石墨烯纳米带功能杂化材料电化学传感器的构建及其转基因大豆检测的研究
发布时间：2018-07-28 15:46
【摘要】：转基因大豆品种及种植面积日渐增加,其安全性已成为了人们关注的焦点。因此,建立灵敏快速、简单便捷的转基因大豆鉴别方法,对推动转基因产品安全监管和保障公民健康具有重要意义。电化学传感技术因具有分析速度快、灵敏度高、易于操作等优点,已成为食品分析领域的热点。本论文基于石墨烯纳米带功能杂化材料优异的电化学性质,构建了新颖、便捷、免标记的电化学传感器,并应用于转基因大豆CaMV 35S启动子(P35S)的检测。主要研究内容如下:1、采用一步还原法制备纳米金/多壁碳纳米管-还原石墨烯纳米带(Au NP/MWCNT-rGONR)杂化材料。捕获探针通过π-π作用固定到杂化材料表面,构建了电化学阻抗传感界面。由于单双链DNA的电子密度不同,当目标DNA与捕获探针杂交形成双链后,传感界面的电负性增大并阻碍了电子传递,进而导致阻抗值增加,基于此原理发展了免标记的电化学阻抗传感器。以P35S序列为检测目标,该传感器的阻抗变化值与P35S的浓度在0.1 fmol/L~500 pmol/L范围内呈良好的线性关系,检出限低至0.03 fmol/L(S/N=3)。该传感器具有很好的选择性、稳定性和重现性,可应用于转基因大豆实样鉴别。2、利用一步热处理法制备了二氧化钛/氮杂石墨烯纳米带(TiO_2/NGNRs)杂化材料。研究发现,TiO_2/NGNRs的光电流强度约是TiO_2的6倍,表明NGNRs的引入,提高了杂化材料的导电性,促进了TiO_2电荷转移并抑制了光生电子-空穴的复合。进一步以TiO_2/NGNRs为载体,通过戊二醛化学交联法,固定基因探针,构建了转基因大豆中P35S的光电化学(PEC)传感器。捕获探针与P35S目标基因杂交形成惰性双链,抑制抗坏血酸(AA)捕获空穴,引起光电流降低,实现目标基因的检测。在优化条件下,该传感器线性范围为0.5~100 nmol/L,检测限为0.17 nmol/L。该传感器有良好的选择性及稳定性,并实现了对转基因大豆的鉴别。3、进一步采用一步热处理法制备了Ag-TiO_2/NGNRs三元纳米杂化材料。研究发现,Ag-TiO_2/NGNRs光电流强度分别是TiO_2/NGNRs和TiO_2的2倍和10倍,表明Ag的表面等离子体效应,进一步改善了三元纳米杂化材料中TiO_2的PEC性能。以Ag-TiO_2/NGNRs为载体,通过纳米Ag连接带有巯基的基因探针,构建了转基因大豆中P35S的PEC传感器。依据上述PEC检测机理,实现目标基因的检测。在优化条件下,构建的PEC传感器具有较宽的检测范围(0.01 nmol/L~500 nmol/L),较低的检出限(0.0033 nmol/L,S/N=3)和优异的选择性、稳定性和重现性,并实现了转基因大豆实样的鉴别。
[Abstract]:The safety of transgenic soybean varieties and planting area has become the focus of attention. Therefore, it is of great significance to establish a sensitive, rapid, simple and convenient identification method for genetically modified soybean to promote the safety supervision of transgenic products and to protect the health of citizens. Electrochemical sensing technology has become a hotspot in the field of food analysis because of its advantages of fast analysis speed, high sensitivity and easy operation. Based on the excellent electrochemical properties of functional hybrid materials of graphene nanobelts, a novel, convenient and labeling free electrochemical sensor was constructed and applied to the detection of CaMV 35s promoter (P35S) in transgenic soybean. The main research contents are as follows: one step reduction method was used to prepare au NP/MWCNT-rGONR hybrid materials. The interface of electrochemical impedance sensing was constructed by immobilization of the probe to the surface of hybrid material by 蟺-蟺 interaction. Because of the different electron density of single and double stranded DNA, the electronegativity of the sensing interface increases after the target DNA is hybridized with the capture probe, which hinders the electron transfer, which leads to the increase of the impedance value. Based on this principle, a label free electrochemical impedance sensor was developed. Using P35S sequence as the detection target, the impedance change of the sensor has a good linear relationship with the concentration of P35S in the range of 0.1 fmol/L~500 pmol/L, and the detection limit is as low as 0.03 fmol/L (S/N=3). The sensor has good selectivity, stability and reproducibility. It can be used in the identification of transgenic soybean samples. Titanium dioxide / aza-graphene nanospheres (TiO_2/NGNRs) hybrid materials were prepared by one-step heat treatment. It is found that the photocurrent intensity of TiO2 / NGNRs is about 6 times of that of TiO_2, which indicates that the introduction of NGNRs improves the conductivity of hybrid materials, accelerates the charge transfer of TiO_2 and inhibits the photogenerated electron-hole recombination. Furthermore, the photoelectrochemical (PEC) sensor of P35S in transgenic soybean was constructed by glutaraldehyde chemical crosslinking method and immobilized gene probe with TiO_2/NGNRs as carrier. The hybridization of the capture probe with the P35S target gene forms inert double strand, which inhibits the trap hole of ascorbic acid (AA), and results in the reduction of photocurrent and the detection of the target gene. Under the optimized conditions, the linear range of the sensor is 0.5 nm / L and the detection limit is 0.17 nmol / L. The sensor has good selectivity and stability, and has achieved the identification of transgenic soybean. 3. Further, Ag-TiO_2/NGNRs ternary nano-hybrid materials were prepared by one-step heat treatment. It is found that the photocurrent intensity of Ag-TiORs is 2 times and 10 times higher than that of TiO_2/NGNRs and TiO_2 respectively, which indicates that the surface plasma effect of Ag can further improve the PEC performance of TiO_2 in ternary nano-hybrid materials. The PEC sensor of P35S in transgenic soybean was constructed by using Ag-TiO_2/NGNRs as carrier and DNA probe with sulfhydryl group ligated with nano-Ag. According to the mechanism of PEC detection, the target gene was detected. Under the optimized conditions, the constructed PEC sensor has a wide detection range (0. 01 nmol/L~500 nmol/L), a lower detection limit (0.0033 nmol / L) and excellent selectivity, stability and reproducibility.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS214.2;O657.1
本文编号：2150768
[Abstract]:The safety of transgenic soybean varieties and planting area has become the focus of attention. Therefore, it is of great significance to establish a sensitive, rapid, simple and convenient identification method for genetically modified soybean to promote the safety supervision of transgenic products and to protect the health of citizens. Electrochemical sensing technology has become a hotspot in the field of food analysis because of its advantages of fast analysis speed, high sensitivity and easy operation. Based on the excellent electrochemical properties of functional hybrid materials of graphene nanobelts, a novel, convenient and labeling free electrochemical sensor was constructed and applied to the detection of CaMV 35s promoter (P35S) in transgenic soybean. The main research contents are as follows: one step reduction method was used to prepare au NP/MWCNT-rGONR hybrid materials. The interface of electrochemical impedance sensing was constructed by immobilization of the probe to the surface of hybrid material by 蟺-蟺 interaction. Because of the different electron density of single and double stranded DNA, the electronegativity of the sensing interface increases after the target DNA is hybridized with the capture probe, which hinders the electron transfer, which leads to the increase of the impedance value. Based on this principle, a label free electrochemical impedance sensor was developed. Using P35S sequence as the detection target, the impedance change of the sensor has a good linear relationship with the concentration of P35S in the range of 0.1 fmol/L~500 pmol/L, and the detection limit is as low as 0.03 fmol/L (S/N=3). The sensor has good selectivity, stability and reproducibility. It can be used in the identification of transgenic soybean samples. Titanium dioxide / aza-graphene nanospheres (TiO_2/NGNRs) hybrid materials were prepared by one-step heat treatment. It is found that the photocurrent intensity of TiO2 / NGNRs is about 6 times of that of TiO_2, which indicates that the introduction of NGNRs improves the conductivity of hybrid materials, accelerates the charge transfer of TiO_2 and inhibits the photogenerated electron-hole recombination. Furthermore, the photoelectrochemical (PEC) sensor of P35S in transgenic soybean was constructed by glutaraldehyde chemical crosslinking method and immobilized gene probe with TiO_2/NGNRs as carrier. The hybridization of the capture probe with the P35S target gene forms inert double strand, which inhibits the trap hole of ascorbic acid (AA), and results in the reduction of photocurrent and the detection of the target gene. Under the optimized conditions, the linear range of the sensor is 0.5 nm / L and the detection limit is 0.17 nmol / L. The sensor has good selectivity and stability, and has achieved the identification of transgenic soybean. 3. Further, Ag-TiO_2/NGNRs ternary nano-hybrid materials were prepared by one-step heat treatment. It is found that the photocurrent intensity of Ag-TiORs is 2 times and 10 times higher than that of TiO_2/NGNRs and TiO_2 respectively, which indicates that the surface plasma effect of Ag can further improve the PEC performance of TiO_2 in ternary nano-hybrid materials. The PEC sensor of P35S in transgenic soybean was constructed by using Ag-TiO_2/NGNRs as carrier and DNA probe with sulfhydryl group ligated with nano-Ag. According to the mechanism of PEC detection, the target gene was detected. Under the optimized conditions, the constructed PEC sensor has a wide detection range (0. 01 nmol/L~500 nmol/L), a lower detection limit (0.0033 nmol / L) and excellent selectivity, stability and reproducibility.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS214.2;O657.1
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