CN113899900A - 一种用于mcf7外泌体检测的适配体传感器及其制备方法及mcf7外泌体的检测方法 - Google Patents
一种用于mcf7外泌体检测的适配体传感器及其制备方法及mcf7外泌体的检测方法 Download PDFInfo
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Abstract
本发明公开了一种用于MCF7外泌体检测的适配体传感器及其制备方法及MCF7外泌体的检测方法,所述制备方法为:将AptCD63溶液包被于96孔板中然后封闭;向包被有AptCD63的孔中加入MCF7外泌体溶液,培育,洗涤;将适配体EpCAM溶液与辣根过氧化物酶溶液混合孵育,得到AptEpCAM‑HRP溶液;加入AptEpCAM‑HRP溶液,培育,洗涤;当向所述适配体传感器中加入络氨酸包裹的金纳米颗粒溶液和过氧化氢水溶液,静置5分钟后络氨酸包裹的金纳米颗粒会产生聚集,反应液由红色转变为蓝色,利用紫外分光光度计即可实现对于MCF7外泌体的定量检测,且检测的灵敏度高。
Description
技术领域
本发明属于癌细胞检测技术领域,具体涉及一种用于MCF7外泌体检测的适配体传感器及其制备方法及MCF7外泌体的检测方法。
背景技术
外泌体是大多数细胞类型分泌的膜包裹的囊泡(直径30-100nm)。它们携带着母细胞的分子信息,富含细胞膜蛋白和用于细胞间通讯的遗传物质。最近的报道表明肿瘤相关的外泌体有助于肿瘤的转移、进展和免疫调节。由于外泌体在体液中相对丰富,且在采集标本时侵袭性小,因此外泌体被认为在癌症液体活检中具有很大的潜力。
传统的外泌体检测方法有Western blot和酶联免疫吸附法。但是,这些方法存在需要大量样品、灵敏度低、操作时间长等缺点。在粒子跟踪检测方法上,缺乏特异性是其不足之处。由于外泌体在癌症早期的浓度相对较低,因此急需开发更加灵敏的检测方法来区分病人和健康人。最近,一些全新的方法被应用于外泌体的检测,如表面增强拉曼散射法,表面等离子体共振法,荧光法,比色法和电化学法。但是,这些等量检测Exosome的方法,其精细的操作步骤对于实验人员的操作技巧要求较高,难以普及,这样限制了对Exosome定量检测的推广和普及。
因此,有关Exosome的定量检测已经成为癌症检测的研究热点,能够实现对Exosome的操作简便、灵敏度高的定量检测是当下需要解决的问题。
发明内容
为解决上述技术问题,本发明提供了一种用于MCF7外泌体检测的适配体传感器及其制备方法及MCF7外泌体的检测方法,利用此适配体传感器可实现对于MCF7外泌体的定量检测,且操作简单,灵敏度高。
本发明采取的技术方案如下:
一种用于MCF7外泌体检测的适配体传感器的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)将AptCD63溶液包被于96孔板中;
(2)以BSA溶液对AptCD63包被后的96孔板进行封闭;
(3)向包被有AptCD63的孔中加入MCF7外泌体溶液,培育,洗涤;
(4)将适配体EpCAM溶液、sulfo-SMCC与辣根过氧化物酶溶液混合孵育,得到AptEpCAM-HRP溶液;
(5)向步骤(3)得到的包被有AptCD63的孔中加入AptEpCAM-HRP溶液,培育,洗涤,即可得到所述用于MCF7外泌体检测的适配体传感器;
当向所述适配体传感器中加入络氨酸包裹的金纳米颗粒溶液和过氧化氢水溶液,静置5分钟后络氨酸包裹的金纳米颗粒会产生聚集,反应液由红色转变为蓝色,利用紫外分光光度计即可实现对于MCF7外泌体的定量检测。
所述AptCD63的基因序列如下:
AptCD63:5′-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA-3′。
所述适配体EpCAM(AptEpCAM)的基因序列如下:
AptEpCAM:5′-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGCCTG-3′。
所述制备方法具体包括以下步骤:
(1)将15uL 1μM的AptCD63溶液包被于96孔板中,并在4℃的黑暗中保存10小时,然后以PBS缓冲液洗涤;
(2)以15μL 1%BSA溶液对AptCD63包被后的96孔板进行封闭,固定时间为10分钟,然后以PBS缓冲液洗涤;
(3)向包被有AptCD63的孔中加入15μL MCF7外泌体溶液,培育30分钟,然后以PBS缓冲液洗涤;
(4)将2μL 5μM的适配体EpCAM溶液、2mg sulfo-SMCC与8μL 100μM的辣根过氧化物酶溶液混合孵育10分钟,得到AptEpCAM-HRP溶液;
(5)向步骤(3)得到的包被有AptCD63的孔中加入10μL AptEpCAM-HRP溶液,培育30分钟,然后以PBS缓冲液洗涤,即可得到所述用于MCF7外泌体检测的适配体传感器。
所述络氨酸包裹的金纳米颗粒溶液的制备方法为:将络氨酸水溶液与金纳米溶液混合,加入盐酸溶液进行官能化反应,即可得到。
所述AptCD63溶液、适配体EpCAM溶液、MCF7外泌体溶液、辣根过氧化物酶溶液分别是通过将AptCD63、适配体EpCAM、MCF7分泌的外泌体、辣根过氧化物酶溶解在PH7.4 10mM的PBS缓冲溶液中得到。
根据上述制备方法可制备得到用于MCF7外泌体检测的适配体传感器,其操作简单,且灵敏度高。
本发明提供的一种MCF7外泌体的定量检测方法,所述检测方法包括以下步骤:
A、将AptCD63溶液包被于96孔板中的多个孔中;
B、以BSA溶液对AptCD63包被后的各孔进行封闭;
C、向各孔中加入系列浓度的MCF7外泌体溶液,培育,洗涤;
D、向各孔中加入AptEpCAM-HRP溶液,培育,洗涤;
E、向各孔中加入络氨酸包裹的金纳米颗粒和过氧化氢,静置5分钟,利用紫外分光光度计测试各孔反应液的紫外光谱;
F、以MCF7外泌体溶液的浓度为横坐标,反应液在520nm、650nm波长处的紫外吸收强度比I520nm/I650nm为纵坐标构建曲线,得出线性方程,根据线性方程即可得到任意I520nm/I650nm下所对应的待测MCF7外泌体溶液的浓度。
步骤C中,MCF7外泌体溶液的浓度分别为1.56×102、3.125×102、6.25×102、12.5×102、25×102颗粒/μL。
所述线性方程为Y=-1.397×lnX+12.988,其相关系数为R2=0.9842,其中Y为I520nm/I650nm,X为MCF7外泌体溶液的浓度,单位为颗粒/μL。
本发明通过一种基于适配体和辣根过氧化物酶共轭物(AptEpCAM-HRP)诱导络氨酸包裹的金纳米颗粒(Tyr-AuNPs)的聚集,这使得宏观上金纳米颗粒溶液的颜色会由原先的红色转变为紫色,肉眼可观察到从红色到蓝色的鲜明颜色变化。基于该适配体平台构建的传感器定量检测外泌体MCF7 Exosome浓度的方法,可以实现对癌细胞分泌的外泌体MCF7Exosome的仪器简易化的高灵敏检测。在该系统中,适配体CD63和适配体EpCAM与过氧化物酶共轭物(AptEpCAM-HRP)将结合到不同的外泌体表面蛋白上,形成三明治结构,从而使辣根过氧化物酶(HRP)在外泌体表面间接修饰。过氧化氢H2O2加入反应溶液中,辣根过氧化物酶共轭物诱导L-络氨酸包裹的金纳米颗粒(Tyr-AuNPs)聚集,该反应可以在5分钟内完成比色检测。这使得宏观上金纳米颗粒溶液的颜色会由原先的红色转变为蓝色,肉眼可观察到从红色到蓝色的鲜明颜色变化。进而使用紫外分光光度计即可实现对于外泌体MCF7 Exosome浓度的定量检测,因此,基于适配体和辣根过氧化物酶平台可在最佳条件下,以2.5×103(个每微升)的低检测限,该检测方法的灵敏度高,检测限低,且操作方便。
附图说明
图1为本发明中的用于MCF7外泌体检测的适配体传感器构建过程的示意图;
图2为金纳米颗粒、氨酸包裹的金纳米颗粒的DLS图;
图3为辣根过氧化物酶、适配体与辣根过氧化物酶共轭物(AptEpCAM-HRP)的紫外图;
图4为不含MCF7外泌体(a)及含有MCF7外泌体(b)的适配体传感器在加入络氨酸包裹的金纳米颗粒溶液和过氧化氢水溶液后的反应体系的紫外光谱图;
图5为不同MCF7外泌体溶液浓度下适配体传感器的紫外光谱图;
图6为以MCF7外泌体溶液浓度为横坐标,I520nm/I650nm为纵坐标构建的线性曲线。
具体实施方式
下面结合实施例对本发明进行详细说明。
本发明中的络氨酸包裹的金纳米颗粒的制备方法如下:
(a)将5mL 38.8mM柠檬酸三钠水溶液快速添加到50mL 1mM沸腾的HAuCl4水溶液中,并将该混合物溶液在沸腾条件下再搅拌30分钟,得到樱桃红色溶液,通过0.45-μmMillipore注射器过滤溶液以除去沉淀物后,即得到金纳米溶液,将滤液储存在4℃的冰箱中;
(b)将100μL 1.5mM L-酪氨酸水溶液与100μL步骤(a)得到的金纳米溶液混合,再加入100μL pH=3的HCl水溶液搅拌混合均匀,即可得到络氨酸包裹的金纳米颗粒溶液。络氨酸在某一特定pH值的条件下,其所带正电荷与负电荷恰好相等(总净电荷为零),当加入定量的盐酸溶液,溶液酸性充满质子,会使络氨酸氨基带正电,羧基带氢,整个分子带一个正电。之后络氨酸通过静电自组装结合到带负电的金纳米颗粒上。
本发明中的AptCD63溶液、适配体EpCAM溶液、MCF7外泌体溶液、辣根过氧化物酶溶液分别是通过将AptCD63、适配体EpCAM、MCF7分泌的外泌体、辣根过氧化物酶溶解在10mMPH7.4的PBS缓冲溶液中得到。
MCF7分泌的外泌体(MCF7 Exosome)购买自艾博抗(上海)贸易有限公司。
实施例1
一种用于MCF7外泌体检测的适配体传感器的制备方法,包括以下步骤:
(1)将15uL 1μM的AptCD63溶液包被于96孔板中,并在4℃的黑暗中保存10小时,然后使用10mM pH 7.4的PBS缓冲液洗涤3次;AptCD63的基因序列为:5′-CAC CCC ACC TCG CTCCCG TGA CAC TAA TGC TA-3′;
(2)以15μL 1%BSA溶液对AptCD63包被后的96孔板进行封闭,使未与AptCD63结合的96孔板的位点由BSA固定,固定时间为10分钟,然后使用10mM pH 7.4的PBS缓冲液洗涤3次;
(3)向包被有AptCD63的孔中加入15μL MCF7外泌体溶液,培育30分钟使MCF7外泌体与AptCD63结合,然后使用10mM pH 7.4的PBS缓冲液洗涤3次;
(4)将2μL 5μM的适配体EpCAM溶液、2mg sulfo-SMCC与8μL 100μM的辣根过氧化物酶溶液混合孵育10分钟,得到AptEpCAM-HRP溶液;适配体EpCAM的基因序列为:5′-CAC TACAGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3′;辣根过氧化物酶溶液、AptEpCAM-HRP溶液的紫外图如图3所示,从图中可见,辣根过氧化物酶的紫外吸收光谱在399nm处有个吸收峰,当通过异双功能交联剂Sulfo-SMCC将辣根过氧化物酶和适配体EpCAM交联起来后的紫外吸收光谱吸收峰向右偏移,表明以成功合成了适配体EpCAM和辣根过氧化物酶的共轭物AptEpCAM-HRP;
(5)向步骤(3)得到的包被有AptCD63的孔中加入10μL AptEpCAM-HRP溶液,培育30分钟,然后使用10mM pH 7.4的PBS缓冲液洗涤3次,即可得到所述用于MCF7外泌体检测的适配体传感器。
当向所述适配体传感器中加入100μL络氨酸包裹的金纳米颗粒溶液和50μL 100μM的过氧化氢水溶液,静置5分钟后络氨酸包裹的金纳米颗粒会产生聚集,肉眼可观察到反应液由红色转变为蓝色,利用紫外分光光度计即可实现对于MCF7外泌体的高灵敏检测。
其紫外光谱图如图4中的a所示,当步骤(3)中的MCF7外泌体溶液替换为10mMPH7.4的PBS缓冲溶液后,其紫外光谱图如图4中的b所,从图中可以看出含有MCF7外泌体的适配体传感器相对于不含MCF7外泌体的适配体传感器,在加入络氨酸包裹的金纳米颗粒溶液和过氧化氢水溶液后,反应体系在520nm处的吸收强度降低,但是在650nm处的吸收强度增强。
实施例2
一种MCF7外泌体的定量检测方法,包括以下步骤:
A、分别将15uL 1μM的AptCD63溶液包被于96孔板的多个孔中,并在4℃的黑暗中保存10小时,然后使用10mM pH 7.4的PBS缓冲液洗涤3次;
B、以15μL 1%BSA溶液对AptCD63包被后的各孔进行封闭,使未与AptCD63结合的96孔板的位点由BSA固定,固定时间为10分钟,然后使用10mM pH 7.4的PBS缓冲液洗涤3次;
C、向包被有AptCD63的各孔中分别加入15μL系列浓度的MCF7外泌体溶液,培育30分钟使MCF7外泌体与AptCD63结合,然后使用10mM pH 7.4的PBS缓冲液洗涤3次;MCF7外泌体溶液的浓度分别为1.56×102、3.125×102、6.25×102、12.5×102、25×102。
D、向各孔中加入10μL AptEpCAM-HRP溶液,培育30分钟,然后使用10mM pH 7.4的PBS缓冲液洗涤3次;
E、向各孔中加入入100μL络氨酸包裹的金纳米颗粒溶液和50μL 100μM的过氧化氢水溶液,静置5分钟,利用紫外分光光度计测试各孔反应液的紫外光谱,如图5所示,从图中可以看出随着MCF7外泌体溶液浓度的增大,检测体系在520nm处的吸收强度逐渐降低,但是在650nm处的吸收强度逐渐增强;
F、以MCF7外泌体溶液的浓度为横坐标,反应液在520nm、650nm波长处的紫外吸收强度比I520nm/I650nm为纵坐标构建曲线,如图6所示,得出线性方程Y=-1.397×lnX+12.988,其相关系数为R2=0.9842,其中Y为I520nm/I650nm,X为MCF7外泌体溶液的浓度,单位为颗粒/μL;根据线性方程即可得到任意I520nm/I650nm下所对应的待测MCF7外泌体溶液的浓度。
上述参照实施例对一种用于MCF7外泌体检测的适配体传感器及其制备方法及MCF7外泌体的检测方法进行的详细描述,是说明性的而不是限定性的,可按照所限定范围列举出若干个实施例,因此在不脱离本发明总体构思下的变化和修改,应属本发明的保护范围之内。
Claims (10)
1.一种用于MCF7外泌体检测的适配体传感器的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)将AptCD63溶液包被于96孔板中;
(2)以BSA溶液对AptCD63包被后的96孔板进行封闭;
(3)向包被有AptCD63的孔中加入MCF7外泌体溶液,培育,洗涤;
(4)将适配体EpCAM溶液、sulfo-SMCC与辣根过氧化物酶溶液混合孵育,得到AptEpCAM-HRP溶液;
(5)向步骤(3)得到的包被有AptCD63的孔中加入AptEpCAM-HRP溶液,培育,洗涤,即可得到所述用于MCF7外泌体检测的适配体传感器;
当向所述适配体传感器中加入络氨酸包裹的金纳米颗粒溶液和过氧化氢水溶液,静置5分钟后络氨酸包裹的金纳米颗粒会产生聚集,反应液由红色转变为蓝色,利用紫外分光光度计即可实现对于MCF7外泌体的定量检测。
2.根据权利要求1所述的制备方法,其特征在于,所述AptCD63的基因序列如下:
AptCD63:5′-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA-3′。
3.根据权利要求1所述的制备方法,其特征在于,所述适配体EpCAM(AptEpCAM)的基因序列如下:
AptEpCAM:5′-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGCCTG-3′。
4.根据权利要求1-3任意一项所述的制备方法,其特征在于,所述制备方法具体包括以下步骤:
(1)将15uL 1μM的AptCD63溶液包被于96孔板中,并在4℃的黑暗中保存10小时,然后以PBS缓冲液洗涤;
(2)以15μL 1%BSA溶液对AptCD63包被后的96孔板进行封闭,固定时间为10分钟,然后以PBS缓冲液洗涤;
(3)向包被有AptCD63的孔中加入15μL MCF7外泌体溶液,培育30分钟,然后以PBS缓冲液洗涤;
(4)将2μL 5μM的适配体EpCAM溶液、2mg sulfo-SMCC与8μL 100μM的辣根过氧化物酶溶液混合孵育10分钟,得到AptEpCAM-HRP溶液;
(5)向步骤(3)得到的包被有AptCD63的孔中加入10μL AptEpCAM-HRP溶液,培育30分钟,然后以PBS缓冲液洗涤,即可得到所述用于MCF7外泌体检测的适配体传感器。
5.根据权利要求1所述的制备方法,其特征在于,所述络氨酸包裹的金纳米颗粒溶液的制备方法为:将络氨酸溶液与金纳米溶液混合,加入盐酸溶液进行官能化反应,即可得到。
6.根据权利要求1所述的制备方法,其特征在于,所述AptCD63溶液、适配体EpCAM溶液、MCF7外泌体溶液、辣根过氧化物酶溶液分别是通过将AptCD63、适配体EpCAM、MCF7分泌的外泌体、辣根过氧化物酶溶解在PH7.4 10mM的PBS缓冲溶液中得到。
7.如权利要求1-6任意一项所述的制备方法制备得到的用于MCF7外泌体检测的适配体传感器。
8.一种MCF7外泌体的定量检测方法,其特征在于,所述检测方法包括以下步骤:
A、将AptCD63溶液包被于96孔板中的多个孔中;
B、以BSA溶液对AptCD63包被后的各孔进行封闭;
C、向各孔中加入系列浓度的MCF7外泌体溶液,培育,洗涤;
D、向各孔中加入AptEpCAM-HRP溶液,培育,洗涤;
E、向各孔中加入络氨酸包裹的金纳米颗粒和过氧化氢,静置5分钟,利用紫外分光光度计测试各孔反应液的紫外光谱;
F、以MCF7外泌体溶液的浓度为横坐标,反应液在520nm、650nm波长处的紫外吸收强度比I520nm/I650nm为纵坐标构建曲线,得出线性方程,根据线性方程即可得到任意I520nm/I650nm下所对应的待测MCF7外泌体溶液的浓度。
9.根据权利要求8所述的检测方法,其特征在于,步骤C中,MCF7外泌体溶液的浓度分别为1.56×102、3.125×102、6.25×102、12.5×102、25×102颗粒/μL。
10.根据权利要求8所述的检测方法,其特征在于,所述线性方程为Y=-1.397×lnX+12.988,其相关系数为R2=0.9842,其中Y为I520nm/I650nm,X为MCF7外泌体溶液的浓度,单位为颗粒/μL。
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