蓝绿色固体，即CyOH，结构如下图所示：
；
2）将CyOH溶解在无水吡啶中，搅拌下逐滴加入三氯氧磷，其中CyOH和三氯氧磷的摩尔比为1:2，室温下搅拌4 h以后，往混合溶液中加入冰，继续在室温条件下搅拌24 h，反应完成，减压蒸馏除去溶剂，粗产品以体积比为2:1的二氯甲烷/甲醇为洗脱剂，柱层析分离提纯，得蓝色固体，即化合物CyP。
3.根据权利要求1所述的一种近红外碱性磷酸酶荧光探针的应用，其特征在于：用于溶
液中碱性磷酸酶的检测；所述荧光探针本身无荧光，与碱性磷酸酶反应生成CyOH，产生强烈
的红色荧光，线性范围为0.01-2.0 U/mL，检测限为0.003 U/mL，该探针表现出很高的灵敏
度；该荧光探针与碱性磷酸酶作用迅速，响应时间在20分钟以内；该荧光探针对碱性磷酸酶
表现出很好的选择性，不受其它酶、生物硫醇、氨基酸以及金属离子的影响；所述CyOH结构
式为。
4.根据权利要求1所述的一种近红外碱性磷酸酶荧光探针在制备成像试剂中的应用，其特征在于：用于检测细胞内的碱性磷酸酶，细胞内的碱性磷酸酶含量非常丰富，因此向细胞内加入探针后，能检测到强的红色荧光信号，但是，再加入一定量的抑制剂Na3VO4时，细胞内荧光信号消失。
5.根据权利要求1所述的一种近红外碱性磷酸酶荧光探针在制备成像试剂中的应用，其特征在于：用于检测组织中的碱性磷酸酶，组织中的碱性磷酸酶含量也非常丰富，因此向组织中加入探针后，能检测到强的红色荧光信号，但是，再加入一定量的抑制剂Na3VO4时，组织内荧光信号消失，另外，该探针的穿透深度范围为40-140 μm，说明该近红外探针具有良好的组织穿透性。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611230342.8A CN106753341B (zh) | 2016-12-27 | 2016-12-27 | 一种近红外碱性磷酸酶荧光探针的制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611230342.8A CN106753341B (zh) | 2016-12-27 | 2016-12-27 | 一种近红外碱性磷酸酶荧光探针的制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106753341A CN106753341A (zh) | 2017-05-31 |
| CN106753341B true CN106753341B (zh) | 2019-03-19 |
Family
ID=58922385
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611230342.8A Expired - Fee Related CN106753341B (zh) | 2016-12-27 | 2016-12-27 | 一种近红外碱性磷酸酶荧光探针的制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106753341B (zh) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312524B (zh) * | 2017-07-19 | 2019-02-26 | 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 |
| CN108219510B (zh) * | 2018-03-21 | 2020-07-03 | 湘潭大学 | 基于半花菁染料的过氧化亚硝基荧光探针的制备和应用 |
| CN108948081B (zh) * | 2018-07-12 | 2020-07-07 | 青岛科技大学 | 一种测定碱性磷酸酶的比率型荧光探针及其合成方法与应用 |
| CN109053802B (zh) * | 2018-09-01 | 2020-06-23 | 青岛科技大学 | 一种比率型近红外荧光探针及其合成方法与应用 |
| CN111257290B (zh) * | 2018-11-30 | 2021-08-31 | 中国科学院大连化学物理研究所 | 近红外荧光探针及其在检测谷胱甘肽硫转移酶中的应用 |
| CN109580958B (zh) * | 2018-12-05 | 2022-02-01 | 中国科学院长春应用化学研究所 | 一种心肌肌钙蛋白i的荧光和比色双信号检测试剂盒及检测方法 |
| CN109913204A (zh) * | 2019-02-22 | 2019-06-21 | 山东师范大学 | 一种检测炎症标志物活性的近红外荧光探针及其合成方法 |
| CN110128414B (zh) * | 2019-05-16 | 2022-04-01 | 湘潭大学 | 一种基于半花菁染料的缺氧荧光探针的制备和应用 |
| CN110105391B (zh) * | 2019-06-12 | 2021-04-27 | 苏州大学 | 碱性磷酸酶响应型分子探针及其应用 |
| CN110511245B (zh) * | 2019-09-03 | 2021-10-19 | 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 |
| CN111825718B (zh) * | 2020-07-21 | 2022-07-29 | 湘潭大学 | 基于喹啉-氧杂蒽的碱性磷酸酶荧光探针的制备和应用 |
| CN113278304B (zh) * | 2021-07-07 | 2022-05-24 | 广西师范大学 | 一种双比率型半花菁类染料分子及其合成方法和应用 |
| WO2024060196A1 (zh) * | 2022-09-23 | 2024-03-28 | 苏州大学 | 一种rna反应型荧光探针及其制备方法和应用 |
| CN116425820A (zh) * | 2023-04-14 | 2023-07-14 | 湘潭大学 | 一种靶向性硝基还原酶荧光探针的制备和应用 |
| CN117186151B (zh) * | 2023-08-23 | 2024-05-17 | 中山大学 | 一种近红外两性离子花菁染料及其制备方法和应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101784897A (zh) * | 2007-05-23 | 2010-07-21 | 应用生物系统有限公司 | 通过从活化的化学发光的基质将能量转移至能量受体染料来检测生物分子的试剂、试剂盒和方法 |
| CN104109176A (zh) * | 2014-06-06 | 2014-10-22 | 浙江工业大学 | 一种化合物及其应用于碱性磷酸酶活性的荧光检测方法 |
| EP2910550A2 (en) * | 2006-11-14 | 2015-08-26 | Enzo Therapeutics, Inc. | Compositions and methods for bone formation and remodeling |
-
2016
- 2016-12-27 CN CN201611230342.8A patent/CN106753341B/zh not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2910550A2 (en) * | 2006-11-14 | 2015-08-26 | Enzo Therapeutics, Inc. | Compositions and methods for bone formation and remodeling |
| CN101784897A (zh) * | 2007-05-23 | 2010-07-21 | 应用生物系统有限公司 | 通过从活化的化学发光的基质将能量转移至能量受体染料来检测生物分子的试剂、试剂盒和方法 |
| CN104109176A (zh) * | 2014-06-06 | 2014-10-22 | 浙江工业大学 | 一种化合物及其应用于碱性磷酸酶活性的荧光检测方法 |
Non-Patent Citations (2)
| Title |
|---|
| A novel sensor for the detection of alkaline phosphatase activity based on the self-assembly of Eu3+-doped oxide nanoparticles andheptamethine cyanine dye;Ben Hao Li et al;《Sensors and Actuators B》;20160422;479–485 |
| Chunmiao Han.Mitochondria-Targeted Near-Infrared Fluorescent Off−On Probe for Selective Detection of Cysteine in Living Cells andin Vivo.《ACS Appl. Mater. Interfaces》.2015,27968−27975. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106753341A (zh) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106753341B (zh) | 一种近红外碱性磷酸酶荧光探针的制备方法和应用 | |
| 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 | |
| Vellaisamy et al. | Cell imaging of dopamine receptor using agonist labeling iridium (III) complex | |
| Lee et al. | Near-infrared fluorescent probes for the detection of glutathione and their application in the fluorescence imaging of living cells and tumor-bearing mice | |
| Qian et al. | A NIR fluorescent probe for the detection and visualization of hydrogen sulfide using the aldehyde group assisted thiolysis of dinitrophenyl ether strategy | |
| Wang et al. | A novel DCM-NBD conjugate fluorescent probe for discrimination of Cys/Hcy from GSH and its bioimaging applications in living cells and animals | |
| Liu et al. | In vivo imaging of alkaline phosphatase in tumor-bearing mouse model by a promising near-infrared fluorescent probe | |
| Yang et al. | A NIR fluorescent probe for the detection of fluoride ions and its application in in vivo bioimaging | |
| Wang et al. | A novel near-infrared styryl-BODIPY fluorescent probe for discrimination of GSH and its application in living cells | |
| Ou-Yang et al. | An infinite coordination polymer nanoparticles-based near-infrared fluorescent probe with high photostability for endogenous alkaline phosphatase in vivo | |
| Huang et al. | A novel near-infrared fluorescent hydrogen sulfide probe for live cell and tissue imaging | |
| Chen et al. | Red-emitting fluorescent probe for detecting hypochlorite acid in vitro and in vivo | |
| Wang et al. | Novel NIR fluorescent probe with dual models for sensitively and selectively monitoring and imaging Cys in living cells and mice | |
| Wang et al. | A reaction-based and highly selective fluorescent probe for hydrogen sulfide | |
| Sun et al. | An efficient TP-FRET-based lysosome-targetable fluorescent probe for imaging peroxynitrite with two well-resolved emission channels in living cells, tissues and zebrafish | |
| Pang et al. | Visualization of endogenous β-galactosidase activity in living cells and zebrafish with a turn-on near-infrared fluorescent probe | |
| Feng et al. | Ratiometric detection and bioimaging of endogenous alkaline phosphatase by a NIR fluorescence probe | |
| Tong et al. | A NIR rhodamine fluorescent chemodosimeter specific for glutathione: Knoevenagel condensation, detection of intracellular glutathione and living cell imaging | |
| Zhang et al. | Development of a novel near-infrared fluorescence light-up probe with a large Stokes shift for sensing of cysteine in aqueous solution, living cells and zebrafish | |
| Ma et al. | A ‘naked-eye’ratiometric and NIR fluorescent detection for hydrogen sulphide with quick response and high selectivity for and its bioimaging | |
| Kong et al. | A near-infrared and two-photon dual-mode fluorescent probe for the colorimetric monitoring of SO 2 in vitro and in vivo | |
| He et al. | A turn-on near-infrared fluorescent probe for detection of cysteine over glutathione and homocysteine in vivo | |
| Guo et al. | Fabrication of a water-soluble near-infrared fluorescent probe for selective detection and imaging of dipeptidyl peptidase IV in biological systems | |
| Wang et al. | Highly selective fluorescent probe based on AIE for identifying cysteine/homocysteine | |
| Li et al. | Development of a two-photon fluorescent probe for the selective detection of β-galactosidase in living cells and tissues |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190319 Termination date: 20211227 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |