CN110470714A - 一种基于dna步行器诱导构象转化和信号放大的电化学发光传感器及其应用 - Google Patents
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Abstract
本发明公开了一种基于DNA步行器诱导构象转化和信号放大的电化学发光传感器及其检测谷胱甘肽(GSH)的分析应用。本发明的技术方案是通过目标GSH将MnO2还原成替代目标Mn2+,Mn2+驱动DNA酶放大反应产生DNA产物。DNA产物驱动电极上内切酶辅助的DNA步行器放大反应，进一步诱导构象转换形成亲和素适体，该适体特异性结合CdS:Mn‑亲和素信号探针，构建了ECL传感器检测GSH。该研究思路为实现GSH的灵敏检测提供了新的策略。
Description
技术领域：
本发明涉及一种基于DNA步行器诱导构象转化和信号放大的电化学发光传感器；本发明还涉及所述传感器的制备方法及其检测谷胱甘肽(GSH)的分析应用。
背景技术：
电化学发光技术具有较低的背景信号、线性范围宽、灵敏度高、选择性好、制备简单以及成本低等优点[Hesari,M.；Swanick,K.N.；Lu,J.-S.；Whyte,R.；Wang,S.；Ding,Z.J.Am.Chem.Soc.2015,137,11266-11269.]，在生物医药、肿瘤标志物的检测、食品安全以及环境监测中都引起了广泛的关注。
将DNA步行器引入生物传感器为提高检测灵敏度带来新的亮点，诸多报道已经证明DNA步行器可以在传感系统中实现信号的扩增和转导，从而进行信号放大[Golub E,Pelossof G,Freeman R,et al.Analytical Chemistry,2009,81(22):9291–9298.]。采用互补碱基配对的原理来创建静态结构，进一步通过DNA步机器进行程序化装配操作，具有“类似机器”功能的步行器因其持续性，方向性，可重复性操作，渐进式操作和自主操作等特点而备受关注。酶促反应的刺激会为DNA 步行器沿着特定路径运行提供动力[Zhang Y,HuJ,Zhang C Y.Analytical Chemistry,2012,84:9544–9549.]。
本发明设计了一种基于DNA步行器引导构象转换形成亲和素适体和利用DNA酶循环放大技术的电化学发光生物传感器，实现对谷胱甘肽GSH的灵敏检测。
发明内容：
本发明的目的是提供一种基于DNA步行器构象转换和信号放大的电化学发光生物传感器，以及利用该生物传感器检测谷胱甘肽GSH的分析应用。它由以下步骤组成：
生物传感器的制备：
步骤1.CdS:Mn-亲和素信号探针的合成：
取100微升CdS:Mn QDs，加入10μL 0.1M EDC和10μL 0.025M NHS活化1小时，加入20μL 1mg/mL亲和素SA于37℃反应6h。
步骤2.GSH将MnO2还原成Mn2+：
20μL不同浓度的GSH与20μL MnO2纳米片混合涡旋3min,离心5min，取上清液得到不同浓度Mn2+的溶液。
步骤3.Mn2+催化的DNA酶循环放大反应：
20mg EDC和10mg NHS加入到50μL的COOH-MB溶液中，37℃下活化1h，加入50μL S1(1μM)37℃反应6h，再加入25μL S2(1μM)反应2h形成DNA酶。磁分离去除多余的DNA，分散到170μL PBS。最后，取5μL上述Mn2+的溶液与20μL MB-S1-S2溶液混合后于37℃反应80min。磁分离后，收集上清液备用。
步骤4.传感器的构建和检测。
ITO电极清洗晾干。Arm和Blocker(A/B)于37℃下反应2h保护Arm，然后6μL退火的H1和A/B按比例混合滴到纳米金修饰的ITO电极，37℃反应过夜,用1mM MCH封板2h。电极冲洗后，6μL步骤3收集的上清液和2U Nt.BbvCI加入电极反应体系中于37℃反应2h。最后与CdS:Mn-SA探针于37℃反应1h。
于100mM PBS(pH 7.4，含有50mM K2S2O8)进行电化学发光检测，PMT是-800V，电位：0～-1.5V，扫速：100mV s-1。
本发明与现有技术相比，主要优点在于：本发明利用CdS:Mn量子点作为信号探针，具有较强的电化学发光信号，提高了检测灵敏度；本发明利用DNA步行器和DNA酶循环放大技术相结合，极大地提高了选择性，放大了电化学发光信号，实现了对GSH的高灵敏、高选择性检测。
本发明的电致化学发光传感器表现出了优良的准确性、高灵敏性、高选择性、稳定性与重现性，分析检测迅速、方便，该生物传感器在生物医学分析检测和早期临床诊断中具有巨大的应用潜力，可用于实际样品的检测。
附图说明：
图1.电化学发光传感器原理示意图：(A)目标GSH将MnO2还原成替代目标Mn2+,(B)替代目标Mn2+驱动DNA酶放大反应产生DNA产物，(C)DNA步行器诱导构象转换的ECL传感器检测GSH。
图2.(A)CdS:Mn QDs的透射电子显微镜(TEM)图，(B)CdS:Mn QDs的高分辨透射电子显微镜图，(C)CdS:Mn QDs的粒径分布图，(D)CdS:Mn QDs的荧光和ECL光谱图。
图3.DNA步行器电泳表征：(a)Arm,(b)Blocker,(c)H1,(d)A/B+H1，(e)步行器诱导的H1剪切产物。
图4.ECL传感器的AFM表征。
图5.(A)不同浓度的GSH对应的ECL信号响应,(B)检测GSH的标准矫正曲线。
具体实施方式：
实施例1.电化学发光传感器的制备及对GSH的检测
Mn2+催化的DNA酶循环放大反应。20mg EDC和10mg NHS加入到50μL的COOH-MB溶液中，37℃下活化1h，加入50μL S1(1μM)37℃反应6h，再加入25μL S2(1μM)反应2h形成DNA酶。磁分离去除多余的DNA，分散到170μL PBS。最后，取5μL上述Mn2+的溶液与20μL MB-S1-S2溶液混合后于37℃反应80min。磁分离后，收集上清液备用。
传感器的构建和检测。ITO电极清洗晾干。Arm和Blocker(A/B)于37℃下反应2h保护Arm，然后6μL退火的H1和A/B按比例混合滴到纳米金修饰的ITO电极，37℃反应过夜,用1mM MCH封板2h。电极冲洗后，6μL步骤3收集的上清液和2U Nt.BbvCI加入电极反应体系中于37℃反应2h。最后与CdS:Mn-SA探针于37℃反应1h。
传感器检测。于100mM PBS(pH 7.4，含有50mM K2S2O8)进行电化学发光检测，PMT是-800V，电位：0～-1.5V，扫速：100mV s-1。
实施例2.电化学发光传感器的制备及对GSH的检测
将“20mg EDC和10mg NHS加入到50μL的COOH-MB溶液中，37℃下活化1h”改为“20mgEDC和10mg NHS加入到50μL的COOH-MB溶液中，37℃下活化1.5h。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对GSH检测的结果同实施例1。
实施例3.电化学发光传感器的制备及对GSH的检测
将“20mg EDC和10mg NHS加入到50μL的COOH-MB溶液中，37℃下活化1h，加入50μLS1(1μM)37℃反应6h。”改为“20mg EDC和10mg NHS加入到50μL的COOH-MB溶液中，37℃下活化1h，加入50μL S1(1μM)37℃反应8h。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对GSH检测的结果同实施例1。
实施例4.电化学发光传感器的制备及对GSH的检测
将“Arm和Blocker(A/B)于37℃下反应2h保护Arm，然后6μL退火的H1和A/B按比例混合滴到纳米金修饰的ITO电极，37℃反应过夜,用1mM MCH封板2h。”改为“Arm和Blocker(A/B)于37℃下反应2h保护Arm，然后6μL退火的H1和A/B按比例混合滴到纳米金修饰的ITO电极，37℃反应10h,用1mM MCH封板2h”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对GSH检测的结果同
实施例1。
实施例5.电化学发光传感器的制备及对GSH的检测
将“电极冲洗后，6μL步骤3收集的上清液和2U Nt.BbvCI加入电极反应体系中于37℃反应2h”改为“电极冲洗后，6μL步骤3收集的上清液和3U Nt.BbvCI加入电极反应体系中于37℃反应2h。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对GSH检测的结果同实施例1。
Claims (3)
1.一种基于DNA步行器诱导构象转化和信号放大的电化学发光传感器，其特征是：利用亲和素标记CdS:Mn量子点构建ECL信号探针，通过目标GSH将MnO2还原生成的Mn2+催化DNA酶循环放大反应，产生DNA产物。DNA产物驱动电极上内切酶辅助的DNA步行器诱导构象转换，结合CdS:Mn-亲和素信号探针，构建了ECL传感器。
2.一种制备权利要求1所述的基于DNA步行器诱导构象转化和信号放大的电化学发光传感器的方法和应用，其特征方法由下列步骤组成：
步骤1.CdS:Mn-亲和素信号探针的合成：
取100微升CdS:Mn QDs，加入10μL 0.1M EDC和10μL 0.025M NHS活化1小时，加入20μL1mg/mL亲和素SA于37℃反应6h。
步骤2.GSH将MnO2还原成Mn2+：
20μL 不同浓度的GSH与20μL MnO2纳米片混合涡旋3min,离心5min，取上清液得到不同浓度Mn2+的溶液。
步骤3.Mn2+催化的DNA酶循环放大反应：
20mg EDC和10mg NHS加入到50μL的COOH-MB溶液中，37℃下活化1h，加入50μL S1(1μM)37℃反应6h，再加入25μL S2(1μM)反应2h形成DNA酶。磁分离去除多余的DNA，分散到170μLPBS。最后，取5μL上述Mn2+的溶液与20μL MB-S1-S2溶液混合后于37℃反应80min。磁分离后，收集上清液备用。
步骤4.传感器的构建和检测。
ITO电极清洗晾干。Arm和Blocker(A/B)于37℃下反应2h保护Arm，然后6μL退火的H1和A/B按比例混合滴到纳米金修饰的ITO电极，37℃反应过夜,用1mM MCH封板2h。电极冲洗后，6μL步骤3收集的上清液和2U Nt.BbvCI加入电极反应体系中于37℃反应2h。最后与CdS:Mn-SA探针于37℃反应1h。
3.根据权利要求2所述的谷胱甘肽GSH的检测方法，其特征是：所述的电化学发光测试是将表面进行反应完成的电极作为工作电极，三电极体系中检测ECL信号。于100mM PBS(pH7.4，含有50mM K2S2O8)进行ECL检测，PMT是-800V，电位：0～-1.5V，扫速：100mV s-1。
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| CN112626242A (zh) * | 2020-12-11 | 2021-04-09 | 宁波大学 | 基于核酸构象引发链取代驱动DNA Walker的双信号检测食源性致病菌的方法 |
| CN112626242B (zh) * | 2020-12-11 | 2022-05-24 | 宁波大学 | 基于核酸构象引发链取代驱动DNA Walker的双信号检测食源性致病菌的方法 |
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| CN114214461A (zh) * | 2021-12-26 | 2022-03-22 | 南京大学 | 一种等温hiv核酸检测试剂盒及检测方法 |
| CN114214461B (zh) * | 2021-12-26 | 2024-03-26 | 南京大学 | 一种等温hiv核酸检测试剂盒及检测方法 |
| CN116023932A (zh) * | 2022-12-09 | 2023-04-28 | 山西大学 | 一种用于谷胱甘肽检测的荧光探针及其制备 |
| CN116023932B (zh) * | 2022-12-09 | 2024-03-12 | 山西大学 | 一种用于谷胱甘肽检测的荧光探针及其制备 |
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