合成生物学 ›› 2022, Vol. 3 ›› Issue (2): 415-427.doi: 10.12211/2096-8280.2021-050

• 研究论文 • 上一篇    

病毒-纳米金杂合导电网络结构在电化学分析的应用

梁晓声1(), 郭永超2, 门冬2,4, 张先恩3,4   

  1. 1.中南民族大学生命科学学院,湖北 武汉 430074
    2.中国科学院武汉病毒研究所,生物安全大科学研究中心,病毒学国家重点实验室,湖北 武汉 430071
    3.中国科学院生物物理研究所,生物大分子科教融合卓越中心,生物大分子国家重点实验室,北京 100101
    4.中国科学院大学,北京 100049
  • 收稿日期:2021-04-26 修回日期:2021-11-14 出版日期:2022-04-30 发布日期:2022-05-11
  • 通讯作者: 梁晓声 E-mail:liangxs@mail.scuec.edu.cn
  • 作者简介:梁晓声(1984—),男,博士,讲师。研究方向为纳米生物技术、纳米材料检测应用等。 E-mail:liangxs@mail.scuec.edu.cn
  • 基金资助:
    国家重点研发计划(2019YFA0904800)

Hybrid systems of virus and nano-gold conducting networks for electrochemical analysis

Xiaosheng LIANG1(), Yongchao GUO2, Dong MEN2,4, Xian’en ZHANG3,4   

  1. 1.College of Life Science,South-Central University for Nationalities,Wuhan 430074,Hubei,China
    2.State Key Laboratory of Virology,Wuhan Institute of Virology,Center for Biosafety Mega-Science,Chinese Academy of Sciences,Wuhan 430071,Hubei,China
    3.National Laboratory of Biomacromolecules,CAS Center for Excellence in Biomacromolecules,Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China
    4.University of Chinese Academy of Sciences,Beijing 100049,China
  • Received:2021-04-26 Revised:2021-11-14 Online:2022-04-30 Published:2022-05-11
  • Contact: Xiaosheng LIANG E-mail:liangxs@mail.scuec.edu.cn

摘要:

本文利用噬菌体展示技术将金结合肽展示在噬菌体M13主要衣壳蛋白(gP8)之上,构建了金结合肽展示的基因改造噬菌体M13(GM M13),并将这种基因改造噬菌体作为矿化成核模板在其表面沉积金,得到金-基因改造噬菌体复合物。利用壳聚糖将金-基因改造噬菌体复合物与辣根过氧化物酶(HRP)包埋修饰到玻碳电极上用于过氧化氢检测。修饰电极对过氧化氢具有高灵敏响应,线性范围2.5 μmol/L~60 mmol/L,检测限为0.32 μmol/L(S/N=3)。HRP/纳米金-噬菌体复合物/壳聚糖修饰玻碳电极对底物信号响应符合Michaelis-Menten动力学方程,Kmapp值经计算为0.3 mmol/L,说明该电极对底物具有高亲和性及高灵敏度。交流阻抗测试表明,HRP/纳米金-噬菌体复合物/壳聚糖修饰电极Ret值显著小于HRP/金纳米颗粒/壳聚糖修饰电极和HRP/壳聚糖修饰电极,说明该电极更有利于电子传递。不同修饰电极对过氧化氢响应信号比较结果表明,金-基因改造噬菌体复合物构建的酶电极与纳米金修饰的同类酶电极相比具有更高的灵敏度,相同底物浓度下可获得数倍的电流信号提升。过氧化氢酶电极的示例证明,金-基因改造噬菌体复合物作为一种酶电极修饰材料可显著提高电极导电面积,增大酶有效固定位点,从而获得显著的信号增益。

关键词: 基因改造噬菌体, 金结合肽, 电化学分析, 生物传感

Abstract:

Genetically modifications of virus coating proteins are attracting broad interest due to their potentials for controllable and designable fabrication of biomaterials. This research aims to constructing a highly sensitive biosensor system by using phage display technology. Phage M13 was genetically modified to display a gold-binding peptide on every copy of its major coat protein (gP8). This genetically modified virus (GM M13) worked as nucleation seeds for gold deposition under proper chemical conditions. The TEM and AFM characterizations showed the resulting complex is a three dimensional network formed by mineralized gold nanowires. The nanoscale particles of the deposited gold on the phages were irregular in shape with a coarse surface. The resultant Au-GM M13 complex was co-immobilized with horseradish peroxidase (HRP) on a glass-carbon electrode by chitosan entrapping, which was employed to H2O2 analysis. The HRP/Au-GM M13 complex modified sensor showed high sensitivity to H2O2, and its responses were proportional to the substrate concentrations ranged from 2.5 μmol/L to 60 mmol/L with a detection limit of 0.32 μmol/L. Such a high sensitivity indicates that the virus-templated nano-gold conducting network exhibits improved electrochemical performance. The enzymatic reaction occurred on the HRP/Au-GM M13 complex modified electrode displayed Michaelis-Menten kinetics, and the apparent Michaelis-Menten constant (Kmapp) value was found to be 0.3 mmol/L, giving solid evidence for the higher affinity and sensitivity of the modified electrode to the substrate. The impedance spectroscopy characterization implied that the HRP/Au-GM M13 complex facilitates the electron transfer compared with enzyme-gold nanoparticles and the embedded enzyme as well, demonstrating its superiority in enzyme electrode modifications for the analysis of H2O2, The GM M13 could serve as a template to form gold nanowires for a multi-dimensional conducting gold network to entrap a large number of enzyme molecules, which helps enlarge the conducting area of the electrode, providing more effective binding sites for the enzyme. As a result, the response signal was increased several folds in detecting H2O2. The proposed method provides insights for developing a variety of electrochemical biosensors.

Key words: genetically modified bacteriophage, gold-binding peptide, electrochemical analysis, biosensor

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