CN110780076B - 一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用 - Google Patents

一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用 Download PDF

Info

Publication number
CN110780076B
CN110780076B CN201911015710.0A CN201911015710A CN110780076B CN 110780076 B CN110780076 B CN 110780076B CN 201911015710 A CN201911015710 A CN 201911015710A CN 110780076 B CN110780076 B CN 110780076B
Authority
CN
China
Prior art keywords
abeta
pcl
pnipam
fluorescent probe
beta
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201911015710.0A
Other languages
English (en)
Other versions
CN110780076A (zh
Inventor
史林启
杨惠茹
黄帆
马如江
安英丽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nankai University
Original Assignee
Nankai University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Nankai University filed Critical Nankai University
Priority to CN201911015710.0A priority Critical patent/CN110780076B/zh
Publication of CN110780076A publication Critical patent/CN110780076A/zh
Application granted granted Critical
Publication of CN110780076B publication Critical patent/CN110780076B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/66—Polyesters containing oxygen in the form of ether groups
    • C08G63/664—Polyesters containing oxygen in the form of ether groups derived from hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
    • 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
    • C09K2211/14—Macromolecular compounds
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Polymers & Plastics (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Materials Engineering (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

一种敏感的β‑淀粉样蛋白(Aβ)纳米荧光探针的制备方法及应用。该探针的构建基于温度响应性的复合胶束，并在疏水微区上接入能够与Aβ蛋白特异性结合的GLVFF短肽以提高复合胶束对Aβ的选择性结合能力，同时接入可与氨基反应的荧光分子——对硝基苯基活性酯修饰的低聚(对苯乙烯撑)衍生物(OPV‑NP)荧光基团来实现复合胶束捕获Aβ前后荧光信号的改变，从而实现对溶液中(例如脑脊液)Aβ蛋白含量的检测。本发明的制备方法简单，无毒，检测方法简便易行，易于操作，耗时短，成本低，提高了检测的安全性和工作效率。该发明检测效果灵敏，对Aβ具有特异性，为低浓度Aβ蛋白的体外检测提供了新的思路，在阿尔兹海默症的早期诊断上具有广泛的应用前景。

Description

一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用
技术领域
本发明属于纳米生物医药材料领域，利用复合壳层胶束化学修饰可靶向Aβ的GLVFF多肽，同时结合具有氨基响应性的对硝基苯基活性酯修饰的低聚(对苯乙烯撑)衍生物(OPV-NP)荧光分子，开发制备了一种高灵敏性的低成本Aβ荧光探针，用于Aβ蛋白的体外检测。
背景技术
阿尔兹海默症(Alzheimer’s disease,AD)，又称老年痴呆症，是老年人最常见的神经退行性疾病之一。该疾病的发展伴随着记忆的逐渐丧失和认知能力的损伤。在AD晚期，难以挽回的病理损伤已经形成，其治疗效果十分有限。因此，尽早诊断、尽早治疗是治疗AD的最好途径。然而目前常用的方法，如：脑部成像(PET，CT等)，神经心理学，认知和神经学测试等的诊断主要基于疾病中晚期大脑发生的病理变化和认知能力的改变，难以适应早期诊断的需要。
不同于大脑的器质性病理变化和认知损伤，存在于体液中的AD生物标志物，β-淀粉样蛋白(Aβ)的含量在AD早期就已经发生明显变化。因此，检测脑脊液中Aβ的含量能够及时有效地进行AD的早期诊断，从而有利于AD的早期治疗。目前针对体液中Aβ的检测方法主要为酶联免疫吸附法(ELISA)。尽管ELISA有着较好的特异性检测能力，但是其操作复杂，检测时间长，用于Aβ识别的酶联抗体的价格十分昂贵，而且其用于化学发光检测的基质具有致癌性，使其应用受到限制，迫切需要寻找一种替代方法。纳米粒子拥有复杂多变的表面结构和可修饰性，通过设计具有适当功能的纳米粒子，可以实现特定的需求，是开发新型Aβ蛋白探针的良好选择。
发明内容
为提高对低浓度Aβ的检测的灵敏度，便捷度和安全性，本发明提供了一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用，为低浓度Aβ蛋白的体外检测提供一种简便、安全、经济的新方法。
本发明利用温度响应性两嵌段聚合物与两亲性嵌段聚合物自组装得到具有表面相分离结构的核-壳-冠结构复合胶束，并在其疏水微区部分化学修饰了LVFF短肽和对硝基苯基活性酯功能化的低聚(对苯乙烯撑)衍生物(OPV-NP)。利用胶束表面疏水微区与亲水链段组成的空腔，捕获Aβ蛋白至疏水微区，同时阻止部分大体积蛋白的进入和干扰。OPV-NP分子上的对硝基苯基活性酯的拉电子作用使其骨架三聚苯乙炔的荧光淬灭。其在疏水微区的引入可以与复合胶束吸附的Aβ蛋白赖氨酸残基的氨基发生反应，通过亲和取代替代对硝基苯基活性酯，导致荧光增强，从而使复合胶束具有荧光探针的功能。LVFF短肽是Aβ蛋白的疏水核心，能够特异性地与Aβ蛋白结合并抑制Aβ聚集。其引入可以进一步提高探针对Aβ蛋白的选择性结合能力，阻止小分子蛋白与OPV-NP分子的反应。该探针避免了传统ELISA方法中昂贵酶联抗体和有毒基质的使用，降低了成本，提高了安全性和工作效率。该纳米探针能够实现对低浓度Aβ蛋白灵敏且特异性的检测，为Aβ的体外检测和阿尔兹海默症的早期诊断提供了新的思路。
本发明的技术方案
一种敏感的β-淀粉样蛋白纳米荧光探针，在温度响应性两嵌段聚合物的响应性链段末端引入能够与Aβ特异性结合的GLVFF短肽，以及能够与Aβ上蛋白氨基反应的具有氨基响应性的荧光分子——对硝基苯基活性酯功能化的低聚(对苯乙烯撑)衍生物(OPV-NP)，然后再通过与两亲性嵌段聚合物的自组装，得到能够敏感且特异性检测低浓度Aβ含量的核-壳-冠结构复合胶束作为Aβ纳米荧光探针。
一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法，具体方法如下：
1)两亲性嵌段聚合物聚环己内酯-b-聚乙二醇(PCL-b-PEG)的合成
在辛酸亚锡的催化下，通过端羟基聚乙二醇(PEG-OH)引发ε-己内酯(ε-CL)开环聚合(ROP)得到PCL-b-PEG两亲性嵌段聚合物。
2)聚环己内酯-三硫代碳酸酯(PCL-TTC)大分子链转移剂的合成
利用丙烯酸羟乙酯(HEA)引发ε-CL开环聚合(ROP)得到聚环己内酯(PCL-OH)；
将链转移剂2-(十二烷基三硫代碳酸酯基)-2-甲基丙酸(DDMAT)经过酰氯化后与PCL-OH反应，得到大分子链转移剂PCL-TTC。
3)温度响应性两嵌段聚合物聚环己内酯-b-聚N-异丙基丙烯酰胺(PCL-b-PNIPAM)的合成
将步骤2)得到的PCL-TTC和N-异丙基丙烯酰胺(NIPAM)单体混合，通过可逆加成-断裂链转移自由基聚合(RAFT)合成PCL-b-PNIPAM。
4)PCL-b-PNIPAM-APM的合成
将N-(2-氨基乙基)丙烯酰胺盐酸盐(APM)单体与步骤3)合成的PCL-b-PNIPAM混合，通过RAFT聚合合成PCL-b-PNIPAM-APM。
5)PCL-b-PNIPAM-GLVFF的合成
利用正己胺和二甲基苯基磷将步骤3)合成的PCL-b-PNIPAM端基的三硫酯键还原为巯基，加入丙烯酰氧基琥珀酰亚胺(NAS)，并通过巯基-双键的点击反应得到PCL-b-PNIPAM-NHS。随后与GLVFF上的氨基反应，纯水透析后，真空冷冻干燥得到PCL-b-PNIPAM-GLVFF。
6)Aβ纳米荧光探针的制备
将步骤1)合成的PCL-b-PEG、步骤5)合成的PCL-b-PNIPAM-GLVFF与步骤4)合成的PCL-b-PNIPAM-APM按照2:1:1的摩尔比溶解在DMF中。加入OPV-NP，其与聚合物中APM的摩尔比大于1:1，37℃下震荡孵育0.5-2h。在电磁搅拌下逐滴滴入4℃纯水中，搅拌过夜，PBS透析后得到粒径40-300nm且分布均匀的复合胶束，在37℃下孵育后即为Aβ纳米荧光探针。
本发明所述Aβ纳米荧光探针能够用于特异性检测溶液中的Aβ——Aβ纳米荧光探针对Aβ检测的特异性；具体步骤如下：
使用人工脑脊液(ACSF)配置Aβ蛋白等多种蛋白溶液[如牛血清蛋白(BSA)，泛素蛋白(ubiquitin)等]。将Aβ纳米荧光探针置于37℃下孵育后，分别加入等质量浓度的上述蛋白(如BSA、ubiquitin和Aβ三种蛋白)溶液，继续在37℃下恒温震荡30min。利用荧光分光光度计检测体系的荧光变化，记录相应荧光光谱和最大发射波长处的荧光强度。发现BSA和ubiquitin对体系荧光无影响，而加入Aβ蛋白后体系荧光明显增强。
本发明所述Aβ纳米荧光探针能够定量检测溶液中Aβ的含量——Aβ纳米荧光探针对Aβ蛋白的检测能力；具体步骤如下：
1)建立Aβ浓度—荧光强度的标准曲线；
将Aβ纳米荧光探针置于37℃下孵育后，分别与不同浓度的Aβ蛋白样品溶液混合，继续在37℃下恒温震荡30min。在相同条件下检测混合液样品的荧光，记录相应荧光光谱和最大发射波长处的荧光强度，得到Aβ浓度与荧光光强的标准曲线。
2)Aβ的含量检测；
按照步骤1)方法检测待测样品与纳米荧光探针混合液的荧光强度，通过步骤1)中建立的Aβ浓度—荧光强度标准曲线即可得到Aβ的含量。
本发明所述Aβ纳米荧光探针的应用能够检测大鼠脑脊液中Aβ含量，具体步骤是：
取SD大鼠脑脊液，分别用ELISA试剂盒和该Aβ纳米荧光探针检测其Aβ含量，结果无显著性差异。
本发明的优点和有益效果：
本发明提供了一种简单有效的纳米荧光探针用于体外Aβ蛋白的检测。该方法制备简单，成本较低，便于操作，避免了昂贵抗体和有毒的致癌发光基底的使用，大大缩短了Aβ蛋白的检测时间，提高了检测的安全性和效率，具有较好的检测灵敏度和特异性，在Aβ的体外检测和阿尔兹海默症的早期诊断上极具价值。
附图说明
图1为Aβ纳米荧光探针的粒径分布及相应的透射电镜图；图中显示，纳米粒子呈球形，粒径在93nm左右，大小均一，具有较窄的粒径分布；
图2为Aβ纳米荧光探针与不同蛋白共同孵育后的荧光强度变化；a)纳米探针分别与三种不同蛋白孵育后的荧光光谱，b)纳米探针与三种不同蛋白孵育后在最大发射波长处的发射光强；
图3为Aβ纳米荧光探针与不同浓度Aβ反应后的荧光发射光谱；
图4为Aβ蛋白浓度相对于Aβ纳米荧光探针荧光强度的标准曲线；
图5为ELISA试剂盒和Aβ纳米荧光探针检测所得大鼠脑脊液中Aβ浓度。
具体实施方式
实施例1：
一种Aβ纳米荧光探针的制备方法，该方法实施步骤如下：
1)两亲性嵌段聚合物PCL-b-PEG的合成
将3.0g干燥处理过的CH3O-PEG114-OH和6.3g减压蒸馏处理过的ε-CL加入到50mL干燥的茄形瓶中混合，用15mL重蒸过的无水甲苯溶解，加入一滴辛酸亚锡(Sn(Oct)2)。随后，通过液氮冷冻—抽真空—充氮气—解冻，循环三次后，在氮气保护下于110℃的油浴中反应12h。反应后加入适量二氯甲烷稀释，然后在十倍体积的冰***中沉淀。待沉淀完全后，经过抽滤，洗涤，真空干燥后所得到两亲性嵌段聚合物PCL-b-PEG。
2)大分子链转移剂PCL-TTC的合成
将0.1g丙烯酸羟乙酯(HEA)和7.0g减蒸后的ε-CL加入到茄形瓶中，加入12mL重蒸过的甲苯溶解，加入一滴Sn(Oct)2。随后，液氮冷冻—抽真空—通氮气—解冻，重复三次。之后在氮气保护下，于110℃的油浴中反应12h。反应结束后，加入适量的二氯甲烷稀释并用十倍体积的冰***沉淀，抽滤，洗涤，真空干燥后得到聚环己内酯PCL-OH。
将0.6g链转移剂DDMAT加入50mL圆底烧瓶，溶于10mL二氯甲烷中。将0.6mL草酰氯溶于5mL二氯甲烷中，在冰水浴和磁力搅拌条件下，10分钟内滴加到圆底烧瓶中。滴加完毕后，升温至25℃，反应2h。30℃条件下旋蒸，除去二氯和过量的草酰氯，加入2mL二氯甲烷再次旋蒸，重复三次。将5g PCL-OH加入圆底烧瓶，溶于20mL二氯甲烷，加入0.17g三乙胺，在25℃条件下反应24h。将反应液浓缩至5mL，在冰***中沉淀，过滤，洗涤，真空干燥后得到PCL-TTC大分子链转移剂。
3)温度响应性嵌段聚合物PCL-b-PNIPAM的合成
首先，将NIPAM用正己烷为溶剂进行重结晶以除去阻聚剂，并真空干燥得到NIPAM单体。将1.2g重结晶后的NIPAM与3.0g PCL-TTC混合，溶于DMF中，加入15mg偶氮二异丁腈(AIBN)作为引发剂，通过液氮冷冻—抽真空—充氮气—解冻，循环三次后，在氮气保护下于70℃的油浴中反应24h，通过RAFT聚合合成PCL-b-PNIPAM，冰***沉淀，抽滤，洗涤后，真空干燥，得到PCL-b-PNIPAM粉末。
4)PCL-b-PNIPAM-APM的合成
将25.5mg APM单体与1.0g PCL-b-PNIPAM混合，溶于DMF中，加入5mg AIBN作为引发剂，通过RAFT反应得到PCL-b-PNIPAM-APM，具体反应条件与步骤3)中PCL-b-PNIPAM合成条件相同。
5)PCL-b-PNIPAM-GLVFF的合成
将0.5g PCL-b-PNIPAM与30mg丙烯酰氧基琥珀酰亚胺(NAS)混合，溶于10mL二氯甲烷，加入35μL二甲基苯基磷(DMPP)作为催化剂，通入N2鼓泡除氧20min。然后加入2.5mL正己胺，反应体系再次通入N2鼓泡除氧15min，然后在避光条件下室温搅拌过夜。反应结束后，用二氯甲烷稀释，冰***沉淀，抽滤，洗涤，真空干燥后得到PCL-b-PNIPAM-NHS。将产物与过量GLVFF混合，溶于5mL DMF，加入100μL三乙胺，30℃水浴条件下电磁搅拌过夜，纯水透析，真空冷冻干燥得到PCL-b-PNIPAM-GLVFF。
6)Aβ纳米荧光探针的制备
将2mg PCL-b-PEG，1mg PCL-b-PNIPAM-GLVFF，1mg PCL-b-PNIPAM-APM溶于DMF，加入0.5mg OPV-NP，得到聚合物浓度为5mg/ml的stock液，37℃下震荡孵育30min。在电磁搅拌下逐滴滴入4℃纯水中，搅拌过夜，经PBS透析后得到0.5mg/ml胶束，37℃下孵育30min，得到Aβ纳米荧光探针。
通过动态光散射(DLS)和透射电镜(TEM)对胶束粒径进行表征，其形貌呈球形，平均粒径为93nm，大小均一，具有较窄的粒径分布，如图1。
7)Aβ纳米荧光探针对Aβ检测的特异性
使用人工脑脊液(ACSF)配置等质量浓度的牛血清蛋白(BSA)，泛素蛋白(ubiquitin)和Aβ蛋白的溶液。将Aβ纳米荧光探针置于37℃下恒温30min后，分别加入等质量浓度的三种蛋白溶液，继续在37℃下恒温震荡30min。利用荧光分光光度计检测体系的荧光变化(Ex＝376nm)，记录相应荧光光谱和最大发射波长(Em＝458nm)处的荧光强度。发现BSA和ubiquitin对体系荧光无影响，而加入Aβ蛋白后体系荧光明显增强，如图2。
8)Aβ纳米荧光探针对Aβ蛋白的检测能力
(1)建立Aβ浓度—荧光强度的标准曲线；
使用人工脑脊液(ACSF)配置一系列浓度的Aβ蛋白溶液。将Aβ荧光探针在37℃下恒温30min后，与不同浓度的Aβ溶液等体积混合，继续在37℃下震荡孵育30min；利用荧光分光光度计检测混合液的荧光(Ex＝376nm)，记录相应荧光光谱(图3)和最大发射波长(Em＝458nm)处的荧光强度，建立Aβ浓度—荧光强度的标准曲线，如图3、图4；
(2)Aβ的含量检测；
按照步骤1)方法检测混合液的荧光强度，通过步骤1)中建立的Aβ浓度—荧光强度标准曲线即可得到Aβ的含量。
9)Aβ纳米荧光探针检测大鼠脑脊液中Aβ含量
取雌性SD大鼠脑脊液，分别用ELISA试剂盒和该Aβ纳米荧光探针检测其Aβ含量，结果无显著性差异，证明了Aβ纳米荧光探针的可靠性，如图5。

Claims (4)

1.一种敏感的β-淀粉样蛋白Aβ纳米荧光探针，其特征在于：在温度响应性两嵌段聚合物聚环己内酯-b-聚N-异丙基丙烯酰胺PCL-b-PNIPAM的响应性链段末端引入能够与Aβ特异性结合的GLVFF短肽，以及能够与Aβ蛋白氨基反应的具有氨基响应性的荧光分子——对硝基苯基活性酯功能化的低聚对苯乙烯撑衍生物OPV-NP，然后再通过与两亲性嵌段聚合物聚环己内酯-b-聚乙二醇PCL-b-PEG的自组装，得到能够敏感且特异性检测Aβ含量的核-壳-冠结构复合胶束作为Aβ纳米荧光探针。
2.一种权利要求1所述的Aβ纳米荧光探针的制备方法，其特征在于步骤如下：
1)两亲性嵌段聚合物聚环己内酯-b-聚乙二醇PCL-b-PEG的合成
在辛酸亚锡的催化下，通过端羟基聚乙二醇PEG-OH引发的ε-己内酯ε-CL开环聚合得到PCL-b-PEG两亲性嵌段聚合物；
2)聚环己内酯-三硫代碳酸酯PCL-TTC大分子链转移剂的合成
利用丙烯酸羟乙酯HEA引发ε-CL开环聚合得到聚环己内酯PCL-OH；将链转移剂2-(十二烷基三硫代碳酸酯基)-2-甲基丙酸DDMAT经过酰氯化后与PCL-OH反应，得到大分子链转移剂PCL-TTC；
3)温度响应性两嵌段聚合物聚环己内酯-b-聚N-异丙基丙烯酰胺PCL-b-PNIPAM的合成
将步骤2)得到的PCL-TTC和N-异丙基丙烯酰胺NIPAM单体混合，通过可逆加成-断裂链转移自由基聚合得到PCL-b-PNIPAM；
4)PCL-b-PNIPAM-APM的合成
将N-(2-氨基乙基)丙烯酰胺盐酸盐APM单体与步骤3)合成的PCL-b-PNIPAM混合，通过可逆加成-断裂链转移自由基聚合得到PCL-b-PNIPAM-APM；
5)PCL-b-PNIPAM-GLVFF的合成
利用正己胺和二甲基苯基磷将步骤3)合成的PCL-b-PNIPAM端基的三硫酯键还原为巯基，加入丙烯酰氧基琥珀酰亚胺NAS，通过巯基-双键的点击反应得到PCL-b-PNIPAM-NHS，随后与短肽GLVFF上的氨基反应，纯水透析后，真空冷冻干燥得到PCL-b-PNIPAM-GLVFF；
6)Aβ纳米荧光探针的制备
将步骤1)合成的PCL-b-PEG、步骤5)合成的PCL-b-PNIPAM-GLVFF与步骤4)合成的PCL-b-PNIPAM-APM按照2:1:1的摩尔比在DMF中溶解，加入OPV-NP，其与APM摩尔比大于1:1，37℃下震荡孵育0.5-2h；在电磁搅拌下逐滴滴入4℃纯水中，搅拌过夜，经PBS透析后得到粒径40-300nm且分布均匀的复合胶束，在37℃下孵育后即得Aβ纳米荧光探针。
3.一种权利要求1所述的Aβ纳米荧光探针的应用，其特征在于所述应用能够定量检测溶液中Aβ的含量，具体步骤如下：
1)建立Aβ浓度—荧光强度的标准曲线；
将Aβ荧光探针在37℃下孵育后，与不同浓度的Aβ样品溶液等体积混合，继续在37℃下震荡孵育30min以上；在相同条件下检测各样品的荧光，记录相应荧光光谱和最大发射波长处的荧光强度，建立Aβ浓度—荧光强度的标准曲线；
2)Aβ的含量检测；
按照步骤1)方法检测待测样品的荧光强度，通过步骤1)中建立的Aβ浓度—荧光强度标准曲线即可得到样品中Aβ的含量。
4.根据权利要求3所述的Aβ纳米荧光探针的应用，其特征在于所述探针能够特异性检测溶液中的Aβ蛋白。
CN201911015710.0A 2019-10-24 2019-10-24 一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用 Active CN110780076B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911015710.0A CN110780076B (zh) 2019-10-24 2019-10-24 一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911015710.0A CN110780076B (zh) 2019-10-24 2019-10-24 一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用

Publications (2)

Publication Number Publication Date
CN110780076A CN110780076A (zh) 2020-02-11
CN110780076B true CN110780076B (zh) 2023-02-17

Family

ID=69386716

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911015710.0A Active CN110780076B (zh) 2019-10-24 2019-10-24 一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用

Country Status (1)

Country Link
CN (1) CN110780076B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113956601B (zh) * 2021-10-28 2024-04-02 南开大学 一种疏水-静电多功能化的纳米复合胶束的制备及在蛋白质酶保护中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6331440B1 (en) * 1995-12-12 2001-12-18 Karolinska Innovations Ab Peptide binding the KLVFF-sequence of amyloid-β
CN103965421A (zh) * 2014-04-24 2014-08-06 浙江大学 一种具有核壳结构的温敏性两亲嵌段共聚物的制备方法及其产品
CN104004149A (zh) * 2014-05-13 2014-08-27 中国科学技术大学 一种可荧光实时监测抗癌药物释放的方法
CN110194822A (zh) * 2019-06-03 2019-09-03 西北师范大学 一种基于单臂TPE分子的温敏型双荧光Pdots的制备及应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180311161A1 (en) * 2015-06-29 2018-11-01 Technion Research & Development Foundation Limited Amphiphilic polymers encapsulating therapeutically active agents

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6331440B1 (en) * 1995-12-12 2001-12-18 Karolinska Innovations Ab Peptide binding the KLVFF-sequence of amyloid-β
CN103965421A (zh) * 2014-04-24 2014-08-06 浙江大学 一种具有核壳结构的温敏性两亲嵌段共聚物的制备方法及其产品
CN104004149A (zh) * 2014-05-13 2014-08-27 中国科学技术大学 一种可荧光实时监测抗癌药物释放的方法
CN110194822A (zh) * 2019-06-03 2019-09-03 西北师范大学 一种基于单臂TPE分子的温敏型双荧光Pdots的制备及应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Engineered peptidic constructs metabolize amyloid β by self-assembly-driven reactions;Tanmay Mondal等;《Chem. Commun.》;20190401;第1-6页 *
Maintenance of Amyloid β Peptide Homeostasis by Artificial Chaperones Based on Mixed-Shell Polymeric Micelles;Fan Huang等;《Angewandte Chemie》;20140701;第126卷;第9131-9136页 *
温度响应性的复合壳层结构胶束对β 淀粉样多肽纤维化的抑制作用;黄帆 等;《2013 年全国高分子学术论文报告会》;20131016;第427页 *

Also Published As

Publication number Publication date
CN110780076A (zh) 2020-02-11

Similar Documents

Publication Publication Date Title
Chen et al. A paper-supported aptasensor based on upconversion luminescence resonance energy transfer for the accessible determination of exosomes
Xu et al. Metal-enhanced fluorescent dye-doped silica nanoparticles and magnetic separation: A sensitive platform for one-step fluorescence detection of prostate specific antigen
Svechkarev et al. Organic fluorescent dye-based nanomaterials: advances in the rational design for imaging and sensing applications
Wei et al. Fe3O4 nanoparticles-loaded PEG–PLA polymeric vesicles as labels for ultrasensitive immunosensors
CN109233810B (zh) 发色聚合物点
Cai et al. MUC-1 aptamer-conjugated dye-doped silica nanoparticles for MCF-7 cells detection
JP5313249B2 (ja) ナノ粒子を用いた蛍光共鳴エネルギ移動検出
CN110128665B (zh) 基于偶氮还原酶响应的两亲性嵌段聚合物近红外荧光探针及应用
CN101260219B (zh) 一种用于实现可逆荧光调控的三嵌段共聚物胶束体系的制备方法
EP2809510B1 (en) Polyelectrolyte-coated polymer dots and related methods
EP3504291B1 (en) Fluorescent particles
CN102725299A (zh) 光致发光纳米粒子及其制备方法与应用
Gubala et al. Kinetics of immunoassays with particles as labels: effect of antibody coupling using dendrimers as linkers
Deng et al. Quantum dots-based multifunctional nano-prodrug fabricated by ingenious self-assembly strategies for tumor theranostic
EP3658648B1 (en) Conjugated polymers and methods of use
CN104146964B (zh) 一种多用途聚赖氨酸荧光自组装纳米微球载体及其制备方法与应用
CN110606859B (zh) 聚集诱导发光化合物、其制备方法及其检测病毒的应用
CN110780076B (zh) 一种敏感的β-淀粉样蛋白纳米荧光探针的制备方法及应用
CN110372829A (zh) 基于偶氮还原响应的聚合物凝胶荧光探针的制备及应用
CN112390786B (zh) 一种具有aie特性的荧光和磁共振造影信号同时增强的两亲性分子、纳米颗粒及制备方法与应用
Zhu et al. Construction of β-cyclodextrin derived CDs-coupled block copolymer micelles loaded with CdSe/ZnS QDs via host-guest interaction for ratiometric fluorescence sensing of metal ions
Aikawa et al. Polystyrene latex particles containing europium complexes prepared by miniemulsion polymerization using bovine serum albumin as a surfactant for biochemical diagnosis
CN102127586A (zh) 一种磁性荧光双功能纳米生物探针及其制备方法
CN115433291A (zh) 海藻酸-g-香豆素衍生物的合成及其制备负载阿霉素的Pickering乳液的方法
CN110776440B (zh) 通过pisa法制备偶氮还原酶响应性聚合物荧光探针及其应用

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