CN106052872B - 一种基于纳米材料自组装的土霉素sers检测方法 - Google Patents
一种基于纳米材料自组装的土霉素sers检测方法 Download PDFInfo
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Abstract
本发明提供了一种基于纳米材料自组装的土霉素SERS检测方法,用于鱼粉中土霉素(OTC)含量的检测。纳米金颗粒(80nm粒径)通过含OTC适配体的碱基序列与纳米金颗粒(13nm粒径)连接在一起,4‑巯基苯甲酸偶联在纳米金颗粒表面,由此产生拉曼信号。当OTC接触检测体系时,OTC适配体与OTC特异性结合,导致80nm纳米金颗粒和13nm纳米金颗粒距离缩小,热点增强,拉曼信号增大,从而实现检测目的。本检测体系能够定量检测土霉素,线性范围是4.60×10‑2‑4.60×102fg/mL,检出限为4.00×10‑3fg/mL。本发明用于土霉素检测具有灵敏度高、快速简便的优点,应用于鱼粉的检测,结果准确可靠。
Description
技术领域
本发明涉及纳米材料和分析化学技术领域,具体涉及一种基于纳米材料自组装的土霉素SERS检测方法,用于对食品中土霉素进行检测。
背景技术
纳米材料是指由纳米结构单元构成的任何类型的材料,其颗粒尺寸一般为0.1~100nm。纳米材料具有不同于宏观材料的物理和化学性质,包括表面效应、小尺寸效应、量子尺寸效应、宏观量子隧道效应、催化性质等,这些特性使其在某些方面具有优良的先天应用优势。
核酸适配体(Aptamer)是通过指数富集配体系统进化(systematic evolutionofligands by exponential enrichment,SELEX)技术从体外筛选得到的,能与相应配体专一性紧密结合的一类单链寡核苷酸序列。这种寡核苷酸序列形成的高级结构能够特异性识别、高亲和力结合与之对应的任何类型的蛋白和低分子等靶物质。与抗体相比,适配体的靶分子范围广,筛选在体外进行,易于人工合成和修饰,分子量较小,稳定性好,对温度不敏感,容易保存。近年来,适配体作为识别分子已经在临床诊断、临床治疗、蛋白质组研究和食品安全检测中逐渐得到广泛应用。
表面增强拉曼光谱(SERS)是检测痕量物质的一种分析方法,可以在分子水平上给出物质的指纹信息。它通常将化合物吸附在粗糙化的贵金属、金属纳米粒子或者金属氧化物纳米粒子的表面,使被测定化合物的散射信号具有了极大的增强效应,其增强系数可达105到1014。SERS光谱具有较高灵敏度、高选择性、可定点研究和无损探测等很多优点,具有广阔的应用前景。
土霉素(Oxytetracycline,OTC),分子式为C22H24N2O9,分子量460.44,是一种被广泛用于治疗奶牛乳房炎等疾病的广谱抗菌药物,同时土霉素又是一种抗生素和生长促进剂,常常被用作饲料添加剂。因此,土霉素极易残留在牛奶和动物组织中。但与此同时,土霉素能与人体内的钙结合,导致人体缺钙,长期使用能产生耐药菌株,给人体健康造成损害。世界粮农组织、世界卫生组织、欧盟及我国政府对牛奶中土霉素的残留都做出了严格的规定,其残留限量不得高于100μg/L。因此,控制土霉素的使用,实施对动物性食品中土霉素及有害代谢产物残留的检测就显得愈发的重要。
本发明首先将80nm粒径纳米金颗粒通过含有OTC适配体的茎环结构的碱基序列与13nm粒径的纳米金颗粒连接在一起,拉曼分子4-巯基苯甲酸以Au-S键结合在13nm纳米金颗粒的表面,由此产生拉曼信号。当OTC接触检测体系时,碱基序列中的OTC适配体一方面与OTC发生特异性结合,另一方面与OTC适配体的互补链解旋,导致80nm粒径纳米金颗粒和15nm粒径纳米金颗粒之间的距离缩小,热点增强,拉曼信号增大,从而实现检测的目的。在一定范围内,OTC浓度与拉曼强度的增强量呈正相关,在632.8nm的激发光源下建立标准曲线,以达到对土霉素定量检测的目的。该发明可以用于鱼粉中土霉素含量的检测。
发明内容:
本发明的目的在于将纳米材料与土霉素适配体结合应用于表面增强拉曼对土霉素进行快速、准确的定量检测,特别涉及到纳米材料与适配体结合应用于土霉素的表面增强拉曼检测中应用。
为了实现上述目的,本发明采用如下技术方案:
(1)通过两步生长法,制备80nm的纳米金颗粒。
(2)土霉素适配体的活化。
(3)取1mL 80nm胶体金向其中加入同体积的1μM的土霉素适配体溶液,于37℃摇床上孵育12h。孵育结束后,使用缓冲液(NaCl 5mM,Tris 5mM)离心清洗3次,分别收集上清和材料,清洗结束后使用缓冲液将反应体系补齐到初始体积。
(4)80nm胶体金表面的钝化。
(5)利用通过亲和素(Avidin)与生物素(Biotin)之间的特异性结合将亲和素(Avidin)与生物素(Biotin)修饰的土霉素适配体DNA连接。
(6)利用亲和素(Avidin)与纳米金的静电吸附作用将修饰80nm纳米金颗粒的土霉素适配体与粒径在13nm左右的纳米金颗粒连接。
(7)利用Au-S键的作用将粒径约为13nm左右的纳米金颗粒与拉曼分子4-巯基苯甲酸结合。
(8)通过土霉素适配体DNA与土霉素特异性结合,茎环结构的DNA碱基互补配对的部分发生解旋,使得80nm胶体金和13nm胶体金的距离拉近,热点增强,拉曼信号强度增大。
(9)对土霉素标准品进行检测,建立标准曲线。配制不同浓度的土霉素标准品加入到纳米复合物体系中,37℃孵育10min,在632.8nm激光激发下,采集时间为15s,循环次数为1次,空白组检测得到的拉曼强度(I0)最小,随着土霉素浓度的增加拉曼强度(I)逐步增加。根据拉曼强度发光差值(△I=I-I0)与对应的土霉素标准品浓度建立标准曲线,实验结果在0.046-460.440fg/mL区间内得到良好线性关系。
(10)对土霉素样品进行检测:对样品做简单的处理,随后直接加入到上述纳米复合物体系中37℃孵育10min后,在632.8nm激光下得到对应的拉曼光谱,从标准曲线中求得对应的土霉素的浓度。
本发明的优点是:
(1)利用适配体对被检测物质实现特异性捕获,有效提高了检测的稳定性和准确性。
(2)利用适配体与抗体相比较,具有可人工合成,不依赖动物和细胞,周期短、成本低、批次间差异小,便于化学修饰,稳定性也好,可长期保存。
(3)本实验灵敏度高,特异性强,为土霉素的快速定量检测提供了新方法。
附图说明
图1:基于纳米材料自组装的土霉素SERS检测方法的实验原理图;
图2:80nm胶体金透射电镜图(A);13nm胶体金透射电镜图(C);
80nm胶体金紫外吸收图(B);13nm胶体金紫外吸收图(D);
图3:土霉素适配体在80nm胶体金表面上孵育前后的紫外吸收图;
图4:亲和素和土霉素适配体孵育前后的紫外吸收图;
图5:土霉素检测的拉曼光谱(A)及标准曲线图(B)
具体实施方式
本发明包括但不限于以上实施例,凡是在本发明的精神和原则下进行的任何等同替换或者局部改进,都将视为在本发明的保护范围之内。
实施例1
通过两步生长法,制备80nm左右的纳米金颗粒包括以下步骤:
1)首先,合成13nm左右的纳米金颗粒,通过以下步骤:取2.5mL氯金酸溶液(0.2%)加入到50mL水中煮沸,剧烈搅拌下加入2mL柠檬酸钠(1%,含0.05%柠檬酸),溶液煮沸5min,自然冷却,进行紫外和透射电镜的表征(图2A、图2B)。
2)其次,进行第一步的生长,具体步骤如下:将3mL纳米金颗粒(13nm左右)稀释至20mL后加入到圆底烧瓶中,在剧烈搅拌下依次向圆底烧瓶中加入10mL的氯金酸溶液(0.04%)和10mL的抗坏血酸(1%)和柠檬酸钠(1%)的混合液,剧烈搅拌45min,然后加热煮沸30min,自然冷却,即得一步生长所得纳米金溶液。
3)最后,进行第二步的生长,也即最后一步的生长。具体步骤如下:将4.5mL第一步生长所得的纳米金颗粒稀释至20mL后加入到圆底烧瓶中,在剧烈搅拌下依次向圆底烧瓶中加入10mL的氯金酸溶液(0.04%)和10mL的抗坏血酸(1%)和柠檬酸钠(1%)的混合液,剧烈搅拌45min,然后加热煮沸30min,自然冷却,即得80nm左右的纳米金颗粒。所得纳米金进行紫外和透射电镜的表征(图2C、图2D)
实施例2
土霉素适配体的活化,具体步骤如下:
首先是将适配体的水溶液置于65℃的水浴中退火30min,待其缓慢地自然冷却至室温后,向其加入适配体体积的1%1mM三(2-羧乙基)膦盐酸盐,室温下静置1h,以活化巯基修饰的茎环DNA。
实施例3
胶体金与土霉素适配体之间的连接,步骤如下:
取1mL 80nm胶体金向其中加入同体积的1μM的土霉素适配体溶液,于37℃摇床上孵育12h。孵育结束后,使用缓冲液(NaCl 5mM,Tris 5mM)3500rpm离心15min,清洗3次,分别收集上清和材料,进行紫外表征(图2A)和核酸含量的测定(图2B)清洗结束后使用缓冲液将反应体系补齐到初始体积。
实施例4
80nm胶体金表面的钝化,具体步骤如下:
向反应体系中加入0.1ml 0.1mM 6-巯基-1-正己醇,钝化1h。钝化结束后使用缓冲液(NaCl 5mM,Tris5mM)离心清洗一次,弃去上清,收集材料,方法同实施例3中的步骤。
实施例5
利用通过亲和素与生物素之间的特异性结合将亲和素与生物素修饰的土霉素适配体DNA连接。具体步骤如下:
向实施例4中离心清洗去掉上清液的反应体系中加入1ml 0.5mg/ml的亲和素水溶液。37℃摇床上孵育12h。孵育结束后上述的离心条件离心清洗3次,分别收集上清和材料,进行紫外的表征(图3)
实施例6
利用亲和素与纳米金的静电吸附作用将修饰80nm纳米金颗粒的土霉素适配体与粒径在13nm左右的纳米金颗粒连接。具体方法为:
向实施例5中经过离心清洗去掉上清的反应体系中,加入1ml的纳米金颗粒,在37℃摇床中孵育12h,孵育结束后用水以10000rpm离心10min,分别收集上清和材料。
实施例7
利用Au-S键的作用将粒径约为13nm左右的纳米金颗粒与拉曼分子4-巯基苯甲酸结合。具体步骤如下:
向实施例6中的材料中加入1ml 1mM 4-巯基苯甲酸,37℃摇床中孵育12h,3500rpm离心15min,收集上清和材料,并且向材料中加入1ml缓冲液(NaCl 5mM,Tris 5mM),4℃储存备用。
实施例8
进行土霉素的SERS检测。通过土霉素适配体DNA与土霉素特异性结合,茎环结构的DNA碱基互补配对的部分发生解旋,使得80nm胶体金和13nm胶体金的距离拉近,热点增强,拉曼信号强度增大。具体方法为:在反应体系与土霉素接触之前进行一次拉曼信号的检测。取1ml 1mM土霉素加入到(6)中所得的材料,37℃水浴10min,相同的参数条件下进行靶标接触后的拉曼检测。
实施例9
对土霉素标准品进行检测,建立标准曲线。具体步骤如下:
将用超纯水依次将土霉素稀释为如下的浓度梯度:0.046fg/mL,0.460fg/mL,4.604fg/mL,46.044fg/mL,460.440fg/mL,分别取1ml的稀释液加入到同等体积的反应体系中,37℃水浴10min,在以下的参数设定下进行拉曼多光谱扫描检测:激发光源为632.8nm,采集时间15s,循环次数1次,拉曼位移1000-1800cm-1,光栅600gr/mm,显微物镜x5vis,狭缝200μm,共焦针孔600μm。
根据拉曼强度值与对应的土霉素标准品浓度建立标准曲线:y=49.51x+649.66,其检测线性范围为0.046fg/ml~460.440fg/ml(R2=0.9979)
实施例10
鱼粉实际样品中土霉素的检测及加标回收率实验
以实施例9得到的3组土霉素浓度数据为本底值,分别向其中加入三种不同浓度的OTC标准品,同样利用本发明方法再次检测其中OTC的含量,得到检测值。回收率%=(检测值-本底值)/添加量×100%。从表一数据可以看到回收率在91.29%~110.98%,说明本发明稳定,灵敏,准确,适用于鱼粉实际样品中OTC的检测。
表一鱼粉实际样品中土霉素的检测及加标回收率
Claims (5)
1.一种基于纳米材料自组装的土霉素SERS检测方法,其特征在于:此拉曼增强基底为金属纳米颗粒和碱基序列的自组装方式,所述的金属纳米颗粒包括直径在80nm的纳米金颗粒和直径在13nm的纳米金颗粒,所述碱基序列为含有土霉素(OTC)适配体的茎环结构的碱基序列,纳米金颗粒通过含有OTC适配体的茎环结构的碱基序列与纳米金颗粒连接在一起,拉曼分子4-巯基苯甲酸以Au-S键偶联到纳米金颗粒的表面,由此产生拉曼信号,当OTC接触检测体系时,碱基序列中的OTC适配体一方面与OTC发生特异性结合,另一方面与OTC适配体的互补链解旋,导致80nm左右粒径纳米金和13nm左右粒径纳米金颗粒之间的距离缩小,热点增强,拉曼信号增大,从而实现检测的目的,OTC的浓度在4.60×10-2fg/mL~4.60×102fg/mL范围内,OTC浓度与拉曼强度的增强量呈正相关,在632.8nm的激发光源下建立标准曲线,以达到对土霉素定量检测的目的。
2.如权利要求1所述的一种基于纳米材料自组装的土霉素SERS检测方法,其特征在于:合成不同粒径的纳米金应用于土霉素的检测。
3.如权利要求1所述的一种基于纳米材料自组装的土霉素SERS检测方法,其特征在于:碱基序列分别以Au-S键和静电吸附作用结合在80nm纳米金颗粒和13nm纳米金颗粒的表面。
4.如权利要求1所述的一种基于纳米材料自组装的土霉素SERS检测方法,其特征在于:土霉素的碱基序列为:5’-生物素-ACC GCA CCA CCG TCA TGA GTG CGA ACT TAC GCA CTCATG ACG GTG GTG CGG TGG TG-3’,第26个和27个碱基之间修饰巯基。
5.如权利要求1所述的一种基于纳米材料自组装的土霉素SERS检测方法,其特征在于:所述方法能够用于鱼粉中土霉素的检测。
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