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题名: 电化学DNA生物传感器对环境有机污染物检测及毒性判别研究
作者: 梁刚
学位类别: 博士后
答辩日期: 2015-06
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 郭良宏
关键词: G-DNA 酶,适体DNA,2-羟基芴,乙醇胺,8-羟基脱氧鸟苷,电化学阻抗谱法,电化学发光法,G-DNAzyme ; Aptamer ; 2-hydroxyfuorene ; Ethanolamine ; 8-hydroxy-2′-deoxyguanosine ; Electrochemical Impendance Spectroscopy ; Electrochemiluminescence
其他题名: APPLICATION OF ELECTROCHEMICAL DNA BIOSENSOR IN DETECTING SMALL MOLECULES AND SCREENING TOXIC ENVIRONMENTAL POLLUTANTS
学位专业: 环境科学
中文摘要:     电化学DNA生物传感器是一种以DNA为识别元件并通过电化学信号转化器将目标物的存在转变为可检测的电信号的传感装置。因其制备成本低,操作简单且具有较高的灵敏性、选择性、便携性及抗干扰性等优点在分析化学领域受到了越来越多的青睐,并被广泛应用于DNA杂交检测、DNA损伤、药物筛选及设计、生物医学、食品安全、医疗诊断、环境污染物监测等领域。采用电化学DNA生物传感器对环境污染物的检测,不仅可以揭示DNA与环境污染物作用的机理,而且有望为我们提供一种研究环境污染物的新方法,具有广阔的应用前景。
本研究设计了电化学DNA生物传感器实现了对2-羟基芴、乙醇胺等小分子的检测;设计了电化学发光DNA阵列传感器对DNA氧化损伤标志物8-羟基脱氧鸟苷进行了检测。该研究为我们提供了采用DNA生物传感技术检测小分子及筛查环境毒性污染物的新方法。研究内容包含以下几部分:
1. 以电化学阻抗谱法为研究手段,构建了高灵敏性的G-DNA酶生物传感器实现了对2-羟基芴选择性检测,并采用比色法、荧光光谱法等对检测机理进行了证明。首先通过Au-硫键作用将富G碱基的DNA序列 (G-DNA)修饰到电极表面,在K+作用下G-DNA形成G-四联体结构,可以与氯高铁血红素分子 (hemin) 结合形成具有类过氧化物酶催化活性的G-DNA酶。该G-DNA酶对双氧水具有较好的催化活性,可以有效的催化双氧水氧化2-羟基芴生成难溶产物并沉积到G-DNA酶膜表面。基于2-羟基芴在G-DNA酶表面原位催化氧化放大反应,DNA膜阻抗 (RCT) 呈现显著变大的趋势,并在催化反应10 min时达到平衡。在Tris-NaClO4 (20 mM, pH=7.4) 缓冲体系中,DNA膜阻抗的变化 (∆RCT) 与2-羟基芴浓度在一定的浓度范围内呈现较好的线性关系:Y (∆RCT) = 3271.3+358.7 lg [C2-HOFlu] (R2=0.99),检测限达到1.2 nM。该电化学G-DNA酶生物传感器对2-羟基芴具有较强的选择性,相同条件下与其它芴的衍生化合物作用后引起的电化学阻抗变化较小。此外,该G-DNA酶生物传感器亦成功用于实际水体系中2-羟基芴的检测,检测限达到3.6 nM。
2. 以电化学阻抗谱法为研究手段,构建了一种对乙醇胺具有高灵敏性、选择性的适体AP-DNA (3) 生物传感器,并采用电化学阻抗谱法、圆二色谱法等分析手段证明了检测乙醇胺的原理。首先,通过Au-硫键作用将适体AP-DNA (3) 修饰到电极表面,当乙醇胺分子与修饰于电极表面的适体AP-DNA (3) 发生作用后引起AP-DNA (3) 构象发生变化——由一条自由态单链DNA变成G-四联体结构。该DNA结构的变化会引起DNA膜层电化学阻抗 (RCT) 显著的增加。根据乙醇胺与适体DNA作用前后DNA膜电化学阻抗的变化 (∆RCT),实现了Tris-NaClO4 (20 mM, pH=7.4) 缓冲体系中乙醇胺的高灵敏检测,检测限达到0.16 nM。电化学阻抗变化 (∆RCT) 与乙醇胺浓度在一定浓度范围内呈现较好的线性关系:Y (∆RCT) = 60.6+5.4 X (R2=0.95)。结果表明,该电化学适体DNA (3) 生物传感器对乙醇胺具有较好的选择性,在相同条件下与其它具有类似分子结构的小分子作用后,DNA膜电化学阻抗变化较小。此外,该电化学适体AP-DNA (3) 生物传感器亦成功用于实际样品中乙醇胺的检测,平均回收率为84 %~93 %。
3. 构建了微孔板阵列传感器实现了对DNA氧化损伤产物标志物8-羟基脱氧鸟苷 (8-oxodGuo) 的高通量检测。首先,采用丝网印刷技术制备了96微孔板阵列电极 (工作电极C,参比电极Ag/AgCl),经NaOH-电活化处理后,利用层层组装技术,通过静电作用依次将PDDA、DNA修饰到C膜表面,得到C/PDDA/DNA传感界面。在弱氧化剂存在条件下,“双功能团”精胺-钌电化学发光探针通过“一步法”特异性反应,标记到电极表面含有8-羟基脱氧鸟苷DNA膜上,从而实现了8-羟基脱氧鸟苷的高特异性、快速检测。研究结果表明,该微孔板阵列传感器具有较高的灵敏性,检测限达到了1/500,即500个正常碱基中可以检测到1个8-羟基脱氧鸟苷损伤。该方法实现了Fenton试剂氧化DNA损伤8-羟基脱氧鸟苷产物的检测。此外,基于8-oxodGuo阵列传感器亦成功用于实际垃圾焚烧飞灰样品有机相提取物毒性识别,这也为我们提供了一种快速、简便可用于导致DNA产生特异性氧化损伤 (8-oxodGuo) 的毒性环境污染物的高通量筛查方法,具有重要的研究意义和广阔的应用前景。
英文摘要:     Electrochemical DNA biosensor is an analytical device that combines the DNA as the biological recognition element with a electrode as the electrochemical transducer to generate a measureable electrochemical signal of the DNA-target binding event, and it has been extensively applied in analytical fields, such as DNA hybridization, DNA damage, drug design and screening, food safety, medical diagnosis and environmental monitoring et al., due to the advantages, such as low cost, easy operation, high sensitivity and selectivity, portability and excellent capacity of resisting disturbance. Furthermore, electrochemical detection of environmental pollutants by the DNA biosensors can uncover the interaction mechenism, and provide us a new technique for monitoring the environmental pollutants, which make it has broad future prospects in many fields.
    In this study, DNA biosensors were designed for the detection of 2-hydroxyfluorene (2-HOFlu) and ethanolamine (EA) by electrochemical impedance spectroscopy (EIS), and detection of 8-hydroxy-2′-deoxyguanosine (8-oxodGuo) by electrochemiluminescence (ECL). It provids us DNA sensing technique for detecting the small molecules and screening environmental toxic pollutans. The main contents are as follows:
1. A sensitive and selective biosensor for the environmental 2-HOFlu was developed. The interaction mechanism was further confirmed by colormetric and fluorenscent spectroscopy. It is based on EIS and was obtained by assembling a thiolated single-stranded DNA on a gold electrode via a S-Au covalent bonding. It was then transformed to a K+-stabilized G-quadruplex-hemin complex which exhibits peroxidase-like activity to catalyze the oxidation of 2-HOFlu by H2O2. This resulted in the formation of insoluble products that precipitated on the gold electrode. As a result, the charge transfer resistance (RCT) between the solution and the electrode surface was strongly increased within 10 min as demonstrated by using the ferro/ferricyanide system as a redox probe. The difference in the charge transfer resistances (ΔRCT) before and after incubation of the DNA film with 2-HOFlu and H2O2 served as the signal for the quantitation of 2-HOFlu with a 1.2 nM detection limit in buffer (Y (∆RCT) = 3271.3+358.7 lg [C2-HOFlu]) (R2=0.99). The assay was highly selective over other selected fluorenes derivatives. It was exploited to determine 2-HOFlu in spiked lake water samples where it displayed a detection limit of 3.6 nM. Conceivably, this method has a wide scope in that it may be applied to other analytes for which respective G-quadruplexes are available.
2. A sensitive electrochemical sensing assay for the detection of EA was reported based on the aptamer AP-DNA (3) modified electrode (via a S-Au covalent bonding) by EIS. The interaction mechanism of the aptamer AP-DNA (3) with EA was further investigated by electrochemical impedance spectroscopy, CD spectroscopy. Specificially, upon EA interacting with the aptamer AP-DNA (3), the conformation of the random coiled G-rich single-strand DNA switched to G-quartet on the gold electrodes in Tris buffer solution. Therefore, the charge transfer resistance (RCT) of the DNA film between the solution and the electrode surface was significantly increased. Depending on the difference in charge transfer resistance (∆RCT) before and after EA interaction with the aptamer AP-DNA (3), EA could be detected with the concentration as low as 0.16 nM, and the linear regression equation was Y (∆RCT) = 60.6+5.4 X (R2=0.95). However, only a much smaller ΔRCT appeared when other selected moleculars with similar structure interacted with the AP-DNA (3) film, which demonstrated that EA could be discriminated from other moleculars by EIS. The performance of the aptamer sensor was further chanllenged in real samples for the detection of EA with good recoveris of 84 %~93 %.
3 A screen-printed carbon biosensor array was constructed for high throughput detection of biomarker 8-oxodGuo (products of oxidatively damaged DNA). First, an array of 8×12 electrode pairs with carbon as working electrode and Ag/AgCl as reference electrode was processed by screen-printing on the designed circuit board. The board was attached to a bottomless 96-well plate to fabricate the 96 electrode well plate. Then PDDA and DNA were sequentially immobilized on the pre-anodized carbon electrode using layer-by-layer assembly technique through electrostatic interaction, building the final microplate-based sensor films (C/PDDA/DNA). The C/PDDA/DNA film was then incubated with spermine-Ru (spermine conjugated ruthenium tris-(bipyridine) derivative) under oxidative condition for 8-oxodGuo labeling. With Spermin-Ru complex as the ECL signal reporter, the rapid, specific detection of 8-OxodGuo was achieved through a specific one-step reaction between 8-OxodGuo and Spermin-Ru. The ECL intensity was found to correlate with the amount of 8-oxodGuo on the surface and the detection limit was estimated to be about 1 lesion in 500 DNA bases. Furthermore, the biosensor array was also successfully applied to detect DNA oxidation product 8-oxodGuo produced by Fenton reagent and organic extracts of ash collected from waste incineration plant. It provides us a simple, rapid electrochemical DNA sensing technique for the screening of environmental toxic pollutants, which has broad future prospects.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34346
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
梁刚. 电化学DNA生物传感器对环境有机污染物检测及毒性判别研究[D]. 北京. 中国科学院研究生院. 2015.
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