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

doi:10.12211/2096-8280.2021-050

Abstract

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 (K_m^app) 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

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，K_m^app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R_et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

CLC Number:

Q 816

Xiaosheng LIANG, Yongchao GUO, Dong MEN, Xian’en ZHANG. Hybrid systems of virus and nano-gold conducting networks for electrochemical analysis[J]. Synthetic Biology Journal, 2022, 3(2): 415-427.

梁晓声, 郭永超, 门冬, 张先恩. 病毒-纳米金杂合导电网络结构在电化学分析的应用[J]. 合成生物学, 2022, 3(2): 415-427.

Figures/Tables 11

Fig. 1 Schematic diagram for developing the enzyme-phage-gold complex

Tab. 1 Primers for constructing of recombinant phage

引物名称	序列（5'→3'）
6264mS	CGCCAAGCTTGCATGCCGCAGGTCCTC
6264dn	GATAGCCTTTGTAGATCTCTC
1381up	GGCATTACGTATTTTACCC
1381S	CTTTCGCTGCAGAGGGTGAGGATC
1381A	CTTTTGCGGGATCCTCACCCTCTGC
1381dn	GCTATTAATTAATTTTCCC
GbS	GTATCGGGTTCTTCTCCTGATTCT
GbA	GATCAGAATCAGGAGAAGAACCCGATACTGCA

Fig. 2 Construction of gold-binding peptide displaying phage and its verificationM—Takara DL15000 DNA ladder. Lane (a) 1—Site 6264 mutated phage genome digested by PstⅠand BglⅡ. Lane (b) 1—Sites 1372 and 1381 mutated phage genome digested by PstⅠ and PacⅠ; 2—Sites 1372 and 1381 mutated phage genome digested by BamHⅠ and PacⅠ. Lane (c) 1—Gold-binding peptide sequence inserted phage genome digested by PstⅠ and PacⅠ; 2—Gold-binding peptide sequence inserted phage genome digested by BamHⅠ and PacⅠ

Fig. 3 Evaluation of gold-binding ability for GM M13 with silver enhancement(1010 GM M13 and 109 GM M13 refer to the genetically modified M13 with the titer of 1010 PFU and 109 PFU, respectively, and the silver enhancement GM M13 without gold nanoparticles is used as the control.)

Fig. 4 AFM and TEM images for the Au-GM M13 complex formed under different reaction conditions(a) AFM images for 0.25 mmol/L HAuCl4, 1012 PFU/mL GM M13, 20 mmol/L Gly-Cl- and 2.5 mmol/L NaBH4; (b) AFM images for 0.375 mmol/L HAuCl4, 1012 PFU/mL GM M13, 20 mmol/L Gly-Cl- and 2.5 mmol/L NaBH4; (c) AFM images for 0.5 mmol/L HAuCl4, 1012 PFU/mL GM M13, 20 mmol/L Gly-Cl- and 2.5 mmol/L NaBH4; (d) TEM image for the Au-GM M13 complex. Complex forming conditions: 0.5 mmol/L HAuCl4, 1012 PFU/mL GM M13, 20 mmol/L Gly-Cl- and 2.5 mmol/L NaBH4

Fig. 5 Voltammograms of the HRP/Au-GM M13 electrode without (a) and with chitosan modifications (b)[Scan rate: 50 mV/s; buffer: PBS (10 mmol/L, pH 7.0) solution containing 0.1 mol/L KCl and 2 mmol/L [Fe(CN)6]3-/4-.]

Fig. 6 Voltammograms of the HRP/Au-GM M13 electrode modified with chitosan at different concentrations of hydrogen peroxide[Scan rate: 20 mV/s for N2-saturated PBS (0.1 mol/L, pH 7.0). The curve peaks were obtained in response to H2O2 concentrations from 2.5 μmol/L to 640 μmol/L. The embedded image shows the calibration curve derived from the curves.]

Fig. 7 Voltammograms of the HRP/Au-GM M13 electrode modified with chitosan at different concentrations of hydrogen peroxide from 640 μmol/L to 60 mmol/L[Scan rate: 20 mV/s for N2-saturated PBS (0.1 mol/L, pH 7.0). The embedded image shows the calibration curve derived from the curves.]

Tab. 2 Comparison of the analytical performance of enzyme-phage-gold modified electrode with other electrochemical biosensors

酶电极修饰方法	检测限	K_m^app	线性范围	来源
Cerasomes with AuNPs@Poly(Ionic Liquid)s	3.3 μmol/L	—	10～70 μmol/L	[22]
Silicahydroxyapatite hybrid film	0.35 μmol/L	21.8 μmol/L	1.0～100 μmol/L	[23]
Methanobactin functionalized gold nanoparticles	—	0.787 mmol/L	52.9 μmol/L～0.64 mmol/L	[24]
Gold nanoparticles on indium/tin oxide electrode	2 μmol/L	0.4 mmol/L	8.0 μmol/L～3.0 mmol/L	[25]
Cerasome	0.83 mmol/L	—	2.5～325 mmol/L	[26]
Carbon nanotubes	0.1 μmol/L	—	0.3～200 μmol/L	[21]
纳米金-噬菌体复合物	0.32 μmol/L	0.3 mmol/L	2.5 μmol/L～60 mmol/L	本研究

Fig. 8 Comparison of voltammograms for the electrode responses to increased H2O2 concentrations[The measurement was performed in the PBS without oxygen. HRP/Au-GM M13 electrode with (a) and without (b) chitosan modification, and HRP/Au nanoparticles electrode with (c) and without (d) chitosan modification]

Fig. 9 Nyquist diagram (Z" vs Z') for Faradaic impedance spectra in the PBS (10 mmol/L, pH 7.0) containing 0.1 mol/L KCl and 2 mmol/L [Fe(CN)6]3-/4-a—HRP/Au-GM M13 electrode modified with chitosan; b—HRP/Au nanoparticles electrode modified with chitosan modified electrode; c—HRP electrode modified with chitosan; d—the glass-carbon electrode without the modification

References 31

1	LUO H, SHI Z, LI N, et al. Investigation of the electrochemical and electrocatalytic behavior of single-wall carbon nanotube film on a glassy carbon electrode[J]. Analytical Chemistry, 2001, 73(5): 915-920.
2	HU M L, ABBASI-AZAD M, MORSALI A, et al. Electrochemical applications of ferrocene-based coordination polymers[J]. Chempluschem, 2021, 85(11): 2397-2418.
3	HUANG X C, ZHANG J R, ZHANG L L, et al. A sensitive H₂O₂ biosensor based on carbon nanotubes/tetrathiafulvalene and its application in detecting NADH[J]. Analytical Biochemistry, 2020, 589: 113493.
4	ITO K, OKUDA-SHIMAZAKI J, KOJIMA K, et al. Strategic design and improvement of the internal electron transfer of heme b domain-fused glucose dehydrogenase for use in direct electron transfer-type glucose sensors[J]. Biosensors and Bioelectronics, 2021, 176: 112911.
5	ZHAO M, GAO Y, SUN J Y, et al. Mediatorless glucose biosensor and direct electron transfer type glucose/air biofuel cell enabled with carbon nanodots[J]. Analytical Chemistry, 2015, 87(5): 2615-2622.
6	袁盛建, 马迎飞. 噬菌体合成生物学研究进展和应用[J]. 合成生物学, 2020, 1(6): 635-655.
	YUAN S J, MA Y F. Advances and applications of phage synthetic biology[J]. Synthetic Biology Journal, 2020, 1(6): 635-655.
7	GUO Y C, ZHOU Y F, ZHANG X E, et al. Phage display mediated immuno-PCR[J]. Nucleic Acids Research, 2006, 34(8): e62.
8	SMITH G P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface[J]. Science, 1985, 228(4705): 1315-1317.
9	KEHOE J W, KAY B K. Filamentous phage display in the new millennium[J]. Chemical Reviews, 2005, 105(11): 4056-4072.
10	SMITH G P, PETRENKO V A. Phage display[J]. Chemical Reviews, 1997, 97(2): 391-410.
11	OH D, QI J, LU Y C, et al. Biologically enhanced cathode design for improved capacity and cycle life for lithium-oxygen batteries[J]. Nature Communications, 2013, 4: 2756.
12	LEE Y J, YI H, KIM W J, et al. Fabricating genetically engineered high-power lithium-ion batteries using multiple virus genes[J]. Science, 2009, 324(5930): 1051-1055.
13	LEE S W, MAO C B, FLYNN C E, et al. Ordering of quantum dots using genetically engineered viruses[J]. Science, 2002, 296(5569): 892-895.
14	张文静, 李明, 周维, 等. 基于病毒组件的纳米材料的自组装合成、功能化及应用[J]. 合成生物学, 2020, 1(3): 298-318.
	ZHANG W J, LI M, ZHOU W, et al. Self-assembly, biosynthesis, functionalization and applications of virus-based nanomaterials[J]. Synthetic Biology Journal, 2020, 1(3): 298-318.
15	ZHANG J T, KANKALA R K, MA J Y, et al. Hollow tobacco mosaic virus coat protein assisted self-assembly of one-dimensional nanoarchitectures[J]. Biomacromolecules, 2021, 22(2): 540-545.
16	ZHANG W J, ZHANG X N, LI F. Virus-based nanoparticles of Simian virus 40 in the field of nanobiotechnology[J]. Biotechnology Journal, 2018, 13(6): 1700619.
17	ZHANG S, YU H M, YANG J, et al. Design of the nanoarray pattern Fe-Ni bi-metal nanoparticles@M13 virus for the enhanced reduction of p-chloronitrobenzene through the micro-electrolysis effect[J]. Environmental Science: Nano, 2017, 4(4): 876-885.
18	YOO P J, NAM K T, QI J, et al. Spontaneous assembly of viruses on multilayered polymer surfaces[J]. Nature Materials, 2006, 5(3): 234-240.
19	CHO W, FOWLER J D, FURST E M, Targeted binding of the M 13 bacteriophage to thiamethoxam organic crystals[J]. Langmuir, 2012, 28(14): 6013-6020.
20	HUANG Y, CHIANG C Y, LEE S K, et al. Programmable assembly of nanoarchitectures using genetically engineered viruses[J]. Nano Letters, 2005, 5(7): 1429-1434.
21	YAMAMOTO K, SHI G Y, ZHOU T S, et al. Study of carbon nanotubes-HRP modified electrode and its application for novel on-line biosensors[J]. The Analyst, 2003, 128(3): 249-254.
22	LIU D L, WU Q, ZOU S, et al. Surface modification of cerasomes with AuNPs@poly(ionic liquid)s for an enhanced stereo biomimetic membrane electrochemical platform[J]. Bioelectrochemistry, 2020, 132: 107411.
23	WANG B, ZHANG J J, PAN Z Y, et al. A novel hydrogen peroxide sensor based on the direct electron transfer of horseradish peroxidase immobilized on silica-hydroxyapatite hybrid film[J]. Biosensors and Bioelectronics, 2009, 24(5): 1141-1145.
24	XIN J Y, DOU B X, WANG Z X, et al. Direct electrochemistry of methanobactin functionalized gold nanoparticles on Au electrode[J]. Journal of Nanoscience and Nanotechnology, 2018, 18(7): 4805-4813.
25	WANG J W, WANG L P, DI J W, et al. Electrodeposition of gold nanoparticles on indium/tin oxide electrode for fabrication of a disposable hydrogen peroxide biosensor[J]. Talanta, 2009, 77(4): 1454-1459.
26	QIAO Y, TAHARA K, ZHANG Q, et al. Cerasomes: soft interface for redox enzyme electrochemical signal transmission[J]. Chemistry - A European Journal, 2016, 22(4): 1340-1348.
27	HAMES B D, HOOPER N M, HOUGHTON J D. Instant Notes in Biochemistry [M]. London: Taylor & Francis, 1997: 67.
28	ZHANG H L, LAI G S, HAN D Y, et al. An amperometric hydrogen peroxide biosensor based on immobilization of horseradish peroxidase on an electrode modified with magnetic dextran microspheres[J]. Analytical and Bioanalytical Chemistry, 2008, 390(3): 971-977.
29	MARCUS R A, SUTIN N. Electron transfers in chemistry and biology[J]. Biochimica et Biophysica Acta, 1985, 811(3): 265-322.
30	CHEN H, JIANG J H, HUANG Y, et al. An electrochemical impedance immunosensor with signal amplification based on Au-colloid labeled antibody complex[J]. Sensors and Actuators B: Chemical, 2006, 117(1): 211-218.
31	HLELI S, MARTELET C, ABDELGHANI A, et al. An immunosensor for haemoglobin based on impedimetric properties of a new mixed self-assembled monolayer[J]. Materials Science and Engineering: C, 2006, 26(2/3): 322-327.

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