发展灵敏快速简便的分析方法检测DNA损伤，对于筛查新化合物潜在的基因毒性、疾病的早期诊断以及环境保护等都具有重要意义。本论文构筑了基于天然双螺旋DNA (dsDNA)层层组装薄膜和电活性指示剂的电化学DNA生物传感器，以循环伏安法(CV)检测DNA的损伤。研究了DNA修复酶对电化学检测DNA损伤的放大作用，以及抗氧化酶对DNA损伤的保护作用。这种将dsDNA层层组装薄膜和与DNA有特异性相互作用的电活性探针相结合的DNA生物传感器，提供了一种普遍适用的、比较灵敏的方法来检测DNA损伤，为体外筛查实验和预测新化合物的潜在基因毒性提出了新的研究思路。第一章 前言简述了电化学检测DNA损伤的研究意义。介绍了DNA的结构和性质，各种因素引起的DNA损伤及DNA损伤的修复方式。简述了电化学检测DNA损伤的研究进展，特别是基于外加指示剂检测DNA损伤的研究现状。DNA的电化学性质、DNA在电极表面的固定方法和电化学检测DNA杂交的研究进展也在本章中做了简要综述。第二章 以亚甲基蓝为探针用循环伏安法检测环氧苯乙烯诱导的天然DNA的损伤构筑了一种基于PSS/{PDDA/dsDNA}3层层组装薄膜的电化学传感器用于检测天然dsDNA的损伤，其中PSS代表聚苯乙烯磺酸钠，PDDA代表聚二甲基二烯丙基氯化铵。将修饰有PSS/{PDDA/dsDNA}3薄膜的热解石墨(PG)电极浸入亚甲基蓝(MB)溶液并吸入MB至稳态，形成的PSS/{PDDA/dsDNA}3-MB薄膜在pH 7.0的空白缓冲溶液中表现出一对MB的可逆的CV还原氧化峰，电位在0.23 V (vs SCE)左右。吸入薄膜的MB在空白缓冲溶液中可以被逐渐释放，而将薄膜再次浸入MB的溶液中一定时间，薄膜又能重新吸入相同量的MB，表明该薄膜能够可逆地结合MB。但是，将薄膜温浴在已知基因毒性的试剂环氧苯乙烯(SO)溶液中，被损伤的PSS/{PDDA/dsDNA}3-MB薄膜不能重新吸入相同量的MB，表现出比未损伤的PSS/{PDDA/dsDNA}3-MB薄膜明显降低的CV峰电流。由于SO损伤dsDNA所形成的加合物不能显著影响dsDNA的构象，因此可能是SO与鸟嘌呤和腺嘌呤的加合物具有较大的空间位阻，阻碍MB嵌入至dsDNA的碱基对之间，导致MB在薄膜中固定量的降低。MB与dsDNA碱基对之间特殊的嵌入作用和MB灵敏的电化学响应，结合MB在DNA层层组装薄膜中可以被可逆吸入的特点，能够显著区别被损伤和未损伤的dsDNA薄膜的CV响应。第三章 核酸外切酶III对电化学检测DNA损伤的放大作用通过层层组装的方法将天然dsDNA固定在电极表面，形成PSS/PDDA/dsDNA薄膜。在Ru(bpy)32的溶液中，利用Ru(III)对DNA中鸟嘌呤(guanine)的电催化氧化峰电流检测了甲磺酸甲酯(MMS)诱导的DNA损伤。由于MMS导致DNA形成无嘌呤位点，使DNA中其余的guanine更加暴露，从而提高了还原Ru(bpy)33+的能力，促使氧化峰电流增加。大肠杆菌外切酶III进一步将MMS损伤的dsDNA中的无嘌呤位点转变成单链DNA区，使更多的guanine露出来，从而显著提高了损伤后薄膜的电催化氧化响应。这一新的思路有望应用于DNA损伤检测的电化学生物传感器，特别是应用于体外检测化合物的基因毒性。第四章 电化学检测由HRP+H2O2+NO2体系产生的活性氮物质NO2•对天然dsDNA的损伤及Catalase的保护作用生命体内的血红素过氧化物酶催化H2O2氧化NO2产生的活性氮物质NO2•能够造成DNA的损伤。选择辣根过氧化物酶HRP作为过氧化物酶的代表，在PG电极上构筑了基于{PDDA/dsDNA}n和PDDA/{dsDNA/HPR}n层层组装薄膜的电化学DNA损伤传感器。以Ru(bpy)32+为探针，用CV法检测了溶液状态和固定在薄膜中的HRP在H2O2作用下，催化氧化NO2产生的NO2•对薄膜中天然dsDNA的损伤。当溶液中或薄膜表面存在catalase时，由于catalase对H2O2的分解作用，可以在很大程度上保护dsDNA不受损伤。这种电化学DNA损伤生物传感器，不仅能够灵敏地检测过氧化物酶+H2O2+NO2体系产生的活性氮物质对DNA的损伤，并模拟生命体内抗氧化酶对DNA损伤的保护，为深刻理解生命体内的DNA损伤机制和抗氧化酶的保护机制提供了模型，同时也为利用电化学手段和层层组装技术在体外研究生命体系的氧化还原过程提供了一种新的思路。

Sensitive, rapid and simple detection of DNA damage is of great significance in toxicity screening of new chemicals, clinical diagnosis and environmental protection. In this dissertation, electrochemical sensors for detecting DNA damage, based on natural double-stranded DNA (dsDNA) layer-by-layer films and electroactive indicators, have been developed. DNA lesions are investigated and detected by cyclic voltammetry (CV). The amplified electrochemical detection of DNA damage by DNA repair enzyme, and the protection effect of antioxidant enzyme on DNA damage are also studied. The electrochemical DNA biosensors, combing dsDNA layer-by-layer films and electroactive probes which have special interactions with DNA, may provide a general and sensitive approach for detection of DNA damage and screening of genetoxic chemicals in vitro.Chapter 1: ForewordA brief introduction of the significance on electrochemical detection of DNA damage is described. The structure and property of DNA, as well as DNA lesion and repair are briefly presented. The research of electrochemical biosensor on DNA damage, especially the method based on indicators, is reviewed. An introduction of electrochemical behaviors of DNA, DNA immobilization methods, and the research development of DNA hybridization by electrochemistry are also presented.Chapter 2: Cyclic voltammetric detection of chemical DNA damage induced by styrene oxide in natural dsDNA layer-by-layer films using methylene blue as electroactive probeIn this work, an electrochemical sensor for detecting damage of natural dsDNA is constructed based on PSS/{PDDA/dsDNA}3 layer-by-layer films, where PSS is poly(styrene sulfonate) and PDDA is poly(diallyldimethylammonium). When the PSS/{PDDA/dsDNA}3 films assembled on pyrolytic graphite (PG) electrodes are immersed into methylene blue (MB) solution and loaded MB into the films, the formed PSS/{PDDA/dsDNA}3-MB films in blank buffers at pH 7.0 show a reversible CV peak pair at 0.23 V vs SCE for MB redox couple. In blank solutions, the MB loaded into the films is released gradually from the films, but the complete reloading of MB into the films can be realized by immersing the films into MB solution again, indicating the good reversibility of MB incorporation. However, after incubation in the solution of known genotoxic agent styrene oxide (SO), the damaged PSS/{PDDA/dsDNA}3-MB films can not return to their original and fully-loaded state with reloading of MB, and show smaller CV peak currents than those of intact PSS/{PDDA/dsDNA}3-MB films. While the formation of SO adducts with dsDNA has no substantial effect on the dsDNA conformation, the steric hindrance of SO adducts with guanine or adenine block the intercalation of MB into the base pairs of dsDNA, resulting in the decrease of CV peak currents of loaded MB. The specific intercalation of MB into dsDNA base pairs and the sensitive electrochemical response of MB, combined with the unique feature of loading reversibility of MB in the DNA layer-by-layer films, make the difference in CV response between the intact and damaged dsDNA films become pronounced in the “loading/release/reloading” procedure. Chapter 3: Using exonuclease III to enhance electrochemical detection of natural DNA damage in layered filmsThe natural dsDNA is immobilized on electrode surface by layer-by-layer assembly, forming PSS/PDDA/dsDNA films, and used to detect DNA damage electrochemically. The DNA lesion induced by the alkylating agent methyl methanesulfonate (MMS) can be detected by cyclic voltammetry with Ru(bpy)32+ in solution, utilizing the electrocatalytic oxidation guanines in dsDNA by Ru(III). Apurinic sites in dsDNA induced by MMS may cause those guanines that originally reside inside the dsDNA double-helix pockets to become more exposed, and then to be rapidly oxidized by Ru(bpy)33+, resulting in higher catalytic current. After treated by E. coli exonuclease III enzyme, the electrocatalytic oxidation peak of the films is further amplified and greatly enhanced, because the enzyme can convert those apurinic sites caused by MMS in the damaged dsDNA into single-stranded DNA regions and make more guanines in the DNA become exposed. This approach provides a novel idea for constructing DNA biosensor in sensitive screening of genetoxic chemicals in vitro.Chapter 4: Electrochemical detection of natural dsDNA damage induced by reactive nitrogen species NO2• from HRP+H2O2+NO2 system and the protection effect of catalase enzymeReactive nitrogen species (RNS) NO2• generated by heme peroxidase-catalyzed oxidation of nitrite (NO2) in the presence of hydrogen peroxide (H2O2) can damage DNA. In this work, an electrochemical sensor for detecting DNA damage is constructed based on {PDDA/dsDNA}n and PDDA/{dsDNA/HPR}n layer-by-layer films on PG electrode, choosing horseradish peroxidase (HRP) as the representative peroxidase. DNA damage induced by HPR in solution or in multilayered films with H2O2 and NO2 can be detected by CV with Ru(bpy)32+ in solution. However，when catalase is added in damage solutions or assembled on the surface of layer-by-layer films, the DNA damage is retarded because H2O2 is rapidly decomposed by catalase. This electrochemical sensor can sensitively detect DNA damage from peroxidase+H2O2+NO2 system and simulate the protection of DNA lesion in vivo, offering a new approach to understand the redox reactions in life process by electrochemistry and layer-by-layer assembly.