Construction of an autonomously concatenated hybridization chain reaction for signal amplification and intracellular imaging,Chemical Science

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Construction of an autonomously concatenated hybridization chain reaction for signal amplification and intracellular imaging
Chemical Science ( IF 7.6 ) Pub Date : 2017-10-23 00:00:00 , DOI: 10.1039/c7sc03939e
Jie Wei _{1,

2,

3,

4,

5} , Xue Gong _{1,

2,

3,

4,

5} , Qing Wang _{1,

2,

3,

4,

5} , Min Pan _{1,

2,

3,

4,

5} , Xiaoqing Liu _{1,

2,

3,

4,

5} , Jing Liu _{4,

5,

6,

7,

8} , Fan Xia _{9,

10,

11,

12,

13} , Fuan Wang _{1,

2,

3,

4,

5}

Affiliation

Biomolecular self-assembly has spurred substantial research efforts for the development of low-cost point-of-care diagnostics. Herein, we introduce an isothermal enzyme-free concatenated hybridization chain reaction (C-HCR), in which the output of the upstream hybridization chain reaction (HCR-1) layer acts as an intermediate input to activate the downstream hybridization chain reaction (HCR-2) layer. The initiator motivates HCR-1 through the autonomous cross-opening of two functional DNA hairpins, yielding polymeric dsDNA nanowires composed of numerous tandem triggers T as output of the primary sensing event. The reconstituted amplicon T then initiates HCR-2 and transduces the analyte recognition into an amplified readout, originating from the synergistic effect between HCR-1 and HCR-2 layers. Native gel electrophoresis, atom force microscopy (AFM) and fluorescence spectra revealed that C-HCR mediated the formation of frond-like branched dsDNA nanowires and the generation of an amplified FRET signal. As a versatile and robust amplification strategy, the unpreceded C-HCR can discriminate DNA analyte from its mutants with high accuracy and specificity. By incorporating an auxiliary sensing module, the integrated C-HCR amplifier was further adapted for highly sensitive and selective detection of microRNA (miRNA), as a result of the hierarchical and sequential hybridization chain reactions, in human serum and even living cells through an easy-to-integrate “plug-and-play” procedure. In addition, the C-HCR amplifier was successfully implemented for intracellular miRNA imaging by acquiring an accurate and precise signal localization inside living cells, which was especially suitable for the ex situ and in situ amplified detection of trace amounts of analyte. The C-HCR amplification provides a comprehensive and smart toolbox for highly sensitive detection of various biomarkers and thus should hold great promise in clinical diagnosis and assessment. The infinite layer of multilayered C-HCR is anticipated to further strengthen the amplification capacity and reliability (anti-invasion performance) of intracellular imaging approach, which is of great significance for its bioanalytical applications.

中文翻译：

用于信号放大和细胞内成像的自主级联杂交链反应的构建

生物分子自组装激发了大量的研究努力，以开发低成本的即时诊断服务。在本文中，我们介绍了一种无等温酶的串联杂交链反应（C-HCR），其中上游杂交链反应（HCR-1）层的输出充当中间输入，以激活下游杂交链反应（HCR- 2）层。引发剂通过两个功能性DNA发夹的自动交叉开放来激发HCR-1，从而产生由大量串联触发器T组成的聚合dsDNA纳米线，作为主要传感事件的输出。重组扩增子T然后启动HCR-2，并将分析物识别转换为放大的读数，该读数源自HCR-1和HCR-2层之间的协同效应。天然凝胶电泳，原子力显微镜（AFM）和荧光光谱表明，C-HCR介导了叶状分支dsDNA纳米线的形成和FRET信号的放大。作为一种通用且强大的扩增策略，无与伦比的C-HCR可以以较高的准确性和特异性将DNA分析物与其突变体区分开。通过集成辅助传感模块，集成的C-HCR放大器进一步适应了人类血清甚至活细胞中的分层和顺序杂交链反应，从而高度敏感和选择性地检测了microRNA（miRNA）。 -集成“即插即用”过程。非原位和原位扩增痕量分析物的检测。C-HCR扩增为各种生物标记物的高灵敏度检测提供了一个全面而智能的工具箱，因此在临床诊断和评估中应具有广阔的前景。多层C-HCR的无限层有望进一步增强细胞内成像方法的扩增能力和可靠性（抗侵袭性能），这对其生物分析应用具有重要意义。

更新日期：2017-11-13

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