CN111041080A - 一种新型的硅球上超支化放大的荧光信号探针及其制备和应用 - Google Patents
一种新型的硅球上超支化放大的荧光信号探针及其制备和应用 Download PDFInfo
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Abstract
本发明公开了一种新型的硅球上超支化放大的荧光信号探针;以及所述荧光信号探针的制备方法及其检测Cu2+的分析应用。本发明的技术方案是利用点击化学反应引发的步行器放大技术结合超支化杂交链式反应(HB‑HCR)构建了树枝状扩增的荧光平台,实现了对Cu2+的灵敏检测。目标Cu2+首先引发点击化学反应和DNA步行器放大过程,将Cu2+转化为大量的DNAS3产物。通过DNA S3与SiO2微球上H1杂交反应,引发了发夹H2、HS‑DNA、H3‑DNA和LT‑DNA之间的超支化杂交链式反应(HB‑HCR),组装成新型的树枝状DNA结构,负载大量的荧光Cy5,实现了对Cu2+放大信号的超灵敏荧光检测。
Description
技术领域:
本发明涉及一种新型的硅球上超支化放大的荧光信号探针;以及所述荧光信号探针的制备方法及其检测Cu2+的分析应用。
背景技术:
荧光法具有背景干扰低、成本低、灵敏度高和线性范围宽等多种优点,是检测Cu2+的理想技术。此外,测定Cu2+还使用了基于化学的识别方法,如Cu(I)催化的点击化学反应[Shen,Q.P.et.al.Biosens.Bioelectron.2013,41,663-668.]以及其他特定试剂[Li,F.;Wang,J.et.al.Biosens.Bioelectron.2013,39,82-87]。从HCR进化而来的超分化杂交链式反应(HB-HCR)可以在等温环境下实现,HB-HCR可以产生许多不同长度的树突状DNA纳米结构,具有更高的扩增效率。脱氧核糖核酸自组装树突状DNA纳米结构因其具有良好的生物相容性、结构稳定性、合成方便,在生物和生物医学领域都备受关注[Hu,Y.;Chen,Z.et.al.DrugDelivery2017,24,1295-1301.]。
本工作开发了一种基于点击化学反应和超支化杂交链式反应(HB-HCR)的多功能荧光平台,并结合DNA步行多重扩增技术,对Cu2+进行了超灵敏检测
发明内容:
本发明的目的之一提供一种新型的硅球上超支化放大的荧光信号探针;以及所述荧光信号探针的制备方法及其检测Cu2+的分析应用。
具体包括以下步骤:
步骤1.基于DNA步行器的放大过程:10μL磁珠(MB)、80μLDNA酶(1μM)、4μL S1(1μM)混合后在37℃下温育12h,磁分离得到的MB-DNA分散到80μL PBS(0.1M,pH 7.4)。然后取8μLS2(1μM)、不同浓度的Cu2+、1μL AA(1μM),37℃下温育90min。离心后分散到50μL PBS,加入6μL Mg2+(10μM),37℃下温育4h。分离后得到大量的DNA(S3)。
步骤2.超支化放大的荧光信号探针的制备用于Cu2+的放大荧光检测:将DNA(S3)和SiO2-H1均匀混合,37℃下温育1.5h,离心除去多余的S3。将H2(50μL0.1μM)加到上述SiO2-H1-S3混合物中温育120分钟,多余的H2离心除去。将50μL 5.0×10-7M SH DNA加到上述混合物中温育120分钟进行杂交反应。再将50μL 5.0×10-7M H3(Cy5-BHQ2)加到上述混合物中温育120min,然后将50μLAS1(5.0×10-7M)加到上述混合物中温育120分钟进行杂交反应。LT和LA温育反应1h获得LT/LA。50μL 5.0×10-7M LT/LA加到上述混合物中温育90分钟进行杂交反应。最后,50μL 5.0×10-7M的AS2加到上述混合物进行HB-HCR反应90分钟。离心除去多余的DNA,沉淀分散到50μL超纯水中,进行荧光检测。
附图说明:
图1(A)基于目标Cu2+引发的点击化反应和步行器放大技术产生DNA S3,(B)二氧化硅微球上超支化放大的荧光探针检测目标Cu2+的原理图。
图2电泳表征:DNA超支化放大的荧光系统构建过程。
图3(A))二氧化硅微球的SEM图像;(B)二氧化硅微球的TEM图像;(C)SiO2微球表面树枝状DNA结构的TEM图像;(D)HB-HCR形成的树枝状DNA的TEM图像。
图4(A)不同浓度目标Cu2+对应的荧光信号。(B)荧光信号变化和Cu2+浓度的关系,插图:测定Cu2+的矫正曲线。
实施例1.超支化放大的荧光信号探针制备及对Cu2+的检测。
基于DNA步行器对Cu2+的放大过程:
10μL磁珠(MB)、80μLDNA酶(1μM)、4μL S1(1μM)混合后在37℃下温育12h,磁分离得到的MB-DNA分散到80μL PBS(0.1M,pH 7.4)。然后取8μL S2(1μM)、一定浓度的、1μLAA(1μM),37℃下温育90min。离心后分散到50μL PBS,加入6μL Mg2+(10μM),37℃下温育4h。分离后得到大量的DNA(S3)。
超支化放大的荧光信号探针的制备用于Cu2+的放大荧光检测:
将DNA(S3)和SiO2-H1均匀混合,37℃下温育1.5h,离心除去多余的S3。将H2(50μL0.1μM)加到上述SiO2-H1-S3混合物中温育120分钟,多余的H2离心除去。将50μL 5.0×10-7MSH加到上述混合物中温育120分钟进行杂交反应。再将50μL 5.0×10-7M H3(Cy5-BHQ2)加到上述混合物中温育120min,然后将50μLAS1(5.0×10-7M)加到上述混合物中温育120分钟进行杂交反应。LT和LA温育反应1h获得LT/LA。50μL 5.0×10-7M LT/LA加到上述混合物中温育90分钟进行杂交反应。最后,50μL 5.0×10-7MAS2加到上述混合物进行HB-HCR反应90分钟。离心除去多余的DNA,沉淀分散到50μL超纯水中,进行荧光检测。
实施例2.超支化放大的荧光信号探针制备及对Cu2+的检测。
将“然后取8μL S2(1μM)、一定浓度的、1μLAA(1μM),37℃下温育90min。”改为“然后取8μL S2(1μM)、一定浓度的、1μLAA(1μM),37℃下温育100min。”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的荧光信号探针。对Cu2+检测的结果同实施例1。
实施例3.超支化放大的荧光信号探针制备及对Cu2+的检测。
将“离心后分散到50μLPBS,加入6μLMg2+(10μM),37℃下温育4h。”改为“离心后分散到50μLPBS,加入6μLMg2+(10μM),37℃下温育3h。”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的荧光信号探针。对Cu2+检测的结果同实施例1。
实施例4.超支化放大的荧光信号探针制备及对Cu2+的检测。
将“将DNA(S3)和SiO2-H1均匀混合,37℃下温育1.5h,离心除去多余的S3。”改为“将DNA(S3)和SiO2-H1均匀混合,37℃下温育2h,离心除去多余的S3。”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的荧光信号探针。对Cu2+检测的结果同实施例1。
实施例5.超支化放大的荧光信号探针制备及对Cu2+的检测。
将“将H2(50μL 0.1μM)加到上述SiO2-H1-S3混合物中温育120分钟,多余的H2离心除去。”改为“将H2(50μL 0.1μM)加到上述SiO2-H1-S3混合物中温育100分钟,多余的H2离心除去。”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的荧光信号探针。对Cu2+检测的结果同实施例1。
Claims (2)
1.一种新型的硅球上超支化放大的荧光信号探针,其特征是:利用点击化学反应引发的步行器放大技术结合超支化杂交链式反应(HB-HCR)构建了树枝状扩增的荧光平台,实现了对Cu2+的灵敏检测。目标Cu2+首先引发点击化学反应和DNA步行器放大过程,将Cu2+转化为大量的DNA S3产物。通过DNA S3与SiO2微球上H1杂交反应,引发了发夹H2、HS-DNA、H3-DNA和LT-DNA之间的超支化杂交链式反应(HB-HCR),组装成新型的树枝状DNA结构,负载大量的荧光Cy5,实现了对Cu2+放大信号的超灵敏荧光检测。
2.一种制备权利要求1所述的硅球上超支化放大的荧光信号探针的方法和应用,其特征方法由下列步骤组成:
步骤1.基于DNA步行器的放大过程:10μL磁珠(MB)、80μLDNA酶(1μM)、4μL S1(1μM)混合后在37℃下温育12h,磁分离得到的MB-DNA分散到80μL PBS(0.1M,pH 7.4)。然后取8μL S2(1μM)、不同浓度的Cu2+、1μL AA(1μM),37℃下温育90min。离心后分散到50μL PBS,加入6μLMg2+(10μM),37℃下温育4h。分离后得到大量的DNA(S3)。
步骤2.超支化放大的荧光信号探针的制备用于Cu2+的放大荧光检测:将DNA(S3)和SiO2-H1均匀混合,37℃下温育1.5h,离心除去多余的S3。将H2(50μL 0.1μM)加到上述SiO2-H1-S3混合物中温育120分钟,多余的H2离心除去。将50μL 5.0×10-7M SH加到上述混合物中温育120分钟进行杂交反应。再将50μL 5.0×10-7M H3(Cy5-BHQ2)加到上述混合物中温育120min,然后将50μLAS1(5.0×10-7M)加到上述混合物中温育120分钟进行杂交反应。LT和LA温育反应1h获得LT/LA。50μL 5.0×10-7M LT/LA加到上述混合物中温育90分钟进行杂交反应。最后,50μL 5.0×10-7M AS2加到上述混合物进行HB-HCR反应90分钟。离心除去多余的DNA,沉淀分散到50μL超纯水中,进行荧光检测。
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CN113358866A (zh) * | 2021-04-22 | 2021-09-07 | 四川大学华西医院 | 基于三重并联杂交链式反应的破伤风抗原的均相超灵敏二维可视化和荧光分析方法及应用 |
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Non-Patent Citations (5)
Title |
---|
LI ZOU ET AL: "Hybridization chain reaction and DNAzyme-based dual signal amplification strategy for sensitive colorimetric sensing of acetylcholinesterase activity and inhibitor screening in rat blood", 《SENSORS AND ACTUATORS B》 * |
MIN QING ET AL: "Click Chemistry Reaction-Triggered 3D DNA Walking Machine for sensitive Electrochemical Detection of Copper ion", 《ANALYTICAL CHEMISTRY》 * |
SAI BI ET AL: "Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine", 《CHEM.SOC.REV.》 * |
SAI BI ET AL: "Hyperbranched Hybridization Chain Reaction for Triggered Signal Amplification and Concatenated Logic Circuits", 《ANGEW.CHEM.INT.ED.》 * |
SEAN D.MASON ET AL: "Emerging bioanalytical applications of DNA walkers", 《TRENDS IN ANALYTICAL CHEMISTRY》 * |
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Application publication date: 20200421 |