【摘要】：轉(zhuǎn)基因大豆品種及種植面積日漸增加,其安全性已成為了人們關(guān)注的焦點(diǎn)。因此,建立靈敏快速、簡(jiǎn)單便捷的轉(zhuǎn)基因大豆鑒別方法,對(duì)推動(dòng)轉(zhuǎn)基因產(chǎn)品安全監(jiān)管和保障公民健康具有重要意義。電化學(xué)傳感技術(shù)因具有分析速度快、靈敏度高、易于操作等優(yōu)點(diǎn),已成為食品分析領(lǐng)域的熱點(diǎn)。本論文基于石墨烯納米帶功能雜化材料優(yōu)異的電化學(xué)性質(zhì),構(gòu)建了新穎、便捷、免標(biāo)記的電化學(xué)傳感器,并應(yīng)用于轉(zhuǎn)基因大豆CaMV 35S啟動(dòng)子(P35S)的檢測(cè)。主要研究?jī)?nèi)容如下:1、采用一步還原法制備納米金/多壁碳納米管-還原石墨烯納米帶(Au NP/MWCNT-rGONR)雜化材料。捕獲探針通過(guò)π-π作用固定到雜化材料表面,構(gòu)建了電化學(xué)阻抗傳感界面。由于單雙鏈DNA的電子密度不同,當(dāng)目標(biāo)DNA與捕獲探針雜交形成雙鏈后,傳感界面的電負(fù)性增大并阻礙了電子傳遞,進(jìn)而導(dǎo)致阻抗值增加,基于此原理發(fā)展了免標(biāo)記的電化學(xué)阻抗傳感器。以P35S序列為檢測(cè)目標(biāo),該傳感器的阻抗變化值與P35S的濃度在0.1 fmol/L~500 pmol/L范圍內(nèi)呈良好的線(xiàn)性關(guān)系,檢出限低至0.03 fmol/L(S/N=3)。該傳感器具有很好的選擇性、穩(wěn)定性和重現(xiàn)性,可應(yīng)用于轉(zhuǎn)基因大豆實(shí)樣鑒別。2、利用一步熱處理法制備了二氧化鈦/氮雜石墨烯納米帶(TiO_2/NGNRs)雜化材料。研究發(fā)現(xiàn),TiO_2/NGNRs的光電流強(qiáng)度約是TiO_2的6倍,表明NGNRs的引入,提高了雜化材料的導(dǎo)電性,促進(jìn)了TiO_2電荷轉(zhuǎn)移并抑制了光生電子-空穴的復(fù)合。進(jìn)一步以TiO_2/NGNRs為載體,通過(guò)戊二醛化學(xué)交聯(lián)法,固定基因探針,構(gòu)建了轉(zhuǎn)基因大豆中P35S的光電化學(xué)(PEC)傳感器。捕獲探針與P35S目標(biāo)基因雜交形成惰性雙鏈,抑制抗壞血酸(AA)捕獲空穴,引起光電流降低,實(shí)現(xiàn)目標(biāo)基因的檢測(cè)。在優(yōu)化條件下,該傳感器線(xiàn)性范圍為0.5~100 nmol/L,檢測(cè)限為0.17 nmol/L。該傳感器有良好的選擇性及穩(wěn)定性,并實(shí)現(xiàn)了對(duì)轉(zhuǎn)基因大豆的鑒別。3、進(jìn)一步采用一步熱處理法制備了Ag-TiO_2/NGNRs三元納米雜化材料。研究發(fā)現(xiàn),Ag-TiO_2/NGNRs光電流強(qiáng)度分別是TiO_2/NGNRs和TiO_2的2倍和10倍,表明Ag的表面等離子體效應(yīng),進(jìn)一步改善了三元納米雜化材料中TiO_2的PEC性能。以Ag-TiO_2/NGNRs為載體,通過(guò)納米Ag連接帶有巰基的基因探針,構(gòu)建了轉(zhuǎn)基因大豆中P35S的PEC傳感器。依據(jù)上述PEC檢測(cè)機(jī)理,實(shí)現(xiàn)目標(biāo)基因的檢測(cè)。在優(yōu)化條件下,構(gòu)建的PEC傳感器具有較寬的檢測(cè)范圍(0.01 nmol/L~500 nmol/L),較低的檢出限(0.0033 nmol/L,S/N=3)和優(yōu)異的選擇性、穩(wěn)定性和重現(xiàn)性,并實(shí)現(xiàn)了轉(zhuǎn)基因大豆實(shí)樣的鑒別。
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TS214.2;O657.1

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