CN111004622B - 一种检测多巴胺的高灵敏荧光探针的制备方法及其应用 - Google Patents
一种检测多巴胺的高灵敏荧光探针的制备方法及其应用 Download PDFInfo
- Publication number
- CN111004622B CN111004622B CN201911191890.8A CN201911191890A CN111004622B CN 111004622 B CN111004622 B CN 111004622B CN 201911191890 A CN201911191890 A CN 201911191890A CN 111004622 B CN111004622 B CN 111004622B
- Authority
- CN
- China
- Prior art keywords
- solution
- aptamer
- seq
- fluorescent probe
- sensitivity
- 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
Links
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
- 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
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
-
- 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
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9406—Neurotransmitters
- G01N33/9413—Dopamine
-
- 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
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- 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
-
- 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
- C09K2211/1074—Heterocyclic compounds characterised by ligands containing more than three nitrogen atoms as heteroatoms
-
- 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
-
- 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
- C09K2211/1408—Carbocyclic compounds
- C09K2211/1425—Non-condensed systems
-
- 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
- C09K2211/1441—Heterocyclic
- C09K2211/145—Heterocyclic containing oxygen as the only 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
- C09K2211/14—Macromolecular compounds
- C09K2211/1441—Heterocyclic
- C09K2211/1466—Heterocyclic containing nitrogen as the only heteroatom
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明属于生物传感器技术领域,特别是指一种检测多巴胺的高灵敏荧光探针的制备方法及其应用。将带有An长度的适体加入TCEP的醋酸缓冲液中,然后与AuNPs溶液混合反应,孵育18h以上,再加入1×PBS调整溶液,孵育6‑8h,然后分两次加入NaCl溶液,静置后离心三次,每次离心混合物用PBS溶液清洗两次,然后将沉淀重新分散在PBS溶液中,加入FAM‑DNA,振荡反应,得高灵敏荧光探针;该方法通过金属增强荧光效应提高传感器的灵敏度,通过加入氧化石墨烯在减少离心过程的同时,实现传感器灵敏度的更高幅度增加。
Description
技术领域
本发明属于生物传感器技术领域,特别是指一种检测多巴胺的高灵敏荧光探针的制备方法及其应用。
背景技术
人类大脑包括数十亿个神经元和多种感官信息传递机制,通过神经元群体密切协调活动控制人或动物的思维和行为。当神经细胞产生功能障碍时,神经递质将会失调,神经异常放电,引起神经性疾病的发生。如多巴胺(dopamine,DA)是中枢神经系统中一种重要的神经递质,其在中枢神经系统、内分泌系统和心血管系统中发挥重要作用,很多疾病都与多巴胺的分泌不足或缺少有关,如:神经性食欲缺乏症、阿尔茨海默病和帕金森病等。在临床实践中,检测大脑中神经递质的微小变化对获取神经信息,预防和诊断神经性疾病有重要意义。
将核酸适体作为识别基团构建的荧光传感器称为荧光适体传感器,该传感器通过换能器将核酸适体与靶目标的强亲和力和特异性结合转换为荧光信号的变化,由荧光信号的变化量来对靶目标进行检测分析,兼具荧光分析和核酸适体的优点,可对多种生物小分子进行定量分析,且价格便宜、操作简单,应用前景广阔。金属增强荧光(Metal EnhancedFluorescence, MEF)现象的研究始于20世纪70年代,是指分布于金属表面、岛状粒子或溶胶粒子附近荧光团的荧光发射强度较之自由态荧光发射强度大大增加的现象。将金属增强荧光效应与荧光适体传感器结合即可得到荧光增强型适体传感器,为生物分子的高灵敏检测提供新方法。专利CN201910341109.4公开了一种基于核酸适配体的超灵敏检测多巴胺的方法,通过多巴胺适配体识别多巴胺,使适配体从双链DNA1中脱离,从而不断引发酶切反应,荧光信号降低,利用此循环放大技术实现了对多巴胺的超灵敏检测。但对比文件1在检测过程中为了实现信号放大,加入微量的多巴胺就需要消耗较多的核酸外切酶、荧光染料、DNA等原料,检测成本较高,且检测过程复杂,而本发明通过金属增强荧光效应提高传感器的灵敏度,通过加入氧化石墨烯在减少离心过程的同时,实现传感器灵敏度的更高幅度增加,不需要消耗过多的原料。
发明内容
本发明提出一种检测多巴胺的高灵敏荧光探针的制备方法及其应用,结合了进金属增强荧光效应与氧化石墨烯对荧光基团的猝灭效应,加入被测物后,荧光信号由增强变为猝灭,相比于一般的荧光增强型传感器,该传感器荧光信号变化幅度更大,检测灵敏度更高。且加入被测物后,不需要进行离心处理,避免了离心过程中所能引入的各种误差,处理过程更为简单。
本发明的技术方案是这样实现的:
一种检测多巴胺的高灵敏荧光探针的制备方法,步骤如下:
(1)将带有An长度的适体加入TCEP的醋酸缓冲液中,然后与AuNPs溶液混合反应,孵育18h以上,得溶液Ⅰ;
(2)向经步骤(1)处理的溶液Ⅰ中加入1×PBS调整溶液的pH值和离子强度,再孵育6-8h,得溶液Ⅱ;
(3)向经步骤(2)处理的溶液Ⅱ中分两次加入NaCl溶液,每次间隔2-3h,然后再分三次加入NaCl溶液,每次间隔3-4h,静置40-50h,得溶液Ⅲ;
(4)将溶液Ⅲ离心三次,每次离心混合物用PBS溶液清洗两次,然后将沉淀重新分散在PBS溶液中,加入FAM-DNA,于37℃条件下振荡反应2-3h,得高灵敏荧光探针。
所述步骤(1)中适体序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ IDNo.4、SEQ ID No.5、SEQ ID No.6或SEQ ID No.7中的任一项所示,适体5'端修饰有-SH。
所述步骤(1)中适体在的TCEP的醋酸缓冲液中的浓度为5-10 μM、适体的TCEP的醋酸缓冲液体积为10-50μL;AuNPs溶液的浓度为0.1-0.5μM、体积为100-300μL,TCEP的醋酸缓冲液的浓度为10 mM。
所述步骤(2)中1×PBS调整溶液的体积为25-30 μL,pH值为3.5-8.4。
所述步骤(3)中NaCl溶液的浓度为2M,前两次每次加入NaCl溶液的体积15-20μL;后三次每次加入NaCl溶液的体积35-40μL。
所述步骤(4)中FAM-DNA序列如SEQ ID No.8所示,其3'端修饰有FAM荧光基团。
利用所述的高灵敏荧光探针检测多巴胺的方法,步骤如下:将待测样品溶液加入到高灵敏荧光探针溶液中,于室温黑暗条件下培养0.5-1.5h,得反应液,向反应液中加入10μg/mL、80μL的氧化石墨烯,等待20-30min后进行荧光检测。
本发明具有以下有益效果:
1、本发明的荧光传感器原理为:金属增强荧光效应和氧化石墨烯对荧光基团的猝灭效应。经过巯基修饰过的目标物的核酸适配体与金纳米颗粒结合后,FAM标记的目标物核酸适配体互补链遵循碱基互补配对原则通过氢键与目标物核酸适配体结合形成荧光增强型结构,荧光信号最强,当PH取6.5,AuNPs@ployAn-aptamer@FAM-DNA复合结构中荧光增强效果最为明显,荧光信号强度是FAM本身强度的3倍。加入多巴胺后,由于核酸适体与靶目标的特异性结合,结合在核酸适体上的FAM-DNA会从纳米结构中脱离出来。此时在溶液中加入一定量的氧化石墨烯,脱离出的FAM-DNA会与氧化石墨烯结合而产生荧光淬灭,荧光信号强度的变化与添加的待测物浓度成正比。该方法通过金属增强荧光效应提高传感器的灵敏度为27.82,相较于常用的离心后检测灵敏度为8.59,提高了三倍以上,通过加入氧化石墨烯在减少离心过程的同时,实现传感器灵敏度的更高幅度增加,检测低灵敏度适配体传感器所不能检测的样本。
2、与现有技术相比,本发明加入被测物后,荧光信号由增强变为猝灭,相比于一般的荧光增强型传感器,该传感器荧光信号变化幅度更大,检测灵敏度更高,最低检测限为0.2nM。且加入被测物后,不需要进行离心处理,避免了离心过程中所能引入的各种误差,处理过程更为简单。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为传感器响应机理图。
图2为pH值对本申请高灵敏荧光探针荧光强度的影响图,其中A为pH值不同时AuNPs@ployAn-aptamer@FAM-DNA样品的发射光谱,B为pH值与荧光521nm峰值处光强的关系图。
图3为pH 6.5环境下,碱基A不同时AuNPs@ployAn-aptamer@FAM-DNA样品的发射光谱图。
图4为离心检测多巴胺结果图,其中左图为不同DA浓度底物的荧光光谱,右图为底物中荧光强度变化与DA浓度之间的线性关系。
图5为不离心时检测多巴胺的结果图,其中A为不同DA浓度未离心样品的荧光光谱,B为底物中荧光强度变化与DA浓度之间的线性关系图。
图6为不离心加石墨烯时检测多巴胺结果图,其中左图为不同DA浓度未离心且加入GO后样品的荧光光谱图,右图为底物中荧光强度变化与DA浓度之间的线性关系图。
图7传感器特异性检测结果。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例
本实施例的一种检测多巴胺的高灵敏荧光探针的制备方法:
(1)仪器与试剂
仪器:荧光强度的检测使用F-7000荧光分光光度计(日立,日本)。所有溶液pH值的检测使用FE-20K-mete(METTLER TOLEDO,Switzerland)。使用HZQ -F200恒温振动器(北京东联海尔仪器有限公司,北京)促进结合。Eppendorfcentrifuge5418(Hamburg,Germany)用于溶液的离心。
试剂:三羟甲基氨基甲烷(2-carboxyethyl),磷化氢液(TCEP) PloyAn-aptamer(DNA1-DNA7)和FAM-DNA购买于上海生工生物工程技术与服务有限公司。多巴胺(DA)购买于北京索莱宝耗材科技股份有限公司。磷酸缓冲液(PBS,10mM, pH7.4)。所有的化学试剂都是分析级的,整个研究过程中使用的超纯水是用PURELAB Option-R(ELGA LabWater,UK)制备。
(2)检测探针的制备
PloyAn-aptamer通过Au-S键结合在纳米金上:用10 mmol/L TCEP在醋酸缓冲液中激活PloyAn-aptamer,浓度定为5 μmol/L;然后将240μL,0.1μmol/L的 AuNPs和30 μL5 μmol/L 的PloyAn-aptamer混合,孵育18小时以上;使用28μL,1×PBS调整溶液的pH值和离子强度,孵育6个小时;再分两次每次隔3个小时向溶液中添加16μL, 2 mol/L 的NaCl溶液,使NaCl的浓度逐渐增加到0.1 mol/L;再分三次每次隔3个小时向溶液中添加36 μL2 mol/L的NaCl溶液,使NaCl溶液的浓度最终达到0.06 mol/L;随后静置48小时,用14000转速离心三次,每次15分钟,每次离心的混合物用200 μL 0.1 mol/L的PBS溶液清洗两次,沉淀重新分散在1L 0.1 mol / L的1×PBS溶液;最后,将100μL , 1μmol / L的FAM-DNA添加进去并在37℃的恒温振荡箱中振荡反应2个小时,得到最终称为探针1-7的复合产物AuNPs@PloyAn- aptamer@FAM-DNA。
PloyAn-aptamer序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6或SEQ ID No.7中的任一项所示,适体5'端修饰有-SH。
FAM-DNA序列如SEQ ID No.8所示,其3'端修饰有FAM荧光基团。
探针的制备原理如图1所示:带有An长度标记的适体(PloyAn - aptamer)通过化学反应产生Au-S键固定在金纳米粒子(AuNPs)的表面,由FAM标记的单链寡核苷酸(FAM-DNA)通过碱基互补配对与对应的适体结合;在AuNPs@PloyAn-aptamer@FAM-DNA的纳米结构中,通过调节FAM和AuNPs之间碱基A的个数,即可实现金属增强荧光的效果,荧光信号最大。
(3)影响因素分析
①、本实验利用碱基A作为隔离层,溶液pH值不同时,碱基A的构想变化可能会影响纳米金与FAM之间的距离,进而影响传感器的灵敏度,为此需要对AuNPs@ployAn-aptamer@FAM-DNA的环境pH值进行优化。从图2可以看出,随着pH值从3.4到6.5,荧光强度增加。而pH为>6.5时,荧光强度相对较低。故当PH取6.5,AuNPs@ployAn-aptamer@FAM-DNA复合结构中荧光增强效果最为明显,荧光信号强度是FAM本身强度的3倍。
②、ployAn的长度对AuNPs@ployAn-aptamer@FAM-DNA的纳米结构的荧光强度影响较大。本实验测量了不同长度的ployAn在AuNPs@ployAn-aptamer@FAM-DNA纳米结构下的荧光光谱图,如图3所示。它们的荧光强度随着碱基A的个数增加,先增强后降低,这是由于荧光基团与纳米金之间发生的现象,由荧光共振能量转移造成的荧光淬灭现象转换为由局域表面等离子共振效应造成的荧光增强现象,最后由于两者距离过远,又转变为FAM自身的荧光发射。其中碱基A的个数为27时,荧光增强效果最为明显。
应用例
(1)多巴胺的检测:
在缓冲液(50mMTris, 30mM NaCl, 50mM KCl)中制备了不同浓度的DA,并加入到实施例制备的探针溶液中。混合物在室温下黑暗中培养1小时。然后,分三种方法进行测试:
①对混合物进行5000 rpm/min离心10分钟,取底物进行荧光检测;
②直接对混合物进行荧光检测;
③在混合液中加入10μg/ml,80μL的氧化石墨烯,等待20分钟后进行荧光检测。
检测多巴胺的原理:加入待测物多巴胺后,由于核酸适体与靶目标的特异性结合,结合在PloyAn– aptamer上的FAM-DNA会从纳米结构中脱离出来。金属增强荧光效应消失,结构AuNPs@PloyAn- aptamer@FAM-DNA的荧光强度将大大降低,此时在溶液中加入一定量的氧化石墨烯,脱离出的FAM-DNA会与氧化石墨烯结合而产生荧光淬灭,溶液中仅剩下AuNPs@PloyAn-aptamer@FAM-DNA结构产生荧光信号,荧光强度的变化与添加的待测物浓度成正比。该方法通过金属增强荧光效应提高传感器的灵敏度,通过加入氧化石墨烯在减少离心过程的同时,实现传感器灵敏度的更高幅度增加。
(2)检测方法分析
在上述最优实验条件下,即PH取6.5,碱基A的个数取27时,进行实验。若采用离心后测量底物的方法,检测结果如图4所示。若直接采用不离心的方法,检测结果如图5所示。若不离心加入10μg/ml,80μL的氧化石墨烯,等待20分钟后测量,检测结果如图6所示。比较三种处理方式下,多巴胺的测量范围,灵敏度与检出限如表1所示。
表1 不同处理方式下,传感器对多巴胺的检测性能参数比较
说明加入氧化石墨烯可不仅可以减少离心处理过程所引入的误差,还可以实现更高灵敏的多巴胺检测。
(3)特异性分析
为了评价传感器的选择性,对一些生物分子和干扰离子进行了测试。结果表明,大多数金属离子(Mg2+、Na+、K+)、葡萄糖、肾上腺素(A)、去甲肾上腺素(Na)、生物素等的荧光强度变化与DA (25nm)相差甚远,强度变化可以忽略不计。从图7可以看出,大多数干扰物(DA浓度相同)的荧光强度小于5%。这表明DA分析系统的特异性。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
<110> 郑州轻工业大学
<120> 一种检测多巴胺的高灵敏荧光探针的制备方法及其应用
<160> 8
<210> 1
<211> 58
<212> DNA
<213> 人工序列
<220>
<221> misc_difference
<222> (1)…(58)
<400> 1
gtctctgtgt gggccagaga acactggggc agatatgggc cagcacagaa tgaggccc 58
<210> 2
<211> 70
<212> DNA
<213> 人工序列
<220>
<221> misc_difference
<222> (1)…(70)
<400> 2
aaaaaaaaaa aagtctctgt gtgggccaga gaacactggg gcagatatgg gccagcacag 60
aatgaggccc 70
<210> 3
<211> 76
<212> DNA
<213> 人工序列
<220>
<221> misc_difference
<222> (1)…(76)
<400> 3
aaaaaaaaaa aaaaaaaagt ctctgtgtgg gccagagaac actggggcag atatgggcca 60
gcacagaatg aggccc 76
<210> 4
<211> 82
<212> DNA
<213> 人工序列
<220>
<221> misc_difference
<222> (1)…(82)
<400> 4
aaaaaaaaaa aaaaaaaaaa aaaagtctct gtgtgggcca gagaacactg gggcagatat 60
gggccagcac agaatgaggc cc 82
<210> 5
<211> 85
<212> DNA
<213> 人工序列
<220>
<221> misc_difference
<222> (1)…(85)
<400> 5
aaaaaaaaaa aaaaaaaaaa aaaaaaagtc tctgtgtggg ccagagaaca ctggggcaga 60
tatgggccag cacagaatga ggccc 85
<210> 6
<211> 88
<212> DNA
<213> 人工序列
<220>
<221> misc_difference
<222> (1)…(88)
<400> 6
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gtctctgtgt gggccagaga acactggggc 60
agatatgggc cagcacagaa tgaggccc 88
<210> 7
<211> 90
<212> DNA
<213> 人工序列
<220>
<221> misc_difference
<222> (1)…(90)
<400> 7
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagtctctgt gtgggccaga gaacactggg 60
gcagatatgg gccagcacag aatgaggccc 90
<210> 8
<211> 38
<212> DNA
<213> 人工序列
<220>
<221> misc_difference
<222> (1)…(38)
<400> 8
gtgttctctg gcgcacacag agacacagaa tgaggccc 38
Claims (5)
1.一种检测多巴胺的高灵敏荧光探针的制备方法,其特征在于,步骤如下:
(1)将带有An长度的适体加入TCEP的醋酸缓冲液中,然后与AuNPs溶液混合反应,孵育18h以上,得溶液Ⅰ;所述步骤(1)中适体序列为SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6或SEQ ID No.7中的任一项,适体5'端修饰有-SH;
(2)向经步骤(1)处理的溶液Ⅰ中加入1×PBS调整溶液的pH值和离子强度,再孵育6-8h,得溶液Ⅱ;
(3)向经步骤(2)处理的溶液Ⅱ中分两次加入NaCl溶液,每次间隔2-3h,然后再分三次加入NaCl溶液,每次间隔3-4h,静置40-50h,得溶液Ⅲ;
(4)将溶液Ⅲ离心三次,每次离心混合物用PBS溶液清洗两次,然后将沉淀重新分散在PBS溶液中,加入FAM-DNA,于37℃条件下振荡反应2-3h,得高灵敏荧光探针;所述步骤(4)中FAM-DNA序列为SEQ ID No.8所示,其3'端修饰有FAM荧光基团。
2.根据权利要求1所述的检测多巴胺的高灵敏荧光探针的制备方法,其特征在于:所述步骤(1)中适体在的TCEP的醋酸缓冲液中的浓度为5-10 μM、适体的TCEP的醋酸缓冲液体积为10-50μL;AuNPs溶液的浓度为0.1-0.5μM、体积为100-300μL,TCEP的醋酸缓冲液的浓度为10 mM。
3.根据权利要求1所述的检测多巴胺的高灵敏荧光探针的制备方法,其特征在于:所述步骤(2)中1×PBS调整溶液的体积为25-30 μL,pH值为3.5-8.4。
4.根据权利要求1所述的检测多巴胺的高灵敏荧光探针的制备方法,其特征在于:所述步骤(3)中NaCl溶液的浓度为2M,前两次每次加入NaCl溶液的体积15-20μL;后三次每次加入NaCl溶液的体积35-40μL。
5.利用权利要求1-4任一项所述的方法制备的高灵敏荧光探针非疾病诊断和检测为目的的检测多巴胺的方法,其特征在于,步骤如下:将待测样品溶液加入到高灵敏荧光探针溶液中,于室温黑暗条件下培养0.5-1.5h,得反应液,向反应液中加入10μg/mL、80μL的氧化石墨烯,等待20-30min后进行荧光检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911191890.8A CN111004622B (zh) | 2019-11-28 | 2019-11-28 | 一种检测多巴胺的高灵敏荧光探针的制备方法及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911191890.8A CN111004622B (zh) | 2019-11-28 | 2019-11-28 | 一种检测多巴胺的高灵敏荧光探针的制备方法及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111004622A CN111004622A (zh) | 2020-04-14 |
CN111004622B true CN111004622B (zh) | 2023-08-15 |
Family
ID=70113621
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911191890.8A Active CN111004622B (zh) | 2019-11-28 | 2019-11-28 | 一种检测多巴胺的高灵敏荧光探针的制备方法及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111004622B (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111537489A (zh) * | 2020-06-09 | 2020-08-14 | 郑州轻工业大学 | 一种核酸适体及调节核酸适体信号强度的方法和应用 |
CN112630439A (zh) * | 2020-12-11 | 2021-04-09 | 郑州轻工业大学 | 基于纳米金的分裂型适体传感器及其制备方法和应用 |
CN112710645B (zh) * | 2020-12-17 | 2022-09-30 | 南京工业大学 | 一种高锰酸根引发的原位荧光反应实时检测多巴胺和碱性磷酸酶的方法及其酶联免疫应用 |
CN113340864B (zh) * | 2021-06-07 | 2023-04-07 | 郑州轻工业大学 | 基于mef效应二次放大ins信号的适体传感器及其制备方法和应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103592292A (zh) * | 2013-10-28 | 2014-02-19 | 江苏大学 | 一种快速灵敏测定多巴胺的方法 |
CN104020199A (zh) * | 2014-06-18 | 2014-09-03 | 青岛科技大学 | 一种基于适体识别作用电化学测定多巴胺的方法 |
CN105136754A (zh) * | 2015-07-24 | 2015-12-09 | 郑州轻工业学院 | 检测多巴胺的荧光适体传感器及方法 |
CN105980855A (zh) * | 2014-03-11 | 2016-09-28 | 哈佛学院院长及董事 | 利用可编程核酸探针的高通量且高度多路复用的成像 |
CN108872173A (zh) * | 2018-06-29 | 2018-11-23 | 郑州轻工业学院 | 一种荧光增强型适体传感器及其制备方法和应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9139614B2 (en) * | 2007-04-25 | 2015-09-22 | The United States Of America, As Represented By The Secretary Of The Navy | Modular linkers for conjugation of organic substances to substantially inorganic substances and methods of manufacture and use thereof |
-
2019
- 2019-11-28 CN CN201911191890.8A patent/CN111004622B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103592292A (zh) * | 2013-10-28 | 2014-02-19 | 江苏大学 | 一种快速灵敏测定多巴胺的方法 |
CN105980855A (zh) * | 2014-03-11 | 2016-09-28 | 哈佛学院院长及董事 | 利用可编程核酸探针的高通量且高度多路复用的成像 |
CN104020199A (zh) * | 2014-06-18 | 2014-09-03 | 青岛科技大学 | 一种基于适体识别作用电化学测定多巴胺的方法 |
CN105136754A (zh) * | 2015-07-24 | 2015-12-09 | 郑州轻工业学院 | 检测多巴胺的荧光适体传感器及方法 |
CN108872173A (zh) * | 2018-06-29 | 2018-11-23 | 郑州轻工业学院 | 一种荧光增强型适体传感器及其制备方法和应用 |
Non-Patent Citations (1)
Title |
---|
姜利英 等.用于检测多巴胺的荧光增强型适体传感器.《光学精密工程》.2019,第27卷(第9期),第1945页左栏. * |
Also Published As
Publication number | Publication date |
---|---|
CN111004622A (zh) | 2020-04-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111004622B (zh) | 一种检测多巴胺的高灵敏荧光探针的制备方法及其应用 | |
Zhang et al. | A ratiometric electrochemical biosensor for the exosomal microRNAs detection based on bipedal DNA walkers propelled by locked nucleic acid modified toehold mediate strand displacement reaction | |
Luo et al. | A ratiometric electrochemical DNA biosensor for detection of exosomal MicroRNA | |
Wang et al. | RNA aptamer-based electrochemical aptasensor for C-reactive protein detection using functionalized silica microspheres as immunoprobes | |
CN106950206B (zh) | 一种基于核酸适配体的荧光传感器检测腺苷的方法 | |
CN107389755B (zh) | 用于检测汞的电化学传感器及其制备方法和应用 | |
Shen et al. | Highly sensitive and simultaneous detection of microRNAs in serum using stir-bar assisted magnetic DNA nanospheres-encoded probes | |
Wang et al. | Multiple signal amplification electrogenerated chemiluminescence biosensors for sensitive protein kinase activity analysis and inhibition | |
Zhang et al. | A fluorescent aptasensor for the femtomolar detection of epidermal growth factor receptor-2 based on the proximity of G-rich sequences to Ag nanoclusters | |
Wang et al. | A competitive electrochemical aptamer-based method for aflatoxin B1 detection with signal-off response | |
CN108802142B (zh) | 基于磁性生物复合材料与铅离子依赖性dna酶的电化学生物传感器检测凝血酶的方法 | |
CN108872173B (zh) | 一种荧光增强型适体传感器及其制备方法和应用 | |
Zhu et al. | Highly sensitive electrochemiluminescent biosensor for adenosine based on structure-switching of aptamer | |
Gao et al. | A fluorescence assay for microRNA let-7a by a double-stranded DNA modified gold nanoparticle nanoprobe combined with graphene oxide | |
CN111579614B (zh) | 基于磁性生物复合材料的dna酶和杂交链式反应的电化学生物传感器检测铅离子的方法 | |
Cao et al. | An ultrasensitive biosensor for virulence ompA gene of Cronobacter sakazakii based on boron doped carbon quantum dots-AuNPs nanozyme and exonuclease III-assisted target-recycling strategy | |
CN109632901B (zh) | 一种检测铅离子的电化学传感器及其制备方法 | |
CN109211997B (zh) | 一种检测β-淀粉样蛋白的基于THMS的电化学发光适体传感器及其制备方法和应用 | |
Zhang et al. | Ultrasensitive glucose detection from tears and saliva through integrating a glucose oxidase-coupled DNAzyme and CRISPR–Cas12a | |
Miao et al. | A novel sensing platform for sensitive cholesterol detection by using positively charged gold nanoparticles | |
Ge et al. | Cocaine detection in blood serum using aptamer biosensor on gold nanoparticles and progressive dilution | |
CN110553991B (zh) | 基于中空金纳米粒-dna复合物的生物/化学检测试剂和检测方法 | |
He et al. | DNA sensor by using electrochemiluminescence of acridinium ester initiated by tripropylamine | |
Wang et al. | Detection of immunoglobulin E using an aptamer based dot-blot assay | |
CN109991297B (zh) | 基于G-四联体DNAzyme信号放大策略的适配体传感器的黄体酮检测方法 |
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 |