JSTA  >> Vol. 5 No. 4 (October 2017)

    基于滚环扩增技术耦合切刻内切酶信号放大技术的新型超灵敏miRNA-122荧光传感器
    A Novel Fluorescent Biosensor for Ultrasensitive Detection of miRNA-122 Based on Rolling Circle Amplification Coupled with Nicking Enzyme Signal Amplification

  • 全文下载: PDF(1325KB) HTML   XML   PP.53-62   DOI: 10.12677/JSTA.2017.54007  
  • 下载量: 39  浏览量: 67  

作者:  

王煦:山东省黄河计量研究院,山东 济南;
徐成功,王敬峰,王玉:济南大学生物科学与技术学院,山东 济南

关键词:
滚环扩增技术切刻内切酶信号放大荧光传感器miRNA-122Rolling Circle Amplification Nicking Endonuclease Signal Amplification Fluorescence Biosensor MiRNA-122

摘要:
MicroRNA-122 (miRNA-122)的异常表达与肝癌的发生、发展及转移紧密相关,发展简单、快速、高灵敏和高特异性的miRNA-122检测方法对于肝癌的早期诊断和预后评估具有非常重要的意义。本文报道了一种基于滚环扩增技术(RCA)耦合切刻内切酶信号放大(NESA)技术的新型荧光传感器并用于超灵敏和高选择性检测miRNA-122。该方法涉及了两个阶段的反应机制,第一阶段,目标物激活切刻内切酶辅助的聚合反应,用以实现目标物与次级目标物循环放大以及生成RCA引物;第二阶段,利用RCA耦合NESA反应,实现信号传导以及多重信号放大。实验结果证明,该传感器表现出对miRNA-122的非常高的灵敏度和特异性,检测下限达到3.9 aM,较之前报道的方法,灵敏度有明显的提高。另外,该方法整个过程只需一步反应,具有操作简便、成本低、检测时间短的优势。所以,所提出的基于RCA耦合NESA的荧光传感器有望建立一个简单而实用的生物传感新平台,并应用于miRNA检测以及癌症的早期诊断和预后评估。

Aberrant expression of microRNA-122 (miRNA-122) is associated with the occurrence, develop-ment and metastasis of hepatocellular carcinoma. It’s of great importance for the development of sensitive and selective method for the detection of miRNA-122 to the early diagnosis and prognosis of hepatocellular carcinoma. A novel fluorescent biosensor has been constructed for ultrasensitive and high specific detection of miRNA-122 based on rolling circle amplification (RCA) coupled with nicking endonuclease signal amplification (NESA). Our assay involves of two stages of reaction mechanism, the first stage is target-activated nicking endonuclease-assisted polymerization, which is used for the realization of the recycle amplification of target and secondary target and the produce of RCA primer. The second stage is to achieve signal transduction and multiple signal amplification by the utilization of RCA coupled with NESA. The results reveal the constructed biosensor display high sensitivity and specificity for miRNA-122 detection, and the limit of detection is as low as 3.9 aM, which is significantly improved compared to the previous methods. Moreover, the proposed method has the advantages of rapidness, low cost, simplicity with only one-step operation. Therefore, the developed RCA coupled with NESA-based fluorescent biosensor might create a simple and practical platform for the detection of miRNA and the early diagnosis and prognosis of cancer.

文章引用:
王煦, 徐成功, 王敬峰, 王玉. 基于滚环扩增技术耦合切刻内切酶信号放大技术的新型超灵敏miRNA-122荧光传感器[J]. 传感器技术与应用, 2017, 5(4): 53-62. https://doi.org/10.12677/JSTA.2017.54007

参考文献

[1] Jopling, C.L., Norman, K.L. and Sarnow, P. (2006) Positive and Negative Modulation of Viral and Cellular mRNAs by Liver-Specific microRNA miRNA-122. Cold Spring Harbor Symposia on Quantitative Biology, 71, 369-376.
https://doi.org/10.1101/sqb.2006.71.022
[2] Shu, X.L., Fan, C.B., Long, B., Zhou, X. and Wang, Y. (2016) The Anti-Cancer Effects of Cisplatin on Hepatic Cancer Are Associated with Modulation of miRNA-21 and miRNA-122 Expression. European Review for Medical and Pharmacological Sciences, 20, 4459-4465.
[3] Bandopadhyay, M., Sarkar, N., Datta, S. and Das, D. (2016) A Pal Hepatitis B virus X Protein Mediated Suppression of miRNA-122 Expression Enhances Hepatoblastoma Cell Proliferation through Cyclin G1-p53 Axis. Infectious Agents and Cancer, 11, 40.
https://doi.org/10.1186/s13027-016-0085-6
[4] Girard, M., Jacquemin, E., Munnich, A., et al. (2008) miRNA-122, a Paradigm for the Role of microRNA in the Liver. Journal of Hepatology, 48, 648-659.
https://doi.org/10.1016/j.jhep.2008.01.019
[5] Tsai, W.C., Hsu, P.W., Lai, T.C., et al. (2009) MicroRNA-122, A Tumor Suppressor microRNA that Regulates Intrahepatic Metastasis of Hepatocellular Carcinoma. Hepatology, 49, 1571-1582.
https://doi.org/10.1002/hep.22806
[6] Dallman, M.J., Porter, A.C., Larsen, C.P., et al. (1989) Lymphokine Production in Al-lografts-Analysis of RNA by Northern Blotting. Transplantation Proceedings, 21, 296-298.
[7] Kwok, S. and Higuchi, R. (1989) Avoiding False Positives with PCR. Nature, 339, 237-238.
[8] Zhou, H., Chen, Q., Tan, W. Qiu, Z. and Li, S. (2017) Integrated Clinicopathological Features and Gene Microarray Analysis of Pancreatic Neuroendocrine Tumors. Gene, 625, 72-77.
https://doi.org/10.1016/j.gene.2017.05.009
[9] Mori, Y. and Notomi, T. (2009) Loop-Mediated Isothermal Amplification (LAMP): A Rapid, Accurate, and Cost-Effective Diagnostic Method for Infectious Diseases. Journal of Infection and Chemotherapy, 15, 62-69.
https://doi.org/10.1007/s10156-009-0669-9
[10] Zhang, Y., Yang, L., Lin, C., Guo, L. et al. (2015) Fluorescence Aptasensor for Ochratoxin A in the Food Samples Based on Hyperbranched Rolling Circle Amplification. Analytical Methods, 53, 250-252.
https://doi.org/10.1039/C5AY01182E
[11] Brasino, M.D. and Cha, J.N. (2015) Isothermal Rolling Circle Amplification of Virus Genomes for Rapid Antigen Detection and Typing. Analyst, 140, 5138-5144.
https://doi.org/10.1039/C5AN00721F
[12] Lee, S.J., Cho, Y.H., Kim, C.S., et al. (2004) Screening for Chlamydia and Gonorrhea by Strand Displacement Amplification in Homeless Adolescents Attending Youth Shelters in Korea. Journal of Korean Medical Science, 19, 495-500.
https://doi.org/10.3346/jkms.2004.19.4.495
[13] Wen, Y.Q., Xu, Y., Mao, X.H., Wei, Y.L., Song, H.Y., Chen, N., Huang, Q., Fan, C.H. and Li, D. (2012) DNAzyme-Based Rolling-Circle Amplification DNA Machine for Ultrasensitive Analysis of MicroRNA in Drosophila Larva. Analytical Chemistry, 84, 7664-7669.
https://doi.org/10.1021/ac300616z
[14] Zhao, Y.X., Chen, F., Li, Q., Wang, L.H. and Fan, C.H. (2015) Isothermal Amplification of Nucleic Acids. Chemical Reviews, 115, 12491-12545.
https://doi.org/10.1021/acs.chemrev.5b00428
[15] Wang, M., Fu, Z.L., Li, B.C., Zhou, Y.L., Yin, H.S. and Ai, S.Y. (2014) One-Step, Ultrasensitive, and Electrochemical Assay of MicroRNAs Based on T7 Exonuclease Assisted Cyclic Enzymatic Amplifi-cation. Analytical Chemistry, 86, 5606-5610.
https://doi.org/10.1021/ac5010376
[16] Shi, X.M., Fan, G.C., Shen, Q.M. and Zhu, J.J. (2016) Photoelectrochemical DNA Biosensor Based on Dual-Signal Amplification Strategy Integrating Inorganic-Organic Na-nocomposites Sensitization with λ-Exonuclease-Assisted Target Recycling. ACS Applied Material & Interfaces, 8, 35091-35098.
https://doi.org/10.1021/acsami.6b14466
[17] Deng, R.J., Zhang, K.X. and Li, J.H. (2017) Isothermal Amplification for Mi-croRNA Detection: From the Test Tube to the Cell. Accounts of Chemical Research, 50, 1059-1068.
https://doi.org/10.1021/acs.accounts.7b00040
[18] Zhuang, J.Y., Lai, W.Q., Xu, M.D., Zhou, Q. and Tang, D.P. (2015) Plas-monic AuNP/g-C3N4 Nanohybrid-Based Photoelectrochemical Sensing Platform for Ultrasensitive Monitoring of Polynucleotide Kinase Activity Accompanying Dnazyme-Catalyzed Precipitation Amplification. ACS Applied Material & Interfaces, 7, 8330-8338.
https://doi.org/10.1021/acsami.5b01923
[19] Qian, Y., Fan, T.T., Wang, P., Zhang, X., Luo, J.J., Zhou, F.Y., et al. (2017) A Novel Label-Free Homogeneous Electrochemical Immunosensor Based on Proximity Hybridization-Triggered Isothermal Exponential Amplification Induced G-Quadruplex Formation. Sensors and Actuators B: Chemical, 248, 187-194.
https://doi.org/10.1016/j.snb.2017.03.152
[20] Yang, C.-T., Pourhassan-Moghaddam, M., Wu, L., Bai, P. and Thierry, B. (2017) Ultrasensitive Detection of Cancer Prognostic miRNA Biomarkers Based on Surface Plasmon Enhanced Light Scattering. ACS Sensors, 2, 635-640.
https://doi.org/10.1021/acssensors.6b00776
[21] Lv, S.F., Chen, F., Chen, C.Y., Chen, X.M., Gong, H. and Cai, C.Q. (2017) A Novel CdTe Quantum Dots Probe Amplified Resonance Light Scattering Signals to Detect MicroRNA-122. Talanta, 165, 659-663.
https://doi.org/10.1016/j.talanta.2017.01.020
[22] Bi, S., Chen, M., Jia, X.Q. and Dong, Y. (2015) A Hot-Spot-Active Magnetic Graphene Oxide Substrate for MicroRNA Detection Based on Cascaded Chemiluminescence Resonance Energy Transfer. Nanoscale, 7, 3745-3753.
https://doi.org/10.1039/C4NR06603K
[23] Bi, S., Yue, S.Z., Song, W.L. and Zhang, S.S. (2016) A Target-Initiated DNA Net-work Caged on Magnetic Particles for Amplified Chemiluminescence Resonance Energy Transfer Imaging of MicroRNA and Targeted Drug Delivery. Chemical Communication, 52, 12841-12844.
https://doi.org/10.1039/C6CC05187A