CN110702756A - 一种检测核酸的电化学方法 - Google Patents
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Abstract
本发明公开了一种检测核酸的电化学方法。本发明的方法包括如下步骤：(1)根据碱基配对原则，设计与目标核酸相匹配的探针DNA或RNA；(2)将探针与待检测对象混合形成核酸杂化溶液；(3)对核酸杂化溶液进行电化学伏安扫描，当出现电化学氧化还原峰时，表明待检测对象包含目标核酸；当未出现电化学氧化还原峰时，表明待检测对象不包含目标核酸。本发明具有特异性高、重复性好、操作经济简单等优点。
Description
技术领域
本发明属于生物传感技术领域，特别是涉及一种检测核酸的电化学方法。
背景技术
miRNA是一类内源性非编码的小分子单链RNA，以基因表达的方式参与调节生物体系的各种生物及病理过程。因此miRNA检测应用引起包括医药、食品、法医和环境等应用领域的广泛兴趣，是目前传感检测应用研究领域的热点。miRNAs在癌变发生、发展过程中发挥关键作用，miRNAs被公认最具潜力的肿瘤标志物。但是，miRNAs链很短，20个碱基左右；序列同源性高，多数序列只相差一个碱基，因此，miRNAs特异性检测存在诸多挑战。miRNAs传统检测方法仍然存在很多无法克服的缺陷，例如微阵列分析方法存在特异性差、重复性差，易出现假阳性问题；而目前最常用的miRNAs检测技术----实时定量PCR(real—time PCR)技术，由于miRNA序列短而逆转录困难，PCR过程中非特异性扩增也容易导致假阳性。
核酸具有电化学活性，核酸的电化学活性来自拥有π电子体系的碱基对。核酸的碱基对能够发生电化学氧化或还原，使得电化学技术应用于癌症miRNAs检测研究成为可能，也为电化学技术进行癌症的早期筛查和伴随监测及遗传性诊断提供了强有力的依据。
发明内容
为了解决现有技术存在的缺陷，本发明的目的在于提供一种检测核酸的电化学方法。本发明具有特异性高、重复性好、操作经济简单等优点。
为了达到上述的目的，本发明采取以下技术方案：
一种检测核酸的电化学方法，包括如下步骤：
(1)根据碱基配对原则，设计与目标核酸相匹配的探针DNA或RNA；
(2)将探针与待检测对象混合形成核酸杂化溶液(即杂化的双链DNA-RNA，DNA-DNA或RNA-RNA)；
(3)对核酸杂化溶液进行电化学伏安扫描，当出现电化学氧化还原峰时，表明待检测对象包含目标核酸；当未出现电化学氧化还原峰时，表明待检测对象不包含目标核酸。
进一步地，上述方法中，所述探针与待检测对象核酸片段按照任意体积比或浓度比进行混合。
进一步地，上述方法中，所述待检测对象为单核酸体系或多核酸混合体系；优选的，所述待检测对象为miRNA或总RNA。
进一步地，上述方法中，所述目标核酸选自DNA、cDNA或RNA；优选的，所述目标核酸选自具有疾病特征或病原体的DNA、cDNA或RNA；更优选的，所述目标核酸为miRNA。
进一步地，上述方法中，所述目标核酸来自人工合成、疾病细胞提取或者血清提取。
进一步地，上述方法中，所述电化学伏安扫描的电解质溶液为pH为7.4的磷酸缓冲溶液(PBS缓冲液)。该pH值模拟人体体液环境。
进一步地，上述方法中，所述电化学伏安扫描的电极为碳电极或金属电极。优选的，所述碳电极为玻碳电极、石墨电极、石墨烯修饰电极；所述金属电极为纳米金属电极或金属氧化物修饰电极。
本发明具有以下技术特点：
1)本发明利用电化学技术实现了核酸的检测，方法简单易行，可以应用于医学、食品、环境等领域，例如，通过对疾病特征的核酸进行检测，实现疾病诊断或病原体检测，具有广泛的应用前景。
2)本发明能够实现对核酸分子单碱基错配检出，具有良好的特异性。
3)本发明的检测结果重复性好。
4)本发明的方法操作过程经济简单，不存在链反应，无需扩增，无需荧光指示剂。
附图说明
图1肝癌miR-122与正配探针及错配探针杂化的电化学伏安扫描图：(a)正扫；(b)负扫。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例的技术方案进行清楚、完整的描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明的保护范围。
以下实施例提供的特异性检测疾病特征miRNA的电化学检测方法的目标核酸样品来源是人工合成的DNA及cDNA或RNA(miRNA)片段，细菌DNA及RNA(miRNA)，病毒DNA及RNA(miRNA)，细胞提取的DNA及RNA(miRNA)，或血清RNA(miRNA)，Total RNA等。
特异性检测疾病特征miRNA的电化学检测方法中核酸杂化形式为杂化的双链DNA-RNA，DNA-DNA或RNA-RNA。
实施例1.RNA-DNA杂化体系
将人工合成的肝癌生物标志物miR-122(5’-UGGAGUGUGACAAUGGUGUUUG-3’)溶液分别等体积地滴入5pM正配探针(5’-CAAACACCATTGTCACACTCCA-3’)溶液和5pM错配探针(5’-CAAACACCATTATCACACTCCA-3’)溶液中，通过RNA-DNA杂化反应，形成正配杂交溶液和错配杂交溶液。以pH＝7.4的模拟体液磷酸缓冲溶液(PBS缓冲液)为电解液，玻碳电极为传感电极，对上述正配杂交溶液和错配杂交溶液进行电化学伏安扫描，电化学传感检测结果如附图1所示：完美配对的杂交溶液伏安曲线在1.2V左右出现电化学氧化峰(正扫，图1a)，1.1V出出现电化学还原峰(负扫，图1b)，错配杂交溶液伏安曲线在测试范围内没有电化学氧化还原峰出现。
实施例2.cDNA-DNA杂化体系
将人工合成的前列腺癌特征标志物miR-141的cDNA(5’-TAACACTGTCTGGTAAAGATGG-3’)溶液分别等体积地滴入5pM正配探针(5’-CCATCTTTACCAGACAGTGTTA-3’)溶液和5pM错配探针(5’-ACATCTTTACCAGACAGTGTTA-3’)溶液中，通过cDNA-DNA杂化反应，形成正配杂交溶液和错配杂交溶液。以pH＝7.4的模拟体液磷酸缓冲溶液(PBS缓冲液)为电解液，石墨碳电极为传感电极，对上述正配杂交溶液和错配杂交溶液进行电化学伏安扫描，电化学传感检测结果显示：完美配对的杂交溶液伏安曲线在1.1V左右出现电化学氧化峰，1.2V出出现电化学还原峰，错配杂交溶液伏安曲线在测试范围内没有电化学氧化还原峰出现。
实施例3.DNA-RNA杂化体系
将人工合成的乳腺癌特征标志物hsa-miR-373(5’-ACUCAAAAUGGGGGCGCUUUCC-3’)溶液分别等体积地滴入5pM正配探针(5’-GGAAAGCGCCCCCATTTTGAGT-3’)溶液和5pM错配探针(5’-GGAAAGCGCCCCCATTTTGGGT-3’)溶液中，通过DNA-RNA杂化反应，形成正配杂交溶液和错配杂交溶液。以pH＝7.4的模拟体液磷酸缓冲溶液(PBS缓冲液)为电解液，金电极为传感电极，对上述正配杂交溶液和错配杂交溶液进行电化学伏安扫描，电化学传感检测结果显示：完美配对的杂交溶液伏安曲线在0.16V左右出现电化学氧化峰，0.14V出出现电化学还原峰，错配杂交溶液伏安曲线在测试范围内没有电化学氧化还原峰出现。
实施例4.胰腺癌DNA-RNA杂化体系
将人工合成的胰腺癌特征标志物miR-1290(5'-UGGAUUUUUGGAUCAGGGA-3')溶液分别等体积地滴入5pM正配探针(5'-TCCCTGATCCAAAAATCCA-3')溶液和5pM错配探针(5'-TCGCTGATCCAAAAATCCA-3')溶液中，通过RNA-DNA杂化反应，形成正配杂交溶液和错配杂交溶液。以pH＝7.4的模拟体液磷酸缓冲溶液(PBS缓冲液)为电解液，纳米二氧化钛修饰电极为传感电极，对上述正配杂交溶液和错配杂交溶液进行电化学伏安扫描，电化学传感检测结果显示：完美配对的杂交溶液伏安曲线在1.5V左右出现电化学氧化峰，0.5V出出现电化学还原峰，错配杂交溶液伏安曲线在测试范围内没有电化学氧化还原峰出现。
实施例5.登革热RNA-RNA杂化体系
将人工合成的登革热特征标志物DENV-2miRNA(5'-GGAAGCUGUACGCAUGGCGUA-3')溶液分别等体积地滴入5pM正配探针(3'-NH2–2-O-Me-(CCUUCGACAUGCG)TACCGCAT-5')溶液和5pM错配探针(3'-NH2–2-O-Me-(CCUUCGACAUGCG)TACCCCAT-5')溶液中，通过RNA-RNA杂化反应，形成正配杂交溶液和错配杂交溶液。以pH＝7.4的模拟体液磷酸缓冲溶液(PBS缓冲液)为电解液，铟锡氧化物薄膜修饰电极为传感电极，对上述正配杂交溶液和错配杂交溶液进行电化学伏安扫描，电化学传感检测结果显示：完美配对的杂交溶液伏安曲线在1.0V左右出现电化学氧化峰，0.8V出出现电化学还原峰，错配杂交溶液伏安曲线在测试范围内没有电化学氧化还原峰出现。
实施例6.肝癌细胞DNA-Total RNA(总miRNA)杂化体系
将从肝癌细胞HePG2中提取的总miRNA溶液分别等体积地滴入5pM肝损伤特征标志物Has-miR-122的正配探针(5'-CAAACACCATTGTCACACTCCA-3')溶液和5pM错配探针(5'-CAAACACCATTCTCACACTCCA-3')溶液中，通过RNA-DNA杂化反应，形成正配杂交溶液和错配杂交溶液。以pH＝7.4的模拟体液磷酸缓冲溶液(PBS缓冲液)为电解液，纳米银修饰电极为传感电极，对上述正配杂交溶液和错配杂交溶液进行电化学伏安扫描，电化学传感检测结果显示：完美配对的杂交溶液伏安曲线在1.2V左右出现电化学氧化峰，1.1V出出现电化学还原峰，错配杂交溶液伏安曲线在测试范围内没有电化学氧化还原峰出现。
实施例7.肺癌患者血清DNA-Total RNA(总miRNA)杂化体系
将从肺癌患者血清中提取的miRNA溶液分别等体积地滴入5pM肺癌特征标志物miR-21的正配探针(5'-TCAACATCAGTCTGATAAGCTA-3')溶液和5pM错配探针(5'-TCAACATCAGTCTGATAAGATA-3')溶液中，通过RNA-DNA杂化反应，形成正配杂交溶液和错配杂交溶液。以pH＝7.4的模拟体液磷酸缓冲溶液(PBS缓冲液)为电解液，石墨烯修饰电极为传感电极，对上述正配杂交溶液和错配杂交溶液进行电化学伏安扫描，电化学传感检测结果显示：完美配对的杂交溶液伏安曲线在0.15V左右出现电化学氧化峰，1.1V出出现电化学还原峰，错配杂交溶液伏安曲线在测试范围内没有电化学氧化还原峰出现。
实施例8.食品或环境检测应用-大肠杆菌E.Coli RNAs检测
将人工合成的目标lacZ,E.coli lacZ miRNA
(nt5’-AUGUGGAUUGGCGAUAAAAAACAA-3’)溶液等体积地滴入5pM正配探针(5’-d(GTTGTTTTTT)-2’-O-Me-RNA(AUCGCCAAUCCACAU)-d(CTGTGAAAGA)-NH2-3’)和错配探针(5’-d(GTTGTTTTTT)-2’-O-Me-RNA(AUCGCCAAUCCACAU)-d(CTGTGAAAGA)-NH2-3’)溶液中，通过RNA-RNA杂化反应，形成正配杂交溶液和错配杂交溶液。以pH＝7.4的模拟体液磷酸缓冲溶液(PBS缓冲液)为电解液，石墨烯修饰电极为传感电极，对上述正配杂交溶液和错配杂交溶液进行电化学伏安扫描，电化学传感检测结果显示：完美配对的杂交溶液伏安曲线在1.7V左右出现电化学氧化峰，1.6V出出现电化学还原峰，错配杂交溶液伏安曲线在测试范围内没有电化学氧化还原峰出现。
除非另作定义，本公开所使用的技术术语或者科学术语应当为本发明所属领域内有一般技能的人士所理解的通常意义。
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求保护范围内。
Claims (10)
1.一种检测核酸的电化学方法，其特征在于，包括如下步骤：
(1)根据碱基配对原则，设计与目标核酸相匹配的探针DNA或RNA；
(2)将探针与待检测对象混合形成核酸杂化溶液；
(3)对核酸杂化溶液进行电化学伏安扫描，当出现电化学氧化还原峰时，表明待检测对象包含目标核酸；当未出现电化学氧化还原峰时，表明待检测对象不包含目标核酸。
2.根据权利要求1所述的检测核酸的电化学方法，其特征在于，所述探针与待检测对象核酸片段按照任意体积比或浓度比进行混合。
3.根据权利要求1所述的检测核酸的电化学方法，其特征在于，所述待检测对象为单核酸体系或多核酸混合体系。
4.根据权利要求3所述的检测核酸的电化学方法，其特征在于，所述待检测对象为miRNA或总RNA。
5.根据权利要求1所述的检测核酸的电化学方法，其特征在于，所述目标核酸选自DNA、cDNA或RNA。
6.根据权利要求5所述的检测核酸的电化学方法，其特征在于，所述目标核酸选自具有疾病特征或病原体的DNA、cDNA或RNA。
7.根据权利要求6所述的检测核酸的电化学方法，其特征在于，所述目标核酸为miRNA。
8.根据权利要求1所述的检测核酸的电化学方法，其特征在于，所述目标核酸来自人工合成、疾病细胞提取或者血清提取。
9.根据权利要求1所述的检测核酸的电化学方法，其特征在于，所述电化学伏安扫描的电解质溶液为pH为7.4的磷酸缓冲溶液。
10.根据权利要求1所述的检测核酸的电化学方法，其特征在于，所述电化学伏安扫描的电极为碳电极或金属电极；优选的，所述碳电极为玻碳电极、石墨电极、石墨烯修饰电极；所述金属电极为纳米金属电极或金属氧化物修饰电极。
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