CN111426833B - 可视化检测肿瘤外泌体的纳米杂化物探针的制备方法 - Google Patents

可视化检测肿瘤外泌体的纳米杂化物探针的制备方法 Download PDF

Info

Publication number
CN111426833B
CN111426833B CN202010263033.0A CN202010263033A CN111426833B CN 111426833 B CN111426833 B CN 111426833B CN 202010263033 A CN202010263033 A CN 202010263033A CN 111426833 B CN111426833 B CN 111426833B
Authority
CN
China
Prior art keywords
probe
stirring
reaction
sample
exosomes
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
CN202010263033.0A
Other languages
English (en)
Other versions
CN111426833A (zh
Inventor
金辉
姜晓文
桂日军
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.)
Qingdao University
Original Assignee
Qingdao 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 Qingdao University filed Critical Qingdao University
Priority to CN202010263033.0A priority Critical patent/CN111426833B/zh
Priority to PCT/CN2020/100534 priority patent/WO2021203568A1/zh
Publication of CN111426833A publication Critical patent/CN111426833A/zh
Application granted granted Critical
Publication of CN111426833B publication Critical patent/CN111426833B/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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • 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

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Plasma & Fusion (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

本发明公开了可视化检测肿瘤外泌体的纳米杂化物探针的制备方法，基于铜离子配位黑磷量子点(Cu‑BPQDs)和有机荧光染料(Cy5)制备了新型双发射纳米杂化物载体探针，该探针可用于乳腺癌外泌体的可视化半定量检测。在探针的水分散液中加入含有外泌体的样品，引起探针表面DNA分子阀门开启，负载的电子受体分子释放，Cu‑BPQDs蓝色荧光从淬灭转为荧光恢复，此过程对Cy5红色荧光影响甚微。随着样品中外泌体浓度的增大，探针‑样品混合分散液的荧光颜色逐渐从红色转为蓝色，包括多种中间过渡色。建立样品中外泌体浓度与探针‑样品混合分散液的荧光颜色类型之间的关联，发展可视化检测肿瘤外泌体的新方法。

Description

可视化检测肿瘤外泌体的纳米杂化物探针的制备方法
技术领域：
本发明属于多功能纳米杂化物和肿瘤外泌体检测探针的制备技术领域，具体涉及一种基于铜离子配位黑磷量子点和有机荧光染料的新型双发射纳米杂化物载体探针的制备方法，其制备的探针可用于乳腺癌外泌体的可视化半定量检测。
背景技术：
近年来的科学研究表明，肿瘤外泌体是一种新型的生物标志物，可广泛用于不同癌症的早期筛查和临床诊断。对肿瘤外泌体的有效检测，在一定程度上可避免癌症常规检测过程中存在的侵入式筛选，克服检测成本高、检测敏感度低等问题。外泌体是一种直径为50～100nm的细胞外囊泡，是经过內溶体途径从多囊体中释放出来的。由于外泌体从亲本细胞中携带了生物大分子，如 mRNA、DNA、跨膜和胞质蛋白等，外泌体可作为介导细胞间信息的信使，在疾病诊断，尤其是在癌症相关生理状态改变的检测方面发挥了重要作用。
现有的肿瘤外泌体检测技术主要包括纳米颗粒追踪分析、流式细胞术、表面等离子共振法、比色分析法、发光分析法、电化学分析法等。金芳芳等公开了一种流式细胞仪检测外泌体的方法(金芳芳；王延博；徐学博；陈张朋；薛姜飞.一种流式细胞仪检测外泌体的方法.国家发明专利.公开号CN110702589A)。何农跃等公开了一种基于分支滚环扩增的外泌体荧光(FL)检测传感器(何农跃；黄蓉蓉；李智洋.一种基于分支滚环扩增的外泌体FL检测传感器.国家发明专利.公开号CN110396536A)。Zhang等报导了Ti3C2 MXenes纳米片催化的电致化学发光生物传感器检测外泌体(Huixin Zhang,Zonghua Wang,Qiuxia Zhang,Feng Wang,Yang Liu.Ti3C2 MXenes nanosheets catalyzed highly efficientelectrogenerated chemiluminescence biosensor for the detection of exosomes.Biosensors andBioelectronics,2019,124–125,184–190)。Doldán等报导了基于表面标志物介导信号放大的电化学三明治免疫传感器用于检测外泌体(Ximena Doldán,Pablo Fagúndez,Alfonso Cayota,Justo Laíz,Juan Pablo Tosar, Electrochemical sandwichimmunosensor for determination of exosomes based on surface marker-mediatedsignal amplification,Analytical Chemistry,2016,88,10466 -10473)。
尽管有关外泌体检测的技术已有文献资料报导，但现有的外泌体检测技术仍然存在瓶颈，急需在纳米级肿瘤外泌体的选择性、灵敏性、可视化分析、定量检测等领域取得进展。基于此，本发明公开了一种基于铜离子配位黑磷量子点(Cu-BPQDs)和五甲川菁类FL染料Cy5的新型绿/红双发射FL纳米杂化物载体探针的制备方法，该纳米探针可用于乳腺癌外泌体的比率FL可视化半定量检测。截止目前，尚未检索到有关基于Cu-BPQDs/Cy5的纳米杂化物载体探针的制备，以及基于纳米杂化物载体探针可视化检测肿瘤外泌体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，设计一种高选择性、高灵敏性、可视化分析和半定量检测乳腺癌外泌体的新方法。
为实现上述目的，本发明涉及的可视化检测肿瘤外泌体的纳米杂化物探针的制备方法，其制备方法包括以下步骤：
1.可视化检测肿瘤外泌体的纳米杂化物探针的制备方法，其特征在于，该方法具体包括以下步骤：
(1)铜离子配位黑磷量子点(Cu-BPQDs)的制备：将25mg黑磷晶体加入 30mL氮甲基吡咯烷酮中，加入1mol/L硝酸铜水溶液5mL，加入溶解了5mg 巯基丙酸的水溶液5mL，磁力搅拌形成均质混合液。将混合液用超声波细胞粉碎仪处理2h，再用超声波清洗器水浴处理6h。将处理后的分散液转入微型磁力高压反应釜中，在N2保护下加热至120℃，连续搅拌反应12h。反应产物冷却至室温，在5000rpm转速下离心10min，取上层清液在14000rpm转速下离心15min。离心后的沉淀物用乙醇和二次蒸馏水冲洗3次，真空干燥得到羧基功能化Cu-BPQDs。
(2)Cy5内包封二氧化硅纳米球(Cy5-SiO2)的制备：将聚乙二醇氨基 PEG-NH2功能化Cy5溶于无水乙醇，加入(3-氨丙基)三乙氧基硅烷(APTS)，搅拌均匀，放置避光处。加入氨水与乙醇，搅拌均匀，加入正硅酸乙酯(TEOS)继续搅拌，再加入TEOS进行反应。反应产物经离心、乙醇洗涤和干燥后制得Cy5 内包封SiO2纳米球。将其分散在APTS与醋酸的混合液中，在室温下执行搅拌反应，产物经离心、洗涤和干燥，得到表面氨基功能化Cy5-SiO2纳米球。
(3)Cy5-SiO2/Cu-BPQDs/mSiO2复合物(Complex)的制备：将N-羟基硫代琥珀酸亚胺(NHS)和1-乙基-(3-二甲基氨基丙基)碳二亚胺(EDC)盐酸盐分散在磷酸盐水(PBS)缓冲液中，加入氨基功能化Cy5-SiO2纳米球，搅拌均匀，放置避光处。磁力搅拌下将羧基功能化Cu-BPQDs水分散液加入混合液中，搅拌反应。产物经离心、洗涤和干燥后制得Cy5-SiO2/Cu-BPQDs杂化物。将此杂化物溶于无水乙醇，加入十二烷基三甲基溴化铵(DTAB)和APTS，搅拌反应，放置避光处。加入氨水与乙醇后搅拌均匀，加入TEOS搅拌，再加入TEOS继续反应。加入NH4NO3洗脱模板DTAB，产物经离心、乙醇洗涤和干燥后制得氨基功能化 Complex。
(4)纳米杂化物载体探针(Complex-5Fu-DNA1/DNA2)的制备：向PBS缓冲液中加入偶联剂NHS和EDC盐酸盐，加入氨基功能化Complex，搅拌反应，添加一端为-COOH另一端为-SH的单链DNA1即HS-DNA1-COOH，在室温下搅拌反应。反应产物经透析、除溶剂、离心、洗涤和干燥制得杂化物 Complex-DNA1。采用水浴孵化，电子受体分子如5-氟尿嘧啶(5Fu)或多巴胺(DA) 进入mSiO2孔道内，完成电子受体分子的负载，如形成Complex-5Fu-DNA1。添加DNA1的互补链DNA2，由于两条DNA单链的碱基互补配对形成双螺旋结构，将电子受体分子封装在mSiO2孔道内，制得Complex-5Fu-DNA1/DNA2。
(5)将此纳米杂化物载体探针分散于PBS缓冲液中，加入含有肿瘤外泌体的水分散液或生物流体样品，磁力搅拌均匀。将此载体探针与含有外泌体的样品形成的混合分散液，用滴管转移至玻璃比色皿中，或滴涂在聚酰亚胺柔性薄膜上。采用365nm波长紫外光激发照射，用智能手机拍摄比色皿中和柔性薄膜上混合分散液的FL颜色。建立混合分散液的FL颜色类型与混合分散液中肿瘤外泌体浓度的关联，构建样品的FL颜色类型变化与样品中肿瘤外泌体浓度的对应关系，进而发展基于新型纳米杂化物探针的可视化检测肿瘤外泌体的新方法。肿瘤外泌体可视化检测的浓度范围为2×101～5×106particles mL–1，检测限为 20～100particles mL–1。
本发明的效果是：公开了一种基于Cu-BPQDs和有机荧光染料Cy5的新型双发射纳米杂化物载体探针(Complex-5Fu-DNA1/DNA2)的制备方法，该探针可用于乳腺癌外泌体的可视化半定量检测。当生物流体样品中含有乳腺癌外泌体时，将该样品加入载体探针的水分散液中。因外泌体与其单链DNA2适体特异性结合形成外泌体/DNA2复合物，使DNA2竞争性挣脱开DNA1，引起Complex 的mSiO2孔道内被负载的电子受体分子5Fu被释放出来。因5Fu释放而远离 Cu-BPQDs，5Fu引发的光诱导电子转移(PIET)效应被抑制，Cu-BPQDs的蓝色 FL从淬灭逐渐转为FL恢复增强。Cy5被实心SiO2包覆形成Cy5-SiO2纳米球，故Cy5的红色FL在样品的添加过程中基本保持不变。因此，Cy5红色FL作为参比，Cu-BPQDs蓝色FL在含有外泌体样品的添加过程中从FL淬灭转为FL 恢复。随着样品中外泌体浓度的增大，探针-样品混合分散液的FL颜色类型逐渐从红色转为蓝色，包括两种颜色之间的过渡色如深红、大红、橘红、橙黄、黄绿、浅绿、深绿、蓝绿、浅蓝、深蓝等。基于样品中肿瘤外泌体的浓度与探针-样品混合分散液的FL颜色类型或色差之间的关联，发展可视化检测肿瘤外泌体的新方法。此外，该探针-样品混合分散液可放置在玻璃比色皿中，实现溶液态(液相)可视化检测外泌体；也可滴涂在柔性薄膜上，实现固态基底(固相)的可视化检测外泌体。
附图说明：
图1为新型纳米杂化物载体探针Complex-5Fu-DNA1/DNA2的制备示意图；
图2为基于该新型纳米杂化物载体探针可视化检测肿瘤外泌体的基本原理和操作过程示意图。
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离子配位黑磷量子点和有机荧光染料的新型双发射纳米杂化物载体探针的制备方法，该纳米杂化物载体探针的制备示意图如图1所示，具体制备步骤如下：
将25mg黑磷晶体加入30mL氮甲基吡咯烷酮中，加入1mol/L硝酸铜水溶液5mL，加入溶解了5mg巯基丙酸的水溶液5mL，磁力搅拌形成均质混合液。将混合液用超声波细胞粉碎仪处理2h，再用超声波清洗器水浴处理6h。将处理后的分散液转入微型磁力高压反应釜中，在N2保护下加热至120℃，连续搅拌反应12h。反应产物冷却至室温，在5000rpm转速下离心10min，取上层清液在14000rpm转速下离心15min。离心后的沉淀物用乙醇和二次蒸馏水冲洗3 次，真空干燥得到羧基功能化Cu-BPQDs。
依据
Figure BDA0002440124800000051
方法制备Cy5-SiO2，将PEG-NH2功能化Cy5溶于无水乙醇，加入APTS，搅拌均匀后放置避光处。加入氨水与乙醇，搅拌均匀后加入TEOS继续搅拌，再加入TEOS进行反应。反应产物经离心、乙醇洗涤和干燥后制得Cy5 内包封SiO2纳米球。将其分散在APTS与醋酸的混合液中，在室温下搅拌反应，产物经离心、洗涤和干燥，得到表面氨基功能化Cy5-SiO2纳米球，平均直径～50 nm。
将偶联剂NHS和EDC盐酸盐分散在PBS缓冲液中，加入氨基功能化 Cy5-SiO2纳米球，搅拌均匀后放置避光处。磁力搅拌下将羧基功能化Cu-BPQDs 水分散液加入混合液中，搅拌反应。产物经离心、洗涤和干燥制得 Cy5-SiO2/Cu-BPQDs杂化物。将此杂化物溶于无水乙醇，加入DTAB和APTS，搅拌反应，放置避光处保存。加入氨水与乙醇并搅拌均匀，加入TEOS继续搅拌，再加入TEOS反应。加入NH4NO3洗脱模板DTAB，产物经离心、乙醇洗涤和干燥处理，制得氨基功能化Cy5-SiO2/Cu-BPQDs/mSiO2复合物Complex，平均直径～100nm。
向PBS缓冲液中加入偶联剂NHS和EDC盐酸盐，加入氨基功能化Complex，搅拌反应，添加HS-DNA1-COOH，在室温下搅拌反应。反应产物经透析、旋蒸、除溶剂、萃取、离心、洗涤和干燥处理制得杂化物Complex-DNA1。通过水浴孵化方式，5Fu进入mSiO2孔道内形成Complex-5Fu-DNA1。添加DNA1互补链 DNA2，两条DNA单链碱基互补配对形成双螺旋结构，将5Fu封装在孔道内，制得纳米杂化物载体探针Complex-5Fu-DNA1/DNA2。
将此纳米杂化物载体探针分散于PBS缓冲液中，加入含有乳腺癌外泌体的水分散液，磁力搅拌均匀。将此载体探针与含有外泌体的水分散液形成的混合分散液，用滴管转移至玻璃比色皿中，或滴涂在聚酰亚胺柔性薄膜上。采用365 nm波长紫外光激发照射，用智能手机拍摄比色皿中和柔性薄膜上混合分散液的 FL颜色。建立混合分散液的FL颜色类型与混合分散液中外泌体浓度的关联，构建样品的FL颜色类型变化与样品中外泌体浓度的对应关系，发展基于该纳米杂化物探针的可视化检测乳腺癌外泌体的新方法。如图2所示，乳腺癌外泌体可视化检测的浓度范围为1×102～1×106particles mL–1，检测限为100particles mL-1。
实施例2：
本实施例涉及的一种基于铜离子配位黑磷量子点和有机荧光染料的新型双发射纳米杂化物载体探针的制备方法，该新型纳米杂化物载体探针 Complex-5Fu-DNA1/DNA2的制备同实施例1，其它具体制备步骤如下：
将此纳米杂化物载体探针分散于PBS缓冲液中，分别加入含有不同乳腺癌外泌体浓度的人血或血浆清样品，磁力搅拌均匀。将此载体探针与含有外泌体的人血清或血浆样品形成的混合分散液，用滴管转移至玻璃比色皿中，或滴涂在聚酰亚胺柔性薄膜上。采用365nm波长紫外光激发照射，用智能手机拍摄比色皿中和柔性薄膜上混合分散液的FL颜色。建立混合分散液的FL颜色类型与混合分散液中外泌体浓度的关联，构建人血清或血浆样品的FL颜色类型变化与人血清或血浆样品中外泌体浓度的对应关系，发展基于该纳米杂化物探针的可视化检测乳腺癌外泌体的新方法。其中，乳腺癌外泌体可视化检测的浓度范围为5×101～2×106particles mL–1，检测限为50particles mL–1。
实施例3：
本实施例涉及的一种基于铜离子配位黑磷量子点和有机荧光染料的新型双发射纳米杂化物载体探针的制备方法，该新型纳米杂化物载体探针 Complex-5Fu-DNA1/DNA2的制备同实施例1，其它具体制备步骤如下：
将此纳米杂化物载体探针分散于PBS缓冲液中，分别加入含有不同乳腺癌外泌体浓度的人尿液样品，磁力搅拌均匀。将此载体探针与含有外泌体的人尿液样品形成的混合分散液，用滴管转移至玻璃比色皿中，或滴涂在聚酰亚胺柔性薄膜上。采用365nm波长紫外光激发照射，用智能手机拍摄比色皿中和柔性薄膜上混合分散液的FL颜色。建立混合分散液的FL颜色类型与混合分散液中外泌体浓度的关联，构建人尿液样品的FL颜色类型变化与人尿液样品中外泌体浓度的对应关系，发展基于该纳米杂化物探针的可视化检测乳腺癌外泌体的新方法。其中，乳腺癌外泌体可视化检测的浓度范围为4×101～5×105particlesmL–1，检测限为40particles mL–1。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (1)

1.可视化检测肿瘤外泌体的纳米杂化物探针的制备方法，其特征在于，该方法具体包括以下步骤：
(1)铜离子配位黑磷量子点Cu-BPQDs的制备：将25mg黑磷晶体加入30mL氮甲基吡咯烷酮中，加入1mol/L硝酸铜水溶液5mL，加入溶解了5mg巯基丙酸的水溶液5mL，磁力搅拌形成均质混合液；将混合液用超声波细胞粉碎仪处理2h，再用超声波清洗器水浴处理6h；将处理后的分散液转入微型磁力高压反应釜中，在N2保护下加热至120℃，连续搅拌反应12h；反应产物冷却至室温，在5000rpm转速下离心10min，取上层清液在14000rpm转速下离心15min；离心后的沉淀物用乙醇和二次蒸馏水冲洗3次，真空干燥得到羧基功能化Cu-BPQDs；
(2)Cy5内包封二氧化硅纳米球Cy5-SiO2的制备：将聚乙二醇氨基PEG-NH2功能化Cy5溶于无水乙醇，加入(3-氨丙基)三乙氧基硅烷APTS，搅拌均匀，放置避光处；加入氨水与乙醇，搅拌均匀，加入正硅酸乙酯TEOS继续搅拌，再加入TEOS进行反应；反应产物经离心、乙醇洗涤和干燥后制得Cy5内包封SiO2纳米球；将其分散在APTS与醋酸的混合液中，在室温下执行搅拌反应，产物经离心、洗涤和干燥，得到表面氨基功能化Cy5-SiO2纳米球；
(3)Cy5-SiO2/Cu-BPQDs/mSiO2复合物的制备：将N-羟基硫代琥珀酸亚胺NHS和1-乙基-(3-二甲基氨基丙基)碳二亚胺EDC盐酸盐分散在磷酸盐水PBS缓冲液中，加入氨基功能化Cy5-SiO2纳米球，搅拌均匀，放置避光处；磁力搅拌下将羧基功能化Cu-BPQDs水分散液加入混合液中，搅拌反应；产物经离心、洗涤和干燥后制得Cy5-SiO2/Cu-BPQDs杂化物；将此杂化物溶于无水乙醇，加入十二烷基三甲基溴化铵DTAB和APTS，搅拌反应，放置避光处；加入氨水与乙醇后搅拌均匀，加入TEOS搅拌，再加入TEOS继续反应；加入NH4NO3洗脱模板DTAB，产物经离心、乙醇洗涤和干燥后制得氨基功能化复合物，简称为Complex；
(4)纳米杂化物载体探针Complex-5Fu-DNA1/DNA2的制备：向磷酸盐水PBS缓冲液中加入偶联剂NHS和EDC盐酸盐，加入氨基功能化Complex，搅拌反应，添加一端为-COOH另一端为-SH的单链DNA1即HS-DNA1-COOH，在室温下搅拌反应；反应产物经透析、除溶剂、离心、洗涤和干燥制得杂化物Complex-DNA1；采用水浴孵化，电子受体分子5-氟尿嘧啶5Fu进入mSiO2孔道内，完成电子受体分子的负载，形成Complex-5Fu-DNA1；添加DNA1的互补链DNA2，由于两条DNA单链的碱基互补配对形成双螺旋结构，将电子受体分子封装在mSiO2孔道内，制得Complex-5Fu-DNA1/DNA2；
(5)将此纳米杂化物载体探针分散于磷酸盐水PBS缓冲液中，加入含有肿瘤外泌体的水分散液或生物流体样品，磁力搅拌均匀；将此载体探针与含有外泌体的样品形成的混合分散液，用滴管转移至玻璃比色皿中，或滴涂在聚酰亚胺柔性薄膜上；采用365nm波长紫外光激发照射，用智能手机拍摄比色皿中或柔性薄膜上混合分散液的FL颜色；建立混合分散液的FL颜色类型与混合分散液中肿瘤外泌体浓度的关联，构建样品的FL颜色类型变化与样品中肿瘤外泌体浓度的对应关系；肿瘤外泌体可视化检测的浓度范围为2×101～5×106particles mL–1，检测限为20～100particles mL–1。
CN202010263033.0A 2020-04-07 2020-04-07 可视化检测肿瘤外泌体的纳米杂化物探针的制备方法 Active CN111426833B (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202010263033.0A CN111426833B (zh) 2020-04-07 2020-04-07 可视化检测肿瘤外泌体的纳米杂化物探针的制备方法
PCT/CN2020/100534 WO2021203568A1 (zh) 2020-04-07 2020-07-07 可视化检测肿瘤外泌体的新型纳米杂化物探针的制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010263033.0A CN111426833B (zh) 2020-04-07 2020-04-07 可视化检测肿瘤外泌体的纳米杂化物探针的制备方法

Publications (2)

Publication Number Publication Date
CN111426833A CN111426833A (zh) 2020-07-17
CN111426833B true CN111426833B (zh) 2021-04-23

Family

ID=71555812

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010263033.0A Active CN111426833B (zh) 2020-04-07 2020-04-07 可视化检测肿瘤外泌体的纳米杂化物探针的制备方法

Country Status (2)

Country Link
CN (1) CN111426833B (zh)
WO (1) WO2021203568A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112782138B (zh) * 2020-12-24 2021-10-29 生物岛实验室 用于检测细胞外囊泡的试剂盒及其应用
CN114539545B (zh) * 2022-01-13 2023-06-13 中国科学院宁波材料技术与工程研究所 一种双金属-有机框架材料及其制备方法和应用
CN114479985A (zh) * 2022-01-27 2022-05-13 西安建筑科技大学 一种包覆黑磷量子点的复合材料及其制备方法和应用
CN114886523A (zh) * 2022-05-18 2022-08-12 福州大学 腹腔穿刺针套管装置
CN115156548B (zh) * 2022-05-19 2023-06-06 青岛大学 纳米带状Cu3P/Cu材料及其制备方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10648959B2 (en) * 2016-05-05 2020-05-12 University Of Southern California Black phosphorus gas sensor
CN108641708B (zh) * 2018-06-13 2019-07-12 青岛大学 基于银纳米簇复合物的三聚氰胺比率荧光探针的制备方法
CN109870438A (zh) * 2019-03-22 2019-06-11 青岛大学 潜指纹荧光可视化检测可卡因的表面分子印迹纳米探针的制备方法
CN109724949B (zh) * 2019-03-25 2019-07-23 青岛大学 一种用于肿瘤标志物可视化检测的柔性杂化膜的制备方法
CN110320260B (zh) * 2019-07-27 2021-07-30 福建师范大学 基于MXenes和黑磷量子点增强的外泌体电致化学发光传感器
CN110846026B (zh) * 2019-12-02 2020-05-19 青岛大学 基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法

Also Published As

Publication number Publication date
WO2021203568A1 (zh) 2021-10-14
CN111426833A (zh) 2020-07-17

Similar Documents

Publication Publication Date Title
CN111426833B (zh) 可视化检测肿瘤外泌体的纳米杂化物探针的制备方法
Mahmoudi et al. Lateral flow assays towards point-of-care cancer detection: A review of current progress and future trends
Arai et al. Exploring the use of upconversion nanoparticles in chemical and biological sensors: from surface modifications to point-of-care devices
Zhu et al. A light-up fluorescence resonance energy transfer magnetic aptamer-sensor for ultra-sensitive lung cancer exosome detection
Liu et al. Core–shell Fe3O4–Au magnetic nanoparticles based nonenzymatic ultrasensitive electrochemiluminescence immunosensor using quantum dots functionalized graphene sheet as labels
CN108414758B (zh) 用于检测肿瘤标志物miRNA-141的SERS生物传感器的制备方法及其应用
Zhu et al. Silver nanoparticles-enhanced time-resolved fluorescence sensor for VEGF165 based on Mn-doped ZnS quantum dots
Gong et al. A sensitive fluorescence “turn on” nanosensor for glutathione detection based on Ce-MOF and gold nanoparticles
Chen et al. Architecting ultra-bright silanized carbon dots by alleviating the spin-orbit coupling effect: a specific fluorescent nanoprobe to label dead cells
Zhang et al. Ratiometric fluorescent probe for ascorbic acid detection based on MnO2 nanosheets, gold nanoclusters and thiamine
Gao et al. A DNA hybridization detection based on fluorescence resonance energy transfer between dye-doped core-shell silica nanoparticles and gold nanoparticles
Karami et al. Intrinsic dual-emissive carbon dots for efficient ratiometric detection of Cu2+ and aspartic acid
Deng et al. Target-triggered signal turn-on detection of prostate specific antigen based on metal-enhanced fluorescence of Ag@ SiO2@ SiO2-RuBpy composite nanoparticles
Bai et al. Semiconducting polymer dots as fluorescent probes for in vitro biosensing
Liao et al. Sensitive fluorescent sensor for the fuzzy exosomes in serum based on the exosome imprinted polymer sandwiched with aggregation induced emission
Yan et al. Carbon dots modified/prepared by supramolecular host molecules and their potential applications: A review
Ma et al. Dual-reverse-signal ratiometric fluorescence method for malachite green detection based on multi-mechanism synergistic effect
Zhong et al. High electrochemical active Au-NP/2D zinc-metal organic frameworks heterostructure-based ECL sensor for the miRNA-522 detection in triple negative breast cancer
Zhu et al. Dual sensing reporter system of assembled gold nanoparticles toward the sequential colorimetric detection of adenosine and Cr (III)
He et al. Ultrasensitive fluorescence detection of microRNA through DNA-induced assembly of carbon dots on gold nanoparticles with no signal amplification strategy
Zhang et al. Janus nanozyme based satellite structure immunosandwich colorimetric strategy for glycoproteins visual detection
Wei et al. Self-assembled electroactive MOF–magnetic dispersible aptasensor enables ultrasensitive microcystin-LR detection in eutrophic water
You et al. Silica-coated triangular gold nanoprisms as distance-dependent plasmon-enhanced fluorescence-based probes for biochemical applications
CN108580919B (zh) 银核介孔金纳米结构材料的制备方法、表面增强拉曼检测探针及其应用
Bao et al. A dual-emission polymer carbon nanoparticles for ratiometric and visual detection of pH value and bilirubin

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