CN108801998B - 一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 - Google Patents
一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 Download PDFInfo
- Publication number
- CN108801998B CN108801998B CN201810605021.4A CN201810605021A CN108801998B CN 108801998 B CN108801998 B CN 108801998B CN 201810605021 A CN201810605021 A CN 201810605021A CN 108801998 B CN108801998 B CN 108801998B
- Authority
- CN
- China
- Prior art keywords
- cluster
- copper nano
- choline
- solution
- piperazine
- 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
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 title claims abstract description 81
- 229910052802 copper Inorganic materials 0.000 title claims abstract description 81
- 239000010949 copper Substances 0.000 title claims abstract description 81
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 229960001231 choline Drugs 0.000 title claims abstract description 77
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 27
- 150000001875 compounds Chemical class 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims description 22
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 35
- UNDVUPUXHOTLAM-UHFFFAOYSA-N N1CCNCC1.NC=1C(=C(C=CC1)O)N Chemical compound N1CCNCC1.NC=1C(=C(C=CC1)O)N UNDVUPUXHOTLAM-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000002360 preparation method Methods 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 19
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims abstract description 17
- 102000016943 Muramidase Human genes 0.000 claims abstract description 15
- 108010014251 Muramidase Proteins 0.000 claims abstract description 15
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims abstract description 15
- 239000004325 lysozyme Substances 0.000 claims abstract description 15
- 229960000274 lysozyme Drugs 0.000 claims abstract description 15
- 235000010335 lysozyme Nutrition 0.000 claims abstract description 15
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 8
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims abstract description 3
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 30
- 239000007864 aqueous solution Substances 0.000 claims description 22
- 239000011259 mixed solution Substances 0.000 claims description 17
- 239000012456 homogeneous solution Substances 0.000 claims description 16
- 239000002953 phosphate buffered saline Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 15
- IYQHCPGJNZBANF-UHFFFAOYSA-N phenazine-1,2-diamine Chemical compound C1=CC=CC2=NC3=C(N)C(N)=CC=C3N=C21 IYQHCPGJNZBANF-UHFFFAOYSA-N 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000006185 dispersion Substances 0.000 claims description 8
- 238000002189 fluorescence spectrum Methods 0.000 claims description 8
- 239000003513 alkali Substances 0.000 claims description 7
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 7
- 210000000232 gallbladder Anatomy 0.000 claims description 7
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 238000003760 magnetic stirring Methods 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 claims description 5
- 150000004987 o-phenylenediamines Chemical class 0.000 claims description 5
- 239000011541 reaction mixture Substances 0.000 claims description 5
- 235000011330 Armoracia rusticana Nutrition 0.000 claims description 4
- 240000003291 Armoracia rusticana Species 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000000703 high-speed centrifugation Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 2
- 230000008020 evaporation Effects 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- 241000894006 Bacteria Species 0.000 claims 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- 239000007975 buffered saline Substances 0.000 claims 1
- 230000001934 delay Effects 0.000 claims 1
- 238000003756 stirring Methods 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 6
- 239000012472 biological sample Substances 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 3
- 108010000659 Choline oxidase Proteins 0.000 abstract description 2
- 239000003638 chemical reducing agent Substances 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 238000001914 filtration Methods 0.000 abstract description 2
- 229910000510 noble metal Inorganic materials 0.000 abstract description 2
- 230000003647 oxidation Effects 0.000 abstract description 2
- 238000007254 oxidation reaction Methods 0.000 abstract description 2
- 239000003381 stabilizer Substances 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 description 5
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 150000002978 peroxides Chemical class 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- PCAXITAPTVOLGL-UHFFFAOYSA-N 2,3-diaminophenol Chemical compound NC1=CC=CC(O)=C1N PCAXITAPTVOLGL-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- MQKPEUAOJLJUMD-UHFFFAOYSA-N phenol;piperazine Chemical compound C1C[NH2+]CCN1.[O-]C1=CC=CC=C1 MQKPEUAOJLJUMD-UHFFFAOYSA-N 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010061998 Hepatic lesion Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- VLBPIWYTPAXCFJ-XMMPIXPASA-O Lyso-PAF C-16-d4 Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](O)COP(O)(=O)OCC[N+](C)(C)C VLBPIWYTPAXCFJ-XMMPIXPASA-O 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700020675 O-deacetyl platelet activating factor Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 239000011173 biocomposite Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000006860 carbon metabolism Effects 0.000 description 1
- 239000002041 carbon nanotube Substances 0.000 description 1
- 229910021393 carbon nanotube Inorganic materials 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000013332 literature search Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- -1 poly(diallyldimethylammonium chloride) Polymers 0.000 description 1
- 229920000371 poly(diallyldimethylammonium chloride) polymer Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000003248 quinolines Chemical class 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- 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
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
-
- 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"
- G01N2021/6432—Quenching
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于贵金属纳米材料和比率荧光探针的制备技术领域,具体涉及一种基于溶菌酶稳定的铜纳米簇复合物及其比率荧光探针的制备方法。以水合肼为还原剂,溶菌酶为稳定剂制备了铜纳米簇。胆碱氧化酶催化胆碱产生过氧化氢,过氧化氢与辣根过氧化物酶氧化邻苯二胺生成二氨基酚嗪。由于二氨基酚嗪与铜纳米簇之间发生内滤效应,随着胆碱浓度的增加,二氨基酚嗪的荧光逐渐增强,而铜纳米簇的荧光被二氨基酚嗪逐渐猝灭,基于此可构建铜纳米簇与二氨基酚嗪荧光峰强度比率与胆碱浓度之间的线性关系,即制得比率荧光探针。该探针的制备方法简单,成本低,产品灵敏度高,可发展成为一种新颖的比率荧光探针,用于生物样品中胆碱的高效检测。
Description
技术领域:
本发明属于贵金属纳米材料和比率荧光探针的制备技术领域,具体涉及一种基于溶菌酶稳定的铜纳米簇复合物及其比率荧光探针的制备方法,其制备的探针可用于胆碱的高效检测。
背景技术:
胆碱是哺乳动物必需的营养物质,胆碱衍生物在人类健康的发展中起着重要的作用。胆碱的前体乙酰胆碱是一种神经递质,它对神经系统和全身主要器官的功能至关重要。胆碱还可作为甲基供体甘氨酸甜菜碱的前体,参与单碳代谢。胆碱的缺乏可导致肝损害、神经系统疾病、细胞死亡、以及癌症。奶制品中含有胆碱,婴幼儿的营养摄入量一般仅限于普通牛奶,所以添加胆碱对人体健康非常重要。胆碱的水平异常在阿尔茨海默病和帕金森病等神经退行性疾病中起重要作用。胆碱含量变化对人体健康和疾病造成很大的影响,因此胆碱的定量测定在临床分析中具有重要意义。胆碱的传统检测方法是色谱法,但该方法存在耗时长、操作复杂、条件苛刻、成本高等问题。相比而言,发展基于生物传感器的分析方法可在一定程度上克服色谱法存在的问题。
当前,基于生物传感器的胆碱分析方法有电化学法、比色法、荧光法、化学发光法等。文献检索表明,Qin等基于金纳米粒子修饰的碳纳米管聚合物构建了电流传感器用于胆碱的测定(Amperometric biosensors based on gold nanoparticles-decorated multi-walled carbon nanotubes-poly(diallyldimethylammonium chloride)biocompositefor the determination of choline,Xia Qin,Huicai Wang,Xinsheng Wang,ZhiyingMiao,Lili Chen,Wei Zhao,Miaomiao Shan,Qiang Chen,Sensors and Actuators B-Chemical,2010,147,593–598);Sanz-Vicente等依据分析酶促反应中荧光信号的变化,发展了水溶性磷脂胆碱的测定方法(Enzymatic methods for choline-containing watersoluble phospholipids based on fluorescence of choline oxidase:Application tolyso-PAF,Isabel Sanz-Vicente,Andres Domínguez,Carlos Ferrandez,Javier Galban,Analytical Biochemistry,2017,519,30–37)。
尽管当前发展的生物传感器方法可用于胆碱的检测,但这些方法需要昂贵的仪器、耗时和繁琐的操作,由于普遍采用了单信号检测模式,导致了测量结果的不稳定性。单信号检测模式依赖信号的强度来定性和定量目标物的浓度,而其强度往往受到体系和环境条件的影响。基于此,采用双信号强度比值处理即获得信号的比率强度,具备自校准功能,有效消除了自体和背景信号产生的干扰,提高了目标物检测结果的准确性和可靠性。贵金属纳米簇是一种重要的发光纳米材料,具有尺寸依赖的理化性质,因其尺寸小、水溶性好、光稳定性高、具备生物相容性和低毒性等优点,已被广泛用于构建荧光探针。迄今为止,尚未有采用比率荧光探针方法检测胆碱,以及基于铜纳米簇复合物的比率荧光探针来检测胆碱的国内外文献和专利报道。
发明内容:
本发明的目的在于克服上述现有技术存在的不足,设计一种操作简单、成本低廉、灵敏度高的基于铜纳米簇复合物的比率荧光探针检测胆碱的方法。
为了实现上述目的,本发明涉及的一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法其制备工艺包括以下步骤:
(1)铜纳米簇的制备:将2毫克五水合硫酸铜加入到5毫升二次蒸馏水中,配制成硫酸铜水溶液,在磁力搅拌下,将0.3克溶菌酶溶于10毫升二次蒸馏水,直至其完全溶解,然后将溶菌酶水溶液放置在45摄氏度的恒温水浴中孵化,在磁力搅拌下,将硫酸铜水溶液缓慢加入溶菌酶水溶液中,制备溶菌酶与硫酸铜的混合水溶液,在45摄氏度水浴中搅拌10分钟,向混合水溶液中加入1摩尔/升的氢氧化钠溶液,将pH调至10-11,滴加质量浓度为50%的水合肼调整混合水溶液的颜色从蓝色变为紫色,然后在45摄氏度水浴中搅拌反应10-24小时;
(2)反应结束后,将制备的铜纳米簇混合水溶液进行高速离心处理,去除不溶物,取上层清液透析5-10小时,透析后取出混合溶液进行旋蒸处理,将混合溶液中90%的水蒸发去除,剩余的溶液放入冷冻干燥箱中进行干燥,将干燥后得到的铜纳米簇分散于0.2摩尔/升的磷酸盐水缓冲液中备用,该缓冲液pH为7.0;
(3)二氨基吩嗪混合溶液的配制:称取162毫克邻苯二胺溶于磷酸盐水缓冲液中,制得3毫摩尔/升的邻苯二胺溶液,量取200微升该邻苯二胺溶液,向其中加入10微升1.5克/升的辣根过氧化物酶和10微升不同浓度的过氧化氢水溶液;
(4)铜纳米簇与二氨基吩嗪混合溶液的配制:将步骤(3)中配制的混合溶液在37摄氏度下避光反应30分钟,然后加入40微升步骤(2)中制得的磷酸盐水缓冲液分散的铜纳米簇,缓慢搅拌均匀,在室温下放置10分钟,得到铜纳米簇与二氨基酚嗪的均质溶液;
(5)测量步骤(4)中不同的过氧化氢摩尔浓度下,铜纳米簇与二氨基酚嗪均质水溶液的荧光发射光谱,拟合铜纳米簇与二氨基酚嗪荧光发射峰强度与过氧化氢浓度之间的线性关系;
(6)量取200微升3毫摩尔/升的邻苯二胺溶液,向其中加入10微升1克/升的辣根过氧化物酶和10微升不同浓度的胆碱水溶液,在37摄氏度下避光搅拌反应30分钟,然后将40微升磷酸盐水缓冲液分散的铜纳米簇加入反应混合物中,室温下放置10分钟,得到铜纳米簇/二氨基酚嗪/胆碱的均质溶液;
(7)测定不同的胆碱浓度下,铜纳米簇与二氨基酚嗪均质溶液的荧光发射光谱,拟合铜纳米簇与二氨基吩嗪荧光发射峰强度比率与胆碱摩尔浓度之间的线性关系,构建出检测胆碱的比率荧光探针,
本发明涉及的步骤(2)中所述的铜纳米簇尺寸为2~5纳米;步骤(3)中所述的过氧化氢摩尔浓度为1-50毫摩尔/升;步骤(4)中所述的铜纳米簇质量浓度为5-30克/升;步骤(7)中所述的胆碱浓度范围为0.01-100微摩尔/升,胆碱的检测极限为5-10纳摩尔/升。
本发明与现有技术相比,以水合肼为还原剂,溶菌酶为稳定剂制备了铜纳米簇。胆碱氧化酶催化胆碱产生过氧化氢,过氧化氢与辣根过氧化物酶氧化邻苯二胺生成二氨基酚嗪。由于二氨基酚嗪与铜纳米簇之间发生内滤效应,随着胆碱浓度的增加,二氨基酚嗪的荧光逐渐增强,而铜纳米簇的荧光被二氨基酚嗪逐渐猝灭,基于此可构建铜纳米簇与二氨基酚嗪荧光峰强度比率与胆碱浓度之间的线性关系,即制得比率荧光探针;其制备方法简单,成本低,产品灵敏度高,可发展成为一种新颖的比率荧光探针,用于生物样品中胆碱的高效检测。
附图说明:
图1为本发明涉及的一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法原理示意图;
图2为本发明涉及的胆碱比率荧光探针随着胆碱摩尔浓度增大对铜纳米簇和二氨基酚嗪荧光发射峰强度的响应,以及荧光峰强度比率与胆碱摩尔浓度之间的线性关系。
具体实施方式:
下面结合附图并通过具体实施例对本发明进行详细说明。
实施例1:
本实施例涉及的一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法其制备工艺与原理如图1所示,具体工艺步骤为:
铜纳米簇的制备:将2毫克五水合硫酸铜加入到5毫升二次蒸馏水中,配制成硫酸铜水溶液。在磁力搅拌下,将0.3克溶菌酶溶于10毫升二次蒸馏水,直至其完全溶解,然后将溶菌酶水溶液放置在45摄氏度的恒温水浴中孵化。在磁力搅拌下,将硫酸铜水溶液缓慢加入溶菌酶水溶液中,制备溶菌酶与硫酸铜的混合水溶液,在45摄氏度水浴中搅拌10分钟。向混合水溶液中加入1摩尔/升的氢氧化钠溶液,将pH调至10-11,滴加质量浓度为50%的水合肼调整混合水溶液的颜色从蓝色变为紫色,然后在45摄氏度水浴中搅拌反应10-24小时。将制备的铜纳米簇混合水溶液进行高速离心处理,去除不溶物,取上层清液透析5-10小时。透析后取出混合溶液进行旋蒸处理,将混合溶液中90%的水蒸发去除,剩余的溶液放入冷冻干燥箱中进行干燥。将干燥后得到的铜纳米簇分散于0.2摩尔/升的磷酸盐水缓冲液中备用,该缓冲液pH为7.0;
二氨基吩嗪混合溶液的配制:称取162毫克邻苯二胺溶于磷酸盐水缓冲液中,制得3毫摩尔/升的邻苯二胺溶液。量取200微升该邻苯二胺溶液,向其中加入10微升1.5克/升的辣根过氧化物酶和10微升1-10毫摩尔/升的过氧化氢水溶液;
铜纳米簇与二氨基吩嗪混合溶液的配制:将配制的混合溶液在37摄氏度下避光反应30分钟,然后加入40微升磷酸盐水缓冲液分散的10克/升铜纳米簇,缓慢搅拌均匀,在室温下放置10分钟,得到铜纳米簇与二氨基酚嗪的均质溶液;
测量不同的过氧化氢摩尔浓度下,铜纳米簇与二氨基酚嗪均质水溶液的荧光发射光谱,拟合铜纳米簇与二氨基酚嗪荧光发射峰强度与过氧化氢浓度之间的线性关系。量取200微升3毫摩尔/升的邻苯二胺溶液,向其中加入10微升1克/升的辣根过氧化物酶和10微升不同浓度的胆碱水溶液,在37摄氏度下避光搅拌反应30分钟,然后将40微升磷酸盐水缓冲液分散的铜纳米簇加入反应混合物中,室温下放置10分钟,得到铜纳米簇/二氨基酚嗪/胆碱的均质溶液;
测定不同的胆碱浓度下,铜纳米簇与二氨基酚嗪均质溶液的荧光发射光谱,拟合铜纳米簇与二氨基吩嗪荧光发射峰强度比率与胆碱摩尔浓度之间的线性关系,构建出检测胆碱的比率荧光探针。分别测定不同的胆碱摩尔浓度下,铜纳米簇与二氨基酚嗪均质溶液的荧光发射光谱(参见图2a),拟合铜纳米簇与二氨基酚嗪荧光发射峰强度比率IDAP/ICuNCs与胆碱摩尔尔浓度CCholine之间的线性关系(参见图2b)即:IDAP/ICuNCs=0.07232+0.03035CCholine(R2=0.9992),构建出胆碱的比率荧光探针,其中胆碱检测的浓度范围为0.01~80微摩尔/升,胆碱的检测极限为5纳摩尔/升。
实施例2:
本实施例中铜纳米簇制备的具体工艺步骤同实施例1。称取162毫克邻苯二胺溶于磷酸盐水缓冲液中,制得3毫摩尔/升的邻苯二胺溶液。量取200微升该邻苯二胺溶液,向其中加入10微升1.5克/升的辣根过氧化物酶和10微升5-20毫摩尔/升的过氧化氢水溶液。将配制的混合溶液在37摄氏度下避光反应30分钟,然后加入40微升制得的磷酸盐水缓冲液分散的20克/升铜纳米簇,缓慢搅拌均匀,在室温下放置10分钟,得到铜纳米簇与二氨基酚嗪的均质溶液。测量不同的过氧化氢摩尔浓度下,铜纳米簇与二氨基酚嗪均质水溶液的荧光发射光谱,拟合铜纳米簇与二氨基酚嗪荧光发射峰强度与过氧化氢浓度之间的线性关系。
量取200微升3毫摩尔/升的邻苯二胺溶液,向其中加入10微升1克/升的辣根过氧化物酶和10微升不同浓度的胆碱水溶液,在37摄氏度下避光搅拌反应30分钟,然后将40微升磷酸盐水缓冲液分散的铜纳米簇加入反应混合物中,室温下放置10分钟,得到铜纳米簇/二氨基酚嗪/胆碱的均质溶液。测定不同的胆碱浓度下,铜纳米簇与二氨基酚嗪均质溶液的荧光发射光谱,拟合铜纳米簇与二氨基吩嗪荧光发射峰强度比率与胆碱摩尔浓度之间的线性关系,构建出检测胆碱的比率荧光探针,胆碱检测的浓度范围为0.02~80微摩尔/升,胆碱的检测极限为8纳摩尔/升。
实施例3:
本实施例中铜纳米簇制备的具体工艺步骤同实施例1。称取162毫克邻苯二胺溶于磷酸盐水缓冲液中,制得3毫摩尔/升的邻苯二胺溶液。量取200微升该邻苯二胺溶液,向其中加入10微升1.5克/升的辣根过氧化物酶和10微升10-50毫摩尔/升的过氧化氢水溶液。将配制的混合溶液在37摄氏度下避光反应30分钟,然后加入40微升制得的磷酸盐水缓冲液分散的25克/升铜纳米簇,缓慢搅拌均匀,在室温下放置10分钟,得到铜纳米簇与二氨基酚嗪的均质溶液。测量不同的过氧化氢摩尔浓度下,铜纳米簇与二氨基酚嗪均质水溶液的荧光发射光谱,拟合铜纳米簇与二氨基酚嗪荧光发射峰强度与过氧化氢浓度之间的线性关系。
量取200微升3毫摩尔/升的邻苯二胺溶液,向其中加入10微升1克/升的辣根过氧化物酶和10微升不同浓度的胆碱水溶液,在37摄氏度下避光搅拌反应30分钟,然后将40微升磷酸盐水缓冲液分散的铜纳米簇加入反应混合物中,室温下放置10分钟,得到铜纳米簇/二氨基酚嗪/胆碱的均质溶液。测定不同的胆碱浓度下,铜纳米簇与二氨基酚嗪均质溶液的荧光发射光谱,拟合铜纳米簇与二氨基吩嗪荧光发射峰强度比率与胆碱摩尔浓度之间的线性关系,构建出检测胆碱的比率荧光探针,胆碱检测的浓度范围为0.05~100微摩尔/升,胆碱的检测极限为10纳摩尔/升。
实施例4:
本实施例涉及到实施例1中制备的比率荧光探针的应用,将其用于生物样品如人血清中胆碱的检测,胆碱摩尔浓度检测范围为0.01-80微摩尔/升,胆碱的检测极限可达5纳摩尔/升。与现有技术相比,如文献Analytical Biochemistry,2017,519,30-37采用单一荧光信号探针,而非双信号比率荧光探针检测胆碱。本发明对人血清样品中胆碱的检测回收率为98.0~99.8%,相对标准偏差为1.7-2.3%。相比现有技术,本发明比率荧光探针对胆碱的检测回收率更高,相对标准偏差更低,且制备工艺简单,成本低,产品灵敏度高,可发展成为一种新颖的胆碱比率荧光探针,用于生物样品中胆碱的高效检测。
Claims (1)
1.一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法,其特征在于,该方法具体包括以下步骤:
(1)铜纳米簇的制备:将2毫克五水合硫酸铜加入到5毫升二次蒸馏水中,配制成硫酸铜水溶液,在磁力搅拌下,将0.3克溶菌酶溶于10毫升二次蒸馏水,直至其完全溶解,然后将溶菌酶水溶液放置在45摄氏度的恒温水浴中孵化,在磁力搅拌下,将硫酸铜水溶液缓慢加入溶菌酶水溶液中,制备溶菌酶与硫酸铜的混合水溶液,在45摄氏度水浴中搅拌10分钟,向混合水溶液中加入1摩尔/升的氢氧化钠溶液,将pH调至10-11,滴加质量浓度为50%的水合肼调整混合水溶液的颜色从蓝色变为紫色,然后在45摄氏度水浴中搅拌反应10-24小时;
(2)将制备的铜纳米簇混合水溶液进行高速离心处理,去除不溶物,取上层清液透析5-10小时,透析后取出混合溶液进行旋蒸处理,将混合溶液中90%的水蒸发去除,剩余的溶液放入冷冻干燥箱中进行干燥,将干燥后得到的铜纳米簇分散于0.2摩尔/升的磷酸盐水缓冲液中备用,该缓冲液pH为7.0;
(3)二氨基吩嗪混合溶液的配制:称取162毫克邻苯二胺溶于磷酸盐水缓冲液中,制得3毫摩尔/升的邻苯二胺溶液,量取200微升该邻苯二胺溶液,向其中加入10微升1.5克/升的辣根过氧化物酶和10微升不同浓度的过氧化氢水溶液;
(4)铜纳米簇与二氨基吩嗪混合溶液的配制:将步骤(3)中配制的混合溶液在37摄氏度下避光反应30分钟,然后加入40微升步骤(2)中制得的磷酸盐水缓冲液分散的铜纳米簇,缓慢搅拌均匀,在室温下放置10分钟,得到铜纳米簇与二氨基酚嗪的均质溶液;
(5)测量步骤(4)中不同的过氧化氢摩尔浓度下,铜纳米簇与二氨基酚嗪均质水溶液的荧光发射光谱,拟合铜纳米簇与二氨基酚嗪荧光发射峰强度与过氧化氢浓度之间的线性关系;
(6)量取200微升3毫摩尔/升的邻苯二胺溶液,向其中加入10微升1克/升的辣根过氧化物酶和10微升不同浓度的胆碱水溶液,在37摄氏度下避光搅拌反应30分钟,然后将40微升磷酸盐水缓冲液分散的铜纳米簇加入反应混合物中,室温下放置10分钟,得到铜纳米簇/二氨基酚嗪/胆碱的均质溶液;
(7)测定不同的胆碱浓度下,铜纳米簇与二氨基酚嗪均质溶液的荧光发射光谱,拟合铜纳米簇与二氨基吩嗪荧光发射峰强度比率与胆碱摩尔浓度之间的线性关系,构建出检测胆碱的比率荧光探针;
步骤(2)中所述的铜纳米簇尺寸为2~5纳米;步骤(3)中所述的过氧化氢摩尔浓度为1-50毫摩尔/升;步骤(4)中所述的铜纳米簇质量浓度为5-30克/升;步骤(7)中所述的胆碱浓度范围为0.01-100微摩尔/升,胆碱的检测极限为5-10纳摩尔/升。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810605021.4A CN108801998B (zh) | 2018-06-13 | 2018-06-13 | 一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 |
PCT/CN2019/081157 WO2019237799A1 (zh) | 2018-06-13 | 2019-04-03 | 一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810605021.4A CN108801998B (zh) | 2018-06-13 | 2018-06-13 | 一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108801998A CN108801998A (zh) | 2018-11-13 |
CN108801998B true CN108801998B (zh) | 2019-07-30 |
Family
ID=64087045
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810605021.4A Expired - Fee Related CN108801998B (zh) | 2018-06-13 | 2018-06-13 | 一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN108801998B (zh) |
WO (1) | WO2019237799A1 (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108801998B (zh) * | 2018-06-13 | 2019-07-30 | 青岛大学 | 一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 |
CN110987881A (zh) * | 2019-11-01 | 2020-04-10 | 江苏大学 | 一种基于酶促反应双发射荧光探针的汞离子检测方法 |
CN113304748B (zh) * | 2020-03-04 | 2023-07-07 | 青岛大学 | 一种具有多种仿酶活性的铜纳米团簇及其制备方法与应用 |
CN114280024B (zh) * | 2021-12-25 | 2023-10-20 | 福州大学 | 一种基于铜纳米簇和氧化反应的特异性亚硝酸盐荧光检测方法 |
CN116970677B (zh) * | 2023-08-03 | 2024-04-30 | 西北大学 | 一种基于框架核酸的铜簇纳米材料在制备检测致病菌产品中的应用 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103073427B (zh) * | 2012-11-16 | 2014-12-31 | 南京工业大学 | 用于检测乙酰胆碱酯酶及其抑制剂活性的探针、用途及制备方法 |
CN105562705B (zh) * | 2015-12-21 | 2018-02-23 | 江苏科技大学 | 一种基于蛋白质合成铜量子点的方法及其应用 |
CN107245333B (zh) * | 2017-06-13 | 2018-07-10 | 陕西师范大学 | 基于亚胺连接的荧光纳米颗粒及其在检测Hg2+和乙酰胆碱酯酶中的应用 |
CN107478621B (zh) * | 2017-06-26 | 2019-11-22 | 南京医科大学 | 定量检测血清中可代谢产生h2o2的生物分子的方法 |
CN107501271B (zh) * | 2017-09-05 | 2019-05-17 | 西北师范大学 | 一种比色荧光双通道识别汞离子的传感器分子及其合成和应用 |
CN108801998B (zh) * | 2018-06-13 | 2019-07-30 | 青岛大学 | 一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 |
-
2018
- 2018-06-13 CN CN201810605021.4A patent/CN108801998B/zh not_active Expired - Fee Related
-
2019
- 2019-04-03 WO PCT/CN2019/081157 patent/WO2019237799A1/zh active Application Filing
Also Published As
Publication number | Publication date |
---|---|
CN108801998A (zh) | 2018-11-13 |
WO2019237799A1 (zh) | 2019-12-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108801998B (zh) | 一种基于铜纳米簇复合物的比率荧光探针检测胆碱的方法 | |
Shi et al. | Naked-eye sensitive detection of alkaline phosphatase (ALP) and pyrophosphate (PPi) based on a horseradish peroxidase catalytic colorimetric system with Cu (ii) | |
Hu et al. | Double-strand DNA-templated synthesis of copper nanoclusters as novel fluorescence probe for label-free detection of biothiols | |
Jiang et al. | Ultra-sensitive fluorescent probes for hypochlorite acid detection and exogenous/endogenous imaging of living cells | |
Huang et al. | A novel one-step colorimetric assay for highly sensitive detection of glucose in serum based on MnO 2 nanosheets | |
Xue et al. | Ultra-small CuS nanoparticles as peroxidase mimetics for sensitive and colorimetric detection of uric acid in human serum | |
Ma et al. | A terbium chelate based fluorescent assay for alkaline phosphatase in biological fluid | |
CN104406971B (zh) | 一种直接胆红素检测试剂 | |
Wang et al. | H 2 O 2-mediated fluorescence quenching of double-stranded DNA templated copper nanoparticles for label-free and sensitive detection of glucose | |
Shaban et al. | A colorimetric alkaline phosphatase biosensor based on p-aminophenol-mediated growth of silver nanoparticles | |
Hu et al. | A ratiometric fluorescence sensor for ultra-sensitive detection of trypsin inhibitor in soybean flour using gold nanocluster@ carbon nitride quantum dots | |
Pan et al. | Turn-on fluorescence measurement of acid phosphatase activity through an aggregation-induced emission of thiolate-protected gold nanoclusters | |
CN110411990B (zh) | 一种基于纳米探针检测过氧化氢和相关目标物的方法 | |
Zhan et al. | Development of iridium (III) phosphorescent probe for hypochlorous acid detection in macrophages cells and cancer cells co-culture system and application in inflamed mouse model | |
Zhang et al. | A new copper mediated on-off assay for alkaline phosphatase detection based on MoOx quantum dots | |
Zuo et al. | A novel ratiometric fluorescent sensing method based on MnO2 nanosheet for sensitive detection of alkaline phosphatase in serum | |
Liang et al. | Rapid and sensitive colorimetric detection of dopamine based on the enhanced-oxidase mimicking activity of cerium (IV) | |
Chen et al. | Responsive methylene blue release from lanthanide coordination polymer for label-free, immobilization-free and sensitive electrochemical alkaline phosphatase activity assay | |
Zhidkova et al. | Determination of superoxide dismutase and SOD-mimetic activities by a chemical system: Co 2/H 2 O 2/lucigenin | |
Lv et al. | A gold nanoparticle based colorimetric and fluorescent dual-channel probe for acetylcholinesterase detection and inhibitor screening | |
Zhen et al. | A NIR fluorescent probe for the specific detection of hypochlorite and its application in vitro and in vivo | |
CN104677897A (zh) | 基于纳米金催化显色体系的pH及尿素的测定方法 | |
Gao et al. | A mitochondria-targeted and deep-red emission ratiometric fluorescent probe for real-time visualization of SO 2 in living cells, zebrafish and living mice | |
Sun et al. | Convenient and sensitive colorimetric determination of alendronate sodium with Ce 4+-triggered oxidation of TMB | |
Li et al. | Poly (cytosine)‐templated Silver Nanoclusters as Fluorescent Biosensor for Highly Sensitive Detection of Uric Acid |
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: 20190730 |