CN106248941A - 一种灵敏检测小肽muc1的方法 - Google Patents
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Abstract
本专利公开了一种灵敏检测肿瘤相关小肽MUC1的方法,涉及电化学发光检测技术领域。利用适配体与目标小肽特异性识别的特点,设计特殊的适配体/引发序列,从而达到目标物特异性识别以及检测目标物转化的目的;通过结合两种发卡结构探针的杂交反应完成信号放大的策略,实现信号的有效放大;最终通过引入电化学探针,采用电化学发光的方法实现MUC1的灵敏检测。本方法检测成本低、灵敏度高。
Description
技术领域
本发明涉及电化学发光检测技术领域,更具体地说是一种以适配体/引发序列作特异性识别和杂交链反应作信号放大的方式构建的电化学发光检测小肽方法。
背景技术
癌症是威胁人类健康的一大杀手,近年来其发病率及死亡率更是呈现逐步走高的趋势。当人体细胞产生癌变后,癌细胞新陈代谢与正常细胞会有很大的差别,同时癌细胞也会产生区别于正常细胞的小分子物质,这主要包括酶、激素、抗原、小肽和小分子RNA等。这些小分子物质就是所谓的肿瘤标志物。相关肿瘤标志物的含量水平是癌症早期预警以及诊断的重要指标。其中,小肽作为新型的肿瘤标志物受到了越来越多的关注,它们在癌症细胞氨基酸的消化、吸收等方面起到重要的作用。因此,通过检测肿瘤相关小肽的含量水平,人们能够及时发现癌变细胞,从而达到癌症早期预警的目的。
小肽MUC1,又叫CA15-3,是一种大的细胞表面糖蛋白,在参与信号传输以及调控免疫细胞生长与凋亡等方面有重要作用。MUC1在胃、结肠、前列腺、乳腺等大多数人类腺癌里都有表达,并且还是已经商品化的血清癌症标志物检测方法里的抗原。肿瘤组织的MUC1在癌细胞表面会产生反常,即糖基化相对于正常细胞不完全。由于MUC1特异性高于一般抗原,敏感性高于癌胚抗原,因而在癌症检测方面被作为肿瘤标记物来应用。
为了解决小肽在低浓度下也能进行检测的问题,小肽识别和信号放大策略变的尤为重要。然而,目前检测小肽的方法,如酶联免疫吸附测定、无需标记的表面等离子共振、液相色谱-质谱的方法虽然具有较高的灵敏度和特异性,但是需要价格昂贵的酶或者一系列的大型仪器。因此发展一种既能检测到低浓度小肽又能降低检测成本的方法具有重要意义。一方面,适配体与靶分子特异性结合的特点以及杂交链反应无需酶即能实现放大信号的优势能很好地解决低成本、灵敏性检测问题,另一方面电化学发光的方法由于具有响应速度快、仪器成本低、检测灵敏度高等优势也可以很好地解决上述问题。
发明内容
本发明要解决的技术问题是构建一种能实现特异性识别以及信号方法的低成本、高灵敏的小肽检测方法。
一种灵敏检测肿瘤相关小肽MUC1的方法,其特征是包括以下步骤:
(1)探针预变性
巯基修饰的捕获探针和发卡探针1、发卡探针2以及适配体/引发序列,适配体的英文名称为aptamer,在95 ºC下分别孵育0.5 h;将这三种溶液在25 ºC下放置1 h;
(2)检测溶液的制备
该步骤中的总反应混合液体积为200 µL,不同浓度的小肽MUC1与一定浓度的适配体/引发序列在工作缓冲溶液中反应,所得混合溶液即为检测溶液;工作缓冲溶液的成分为20mM Tris盐酸, 100 mM 氯化钠, 10 mM 氯化钾, 10 mM 氯化镁,pH 为7.5;
(3)电极修饰过程
该步骤每一步修饰完成后都使用浓度为0.01 M、pH 7.4的磷酸缓冲溶液清洗,孵育温度均为25 ºC;首先,将10 µL 浓度为2 µM的捕获探针滴加到预处理过的金电极上,孵育过夜;滴加5 µL 浓度为1 mM的巯基己醇,孵育2 h;滴加10 µL 步骤(2)中所得检测溶液,孵育1 h;依次滴加5 µL 浓度均为1 µM的发卡探针1和发卡探针2,孵育6 h;滴加10 µL 浓度为2mM的三联吡啶钌溶液;
(4)电化学发光检测
电化学发光检测的实验参数如下:扫描电压范围为-0.1 ~ 0.6 V、扫描速度为50 mV S−1、光电倍增管设置为600 V。
本发明的有益效果
(1)利用适配体技术,能够实现目标物小肽MUC1的特异性识别;
(2)利用发卡探针1和发卡探针2之间的杂交链反应,简洁实现信号快速放大;
(3)本发明所述方法检测成本低、灵敏度高。
附图说明
图1为本文所述方法的实验原理图。
具体实施方式
为了更好地理解本发明,下面结合实施例和附图进一步阐明本发明的内容,但本发明的内容不仅仅局限于下面的实施。
实施例1
一种灵敏检测肿瘤相关小肽MUC1的方法,其特征是包括以下步骤:
(1)探针预变性
巯基修饰的捕获探针和发卡探针1、发卡探针2以及适配体/引发序列,适配体的英文名称为aptamer,在95 ºC下分别孵育0.5 h;将这三种溶液在25 ºC下放置1 h;
(2)检测溶液的制备
该步骤中的总反应混合液体积为200 µL,不同浓度的小肽MUC1与浓度为1 µM的适配体/引发序列在工作缓冲溶液中反应,所得混合溶液即为检测溶液;工作缓冲溶液的成分为20 mM Tris盐酸, 100 mM 氯化钠, 10 mM 氯化钾, 10 mM 氯化镁,pH 为7.5;
(3)电极修饰过程
该步骤每一步修饰完成后都使用浓度为0.01 M、pH 7.4的磷酸缓冲溶液清洗,孵育温度均为25 ºC;首先,将10 µL 浓度为2 µM的捕获探针滴加到预处理过的金电极上,孵育过夜;滴加5 µL 浓度为1 mM的巯基己醇,孵育2 h;滴加10 µL 步骤(2)中所得检测溶液,孵育1 h;依次滴加5 µL 浓度均为1 µM的发卡探针1和发卡探针2,孵育6 h;滴加10 µL 浓度为2mM的三联吡啶钌溶液;
(4)电化学发光检测
电化学发光检测的实验参数如下:扫描电压范围为-0.1 ~ 0.6 V、扫描速度为50 mV S−1、光电倍增管设置为600 V。
实施例2
检测步骤同例1,不同之处是:步骤(4)中扫描电压范围为-0.2 ~ 0.8 V、扫描速度为100 mV S−1、光电倍增管设置为800 V。
SEQUENCE LISTING
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<120> 一种灵敏检测小肽MUC1的方法
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Claims (2)
1.一种灵敏检测小肽MUC1的方法,其特征是包括以下步骤:
(1)探针预变性
巯基修饰的捕获探针和发卡探针1、发卡探针2以及适配体/引发序列,适配体的英文名称为aptamer,在95 ºC下分别孵育0.5 h;将这三种溶液在25 ºC下放置1 h;
(2)检测溶液的制备
该步骤中的总反应混合液体积为200 µL,不同浓度的小肽MUC1与一定浓度的适配体/引发序列在工作缓冲溶液中反应,所得混合溶液即为检测溶液;工作缓冲溶液的成分为20mM Tris盐酸, 100 mM 氯化钠, 10 mM 氯化钾, 10 mM 氯化镁,pH 为7.5;
(3)电极修饰过程
该步骤每一步修饰完成后都使用浓度为0.01 M、pH 7.4的磷酸缓冲溶液清洗,孵育温度均为25 ºC;首先,将10 µL 浓度为2 µM的捕获探针滴加到预处理过的金电极上,孵育过夜;滴加5 µL 浓度为1 mM的巯基己醇,孵育2 h;滴加10 µL 步骤(2)中所得检测溶液,孵育1 h;依次滴加5 µL 浓度均为1 µM的发卡探针1和发卡探针2,孵育6 h;滴加10 µL 浓度为2mM的三联吡啶钌溶液;
(4)电化学发光检测
电化学发光检测的实验参数如下:扫描电压范围为-0.1 ~ 0.6 V、扫描速度为50 mV S−1、光电倍增管设置为600 V。
2.如权利要求1所述的一种灵敏检测肿瘤相关小肽MUC1的方法,其特征是步骤(1)中适配体/引发序列、发卡探针1以及发卡探针2的核苷酸序列,它们的核苷酸序列为说明书核苷酸和氨基酸序列表中所述序列。
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CN114047243A (zh) * | 2021-11-16 | 2022-02-15 | 南开大学 | 一种基于CRISPR/Cas12a检测SARS-CoV-2的电化学适配体传感器 |
CN115980163A (zh) * | 2023-03-17 | 2023-04-18 | 武汉理工大学 | 一种便携式肿瘤dna的电化学发光检测装置及方法 |
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