CN110567946A - 一种核酸染料抑制DNAzyme传感体系背景信号的方法 - Google Patents
一种核酸染料抑制DNAzyme传感体系背景信号的方法 Download PDFInfo
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 18
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 18
- 108091027757 Deoxyribozyme Proteins 0.000 title claims abstract description 15
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 7
- 108020004414 DNA Proteins 0.000 claims abstract description 32
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- 230000003197 catalytic effect Effects 0.000 claims abstract description 7
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000004458 analytical method Methods 0.000 claims description 3
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- 230000000694 effects Effects 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims 4
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 6
- 108700025694 p53 Genes Proteins 0.000 abstract description 4
- 230000003993 interaction Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
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- 108091023037 Aptamer Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
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Abstract
一种核酸染料抑制DNAzyme传感体系背景信号的方法，是利用核酸染料与血红素相互作用，从而抑制血红素的催化性能，降低DNAzyme传感体系的背景信号；当目标DNA存在时，目标DNA和相应的探针链DNA在室温下孵育一段时间形成含有G‑四聚体结构的双链DNA，血红素嵌入G‑四聚体结构中，同时阻止了血红素与核酸染料的作用，形成DNAzyme催化鲁米诺和双氧水反应产生强的化学发光信号，实现高灵敏检测DNA。采用该法，对p53基因的检出限可达2.0pM。使用不用的探针链，该体系可用于不同DNA的测定，故具有广阔的应用前景。
Description
技术领域
本发明涉及一种核酸染料抑制DNAzyme传感体系背景信号的方法，属于生物传感技术领域。
背景技术
近年来，DNA检测已经涉及到临床诊断、基因诊疗、药物筛选、司法鉴定等领域，因而愈来愈多的研究工作者竭力构建多种高灵敏检测DNA的新方法。目前提高生物传感器灵敏度有两个途径：一种是通过信号放大，如聚合酶链式反应、、滚环扩增、目标物循环放大、目标催化发夹自组装等；另外一种是借助纳米材料特殊性或者通过设计特殊的DNA结构来降低体系的背景信号。目前基于信号放大检测的方法多，且较为成熟，但是如何有效降低背景信号的问题没有得到很好的解决。
DNAzyme传感体系是一种极具前景的无标记检测DNA的方法。在现有报道中，主要将血红素适配体的四个GGG重复序列分成两部分，在目标DNA存在下，结合形成不对称分裂的G-四聚体DNA酶(Chem.Commun.,2015,51:12373-12376；Anal.Chim.Acta.,2018,997:1-8)。然而，空白血红素也有催化性能，会产生相对高的背景信号。因此，有必要开发一种有效抑制DNAzyme传感体系背景信号的方法。
发明内容
本发明的目的在于利用核酸染料与血红素相互作用，从而抑制血红素的催化性能，降低DNAzyme传感体系的背景信号；当目标DNA存在时，目标DNA和相应的探针链DNA在室温下孵育一段时间形成含有G-四聚体结构的双链DNA，血红素嵌入G-四聚体结构中，同时阻止了血红素与核酸染料的作用，形成DNAzyme催化鲁米诺和双氧水反应产生强的化学发光信号，实现高灵敏检测。
本发明的技术方案如下：
(1)在96孔板中将探针链DNA与血红素孵育，再加入核酸染料孵育一定的时间，加入鲁米诺和双氧水，测定空白的化学发光信号；
(2)在96孔板中加入目标DNA及探针链DNA、HEPES缓冲溶液，室温孵育一定时间后，加入血红素，再孵育一定时间，使之形成有G-四聚体结构的双链DNA；并测定目标DNA存在时，形成的DNAzyme催化鲁米诺和双氧水反应产生强的化学发光信号
(3)根据对比空白所增强的化学发光信号，对目标DNA进行传感分析。
发明效果
与现有技术相比，本发明具有如下优点：
(1)核酸染料会与血红素相互作用，降低血红素的催化活性，从而降低DNAzyme传感体系的背景信号；
(2)该方法的灵敏度高，可实现对pM级DNA的检测；
(3)本方法无需标记、无需分离，分析成本低。
具体实施方式
实施例1
取20μL浓度为500nM的G3(5’-AGG AGA GAC CTG GGT-3’)探针DNA和20μL浓度为500nM的G9(5’-AGG GCG GGT GGG TGC GCA CAG AGG AA-3’)探针DNA使其终浓度均为10nM在pH为7.4的HEPES缓冲中孵育，加入不同浓度的p53基因，于30℃恒温水浴中孵育2h，然后加入终浓度为50nM的Hemin孵育30min，再加入终浓度为0.3×(0.57μM)的核酸染料SYBRGreen I(SG)，避光室温孵育10min，取孵育后的试样100μL加入50μL2.0mM的luminol和50μL20mM的H2O2，用化学发光仪快速测定并记录其化学发光强度；在无目标基因的情况下，测定空白信号；根据样品信号与空白信号的比值对目标基因进行定量分析。
实施例2
取20μL浓度为500nM的G3(5’-AGG AGA GAC CTG GGT-3’)探针DNA和20μL浓度为500nM的G9(5’-AGG GCG GGT GGG TGC GCA CAG AGG AA-3’)探针DNA使其终浓度均为10nM在pH为8.0的HEPES缓冲中孵育，加入不同浓度的p53基因，于30℃恒温水浴中孵育2h，然后加入终浓度为80nM的Hemin，孵育20min，再加入终浓度为5×的核酸染料Pico Green(PG)，避光室温孵育20min，取孵育后的试样100μL加入50μL 2.0mM的luminol和50μL 20mM的H2O2，用化学发光仪快速测定并记录其化学发光强度；在无目标基因的情况下，测定空白信号；根据样品信号与空白信号的比值对目标基因进行定量分析。
采用实施例1、实施例2均可实现低背景的传感检测p53基因。使用不用的探针链，该体系可用于不同DNA的测定，具有广阔的应用前景。
Claims (6)
1.一种核酸染料抑制DNAzyme传感体系背景信号的方法，其特征在于包括以下几个方面：
(1)将一定浓度的核酸染料加入DNAzyme的辅因子血红素(hemin)中，孵育一段时间后，形成核酸染料-血红素复合物，进而全面抑制血红素的催化性能，具有低的空白信号；
(2)当目标DNA存在时，目标DNA和相应的探针链DNA在室温下孵育一段时间形成含有G-四聚体结构的双链DNA，血红素嵌入G-四聚体结构中，形成DNAzyme，同时阻止了血红素与核酸染料的作用，具有强的催化鲁米诺与双氧水反应的性能；
(3)根据化学发光的强度对可实现对目标DNA的低背景传感分析。
2.按权利要求1所述的方法，与血红素作用的物质是核酸染料，包括SYBR Green I、Pico Green等。
3.按权利要求1所述的方法，其特征在于加入核酸染料孵育的时间为5-20min。
4.按权利要求1所述的方法，其特征在于采用的核酸染料的浓度为0.5-2.5μM。
5.按权利要求1所述的方法，其特征在于孵育形成双链的时间为1-2h；加入血红素后，孵育时间为40-60min。
6.按权利要求1所述的方法，其特征孵育溶液和化学发光反应的pH为7-8。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104845969A (zh) * | 2015-05-08 | 2015-08-19 | 首都师范大学 | 一种调控和提高脱氧核酶催化活性的方法 |
| CN106755460A (zh) * | 2017-01-10 | 2017-05-31 | 北京化工大学 | 一种单碱基突变检测方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104845969A (zh) * | 2015-05-08 | 2015-08-19 | 首都师范大学 | 一种调控和提高脱氧核酶催化活性的方法 |
| CN106755460A (zh) * | 2017-01-10 | 2017-05-31 | 北京化工大学 | 一种单碱基突变检测方法 |
Non-Patent Citations (2)
| Title |
|---|
| LI DENG ET AL.: "Improving the Signal-to-Background Ratio during Catalytic Hairpin Assembly through Both-End-Blocked DNAzyme" * |
| 张后春: "高信背比DNA酶-SYBR Green I化学发光传感体系的研究" * |
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