以还原氧化石墨烯和纳米二氧化锆为DNA探针固定平台电化学测定转基因玉米中特定基因序列

doi:10.11944/j.issn.1000-0518.2019.07.180378

摘要/Abstract

摘要：

草胺膦乙酰转移酶基因(PAT)是一种转基因植物的外源DNA片段。本文以还原氧化石墨烯和纳米二氧化锆(nanoZrO₂)的复合物作为固定DNA探针的平台,建立了一种灵敏地检测PAT基因的方法。首先,氧化石墨烯直接在电极表面进行电化学还原,然后将一层nanoZrO₂涂覆于其表面,利用DNA中的磷酸基团与nanoZrO₂中氧的亲和作用固定DNA探针。通过微分脉冲伏安法检测DNA探针与PAT基因片段的杂交,构建了用于检测PAT基因片段的电化学生物传感器。该传感器具有稳定性好,重复性好的特点,可灵敏地检测转基因玉米中的PAT基因,检测限达2.0×10^-15 mol/L。

关键词: 微分脉冲伏安法, 还原氧化石墨烯, 纳米氧化锆, 草胺膦乙酰转移酶基因, 转基因玉米

Abstract:

The phosphinothricin acetyltransferase(PAT) gene is a kind of exogenous DNA segments indicating genetically modified plants. In this research, a sensitive method for determination of the PAT gene in transgenic maize has been developed using reduced graphene oxide and nano zirconia(nanoZrO₂) composites as a platform for immobilizing DNA probes. First, the graphene oxide was direct electrochemically reduced on a glassy carbon electrode(GCE); then, a layer of nanoZrO₂ was covered on the modified electrode. Last, the DNA probes were immobilized via the affinity between phosphate groups in the DNA probes and the oxygen in nanoZrO₂. The hybridization of the DNA probes with the PAT gene segments was monitored by differential pulse voltammetry(DPV). The sensor was stable, reproducible and sensitive to effectively discriminate the PAT gene in transgenic maize. The detection limit was 2.0×10^-15 mol/L.

Key words: differential pulse voltammetry, reduced graphene oxide, nano zirconia, phosphinothricin acetyltransferase gene, transgenic maize

王学亮, 王朝霞, 汪涛, 戴晓辉. 以还原氧化石墨烯和纳米二氧化锆为DNA探针固定平台电化学测定转基因玉米中特定基因序列[J]. 应用化学, 2019, 36(7): 839-846.

Xueliang WANG, Zhaoxia WANG, Tao WANG, Xiaohui DAI. Electrochemical Determination of the Specific Genetic Sequence in Transgenic Maize Using Reduced Graphene Oxide and Nano Zirconia Composites as a Platform for Immobilizing DNA[J]. Chinese Journal of Applied Chemistry, 2019, 36(7): 839-846.

参考文献

[1]	Tam P D. Genetically Modified Organism(GMO) Detection by Biosensor Based on SWCNT Material[J]. Curr Appl Phys,2015,15(3):397-401.
[2]	Deisingh A K,Badrie N.Detection Approaches for Genetically Modified Organisms in Foods[J]. Food Res Int,2005,38:639-649.
[3]	Arugula M A,Zhang Y,Simonian A L.Biosensors as 21st Century Technology for Detecting Genetically Modified Organisms in Food and Feed[J]. Anal Chem,2014,86(1):119-129.
[4]	European Commission.Regulation(EC) No.1830/2003 Concerning the Traceability and Labelling of Genetically Modified Organisms and the Traceability of Food and Feed Products Produced from Genetically Modified Organisms and Amending Directive 2001/18/EC[J]. Off J Eur Union,2003,268:L24-L28.
[5]	Ahmed F E.Detection of Genetically Modified Organism in Food[J]. Trends Biotechnol,2002,20(5):215-223.
[6]	Vollenhofer S,Burg K,Schmidt J,et al. Genetically Modified Organisms in Food Screening and Specific Detection by Polymerase Chain Reaction[J]. J Agric Food Chem,1999,47(12):5038-5043.
[7]	Mafra I,Ferreira I,Oliveira M.Food Authentication by PCR-Based Methods[J]. Eur Food Res Technol,2008,227(3):649-665.
[8]	Gašpari$\check{c}$ M B,Tengs T,La Paz J L,et al. Comparison of Nine Different Real-Time PCR Chemistries for Qualitative and Quantitative Applications in GMO Detection[J]. Anal Bioanal Chem,2010,396(6):2023-2029.
[9]	Manzanares-Palenzuela C L,Mafra I,Costa J,et al. Electrochemical Magneto-Assay Coupled to PCR as a Quantitative Approach to Detect the Soybean Transgenic Event GTS40-3-2 in Foods[J]. Sens Actuators B,2016,222:1050-1057.
[10]	Ma Y,Jiao K,Yang T,et al. Sensitive PAT Gene Sequence Detection by Nano-SiO2/p-Aminothiophenol Self-assembled Films DNA Electrochemical Biosensor Based on Impedance Measurement[J]. Sens Actuators B,2008,131(2):565-571.
[11]	Yang T,Zhang W,Du M,et al. A PDDA/Poly(2,6-Pyridinedicarboxylic Acid)-CNTs Composite Film DNA Electrochemical Sensor and Its Application for the Detection of Specific Sequences Related to PAT Gene and NOS Gene[J]. Talanta,2008,75(4):987-994.
[12]	Jiang C,Yang T,Jiao K,et al. A DNA Electrochemical Sensor with Poly-L-Lysine/Single-Walled Carbon Nanotubes Films and Its Application for the Highly Sensitive EIS Detection of PAT Dene Fragment and PCR Amplification of NOS Gene[J]. Electrochim Act,2008,53(6):2917-2924.
[13]	Zhang D D,Li L Z,Ma W N,et al. Electrodeposited Reduced Graphene Oxide Incorporating Polymerization of L-Lysine on Electrode Surface and Its Application in Simultaneous Electrochemical Determination of Ascorbic Acid, Dopamine and Uric Acid[J]. Mater Sci Eng C,2017,70:241-249.
[14]	Zhou M,Wang Y L,Zhai Y M,et al. Controlled Synthesis of Large-Area and Patterned Electrochemically Reduced Graphene Oxide Films[J]. Chem Eur J,2009,15:6116-6120.
[15]	Guo H L,Wan X F,Qian Q Y,et al. A Green Approach to the Synthesis of Graphene Nanosheets[J]. ACS Nano,2009,3(9):2653-2659.
[16]	Wang J F,Yang S L,Guo D Y,et al. Comparative Studies on Electrochemical Activity of Graphene Nanosheets and Carbon Nanotubes[J]. Electrochem Comm,2009,11(10):1892-1895.
[17]	Shao Y Y,Wang J,Engelhard M,et al. Facile and Controllable Electrochemical Reduction of Graphene Oxide and Its Applications[J]. J Mater Chem,2010,20:743-748.
[18]	Yang T,Li X,Li Q H,et al. Electrochemically Reduced Graphene Oxide-Enhanced Electropolymerization of Poly-Xanthurenic Acid for Direct[J]. Polym Chem,2013,4:1228-1234.
[19]	Xu Y,Gao M,Zhang G,et al. Electrochemically Reduced Graphene Oxide with Enhanced Electrocatalytic Activity Toward Tetracycline Detection[J]. Chinese J Catal,2015,36(11):1936-1942.
[20]	Sun W,Lu Y,Wu Y,et al. Electrochemical Sensor for Transgenic Maize MON810 Sequence with Electrostatic Adsorption DNA on Electrochemical Reduced Graphene Modified Electrode[J]. Sens Actuators B,2014,202(31):160-166.
[21]	Gao F,Qi X,Cai X,et al. Electrochemically Reduced Graphene Modified Carbon Ionic Liquid Electrode for the Sensitive Sensing of Rutin[J]. Thin Solid Films,2012,520(15):5064-5069.
[22]	Fang M M,David M K,Anthony C S.A “Mix and Match” Ionic-Covalent Strategy for Self-Assembly of Inorganic Multilayer Films[J]. J Am Chem Soc,1997,119(50):12184-12191.
[23]	Zhang W,Yang T,Jiang C,et al. DNA Hybridization and Phosphinothricin Acetyltransferase Gene Sequence Detection Based on Zirconia/Nanogold Film Modified Electrode[J]. Appl Surf Sci,2008,254(15):4750-4756.
[24]	Sun W,Wang X,Wang W,et al. Electrochemical DNA Sensor for Staphylococcus aureus Nuc Gene Sequence with Zirconia and Graphene Modified Electrode[J]. J Solid State Electrochem,2015,19(8):2431-2438.
[25]	Renuka L,Anantharaju K S,Sharma S C,et al. A Comparative Study on the Structural, Optical, Electrochemical and Photocatalytic Properties of ZrO2 Nanooxide Synthesized by Different Routes[J]. J Alloys Comp,2017,695:382-395.
[26]	Yang J,Jiao K,Yang T.A DNA Electrochemical Sensor Prepared by Electrodepositing Zirconia on Composite Films of Single-Walled Carbon Nanotubes and Poly(2,6-Pyridinedicarboxylic Acid), and Its Application to Detection of the PAT Gene Fragment[J]. Anal Bioanal Chem,2007,389(3):913-921.
[27]	Yang J,Wang X L,Shi H Q.An Electrochemical DNA Biosensor for Highly Sensitive Detection of Phosphinothricin Acetyltransferase Gene Sequence Based on Polyaniline-(Mesoporous Nanozirconia)/Poly-Tyrosine Film[J]. Sens Actutators B,2012,162(1):178-183.
[28]	Zou Y J,Sun L,Xu X F.Biosensor Based on Polyaniline-Prussian Blue/Multi-walled Carbon Nanotubes Hybrid Composites[J]. Biosens Bioelectron,2007,22(11):2669-2674.