【作者】 钟桐生；

【导师】 黄杉生；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 博士

【摘要】 土壤是固态地球表面具有生命活动、处于生物与环境间进行物质循环和能量交换的疏松表层,又是能生长植物的复合生物地球化学材料。土壤有机质是土壤具有生物学性质和结构的基本物质,既是生命活动的条件,也是生命活动的产物。腐殖质是一种由动植物部分分解转换而成的复杂的有机大分子化合物,广泛存在于土壤、水体(如湖泊、河流、海洋和地下水等)以及沉积物中,含有大量的羧基、酚羟基、甲氧基等官能团。不同地点、不同年代的腐殖质,其分子大小、化学组成、结构、官能团以及聚电解特性等各不相同。根据在酸和碱溶液中的溶解度,通常可把腐殖质分为三种主要组分：(1)腐殖酸(又称褐腐酸或胡敏酸)：溶于稀碱液,但在酸化碱提取物时絮凝,即溶于碱而不溶于酸的组分；(2)富里酸(或黄腐酸)：对稀碱液提取物酸化后,仍溶于液相的组分,即既溶于碱又溶于酸的组分；(3)腐黑物(胡敏素)：不能被稀碱和稀酸从土壤样品中提取的腐殖质组分。腐殖酸对环境中许多有毒有机或无机化学材料的存在和运输、营养物质的循环过程起着非常重要的作用,对重金属离子和有机污染物的传输和转换有很大的影响。腐殖酸的形成过程非常复杂,对其结构特征的研究有利于了解这些反应和过程,也有助于我们更好地了解腐殖酸与重金属离子的络合、还原、运动和固定,放射性核素,杀虫剂以及其它有毒化学药品等作用的内在机理,这样,反过来有助于提高我们对腐殖酸和环境污染物在自然生态系统中的行为的预知能力。然而,由于腐殖酸具有非均一性和不确定性,因此,没有一种分析仪器可以准确提供腐殖酸的结构和功能信息。对腐殖质的化学和反应的认识的重大发展只可能是通过将各种光谱技术和湿化技术结合起来。许多仪器分析方法已用于腐殖酸特性的研究,如核磁共振、傅立叶变换红外光谱、表面增强拉曼光谱、紫外可见光谱、电子顺磁共振光谱、固定金属离子亲合色谱和高效体积排阻色谱等,在这些分析方法中,以不破坏其结构的光谱技术显得非常重要,它可直接用于固体和液体样品的测定。这些方法具有如下几个优点：(1)不破坏样品结构；(2)样品用量少；(3)操作简单,无需对样品进行特殊处理；(4)可提供有用的分子结构和官能团信息。目前各种荧光光谱技术如荧光激发光谱、荧光发射光谱、同步荧光光谱以及三维荧光光谱(或三维激发-发射阵列荧光光谱)被广泛用于定性或定量描述腐殖酸的物理化学特性。它能够获得激发波长和发射波长同时变化时的荧光强度信息,并且可对多组分复杂体系中荧光光谱(激发／发射)重叠的对象进行光谱识别和表征,是一种很有用的光谱指纹技术。运用本技术及其它相关技术,本文对红壤腐殖酸和黑土腐殖酸的有关性质作了比较详细的研究,找出了其异同点。同时,为了便于今后更好地研究土壤腐殖酸与环境中重金属离子和生物分子的相互作用情况,制备了几种用于检测土壤中重金属离子和生物分子的光电化学传感器。化学传感器是集电子科学、化学科学和材料科学于一体,属国家九五科技攻关项目和鼓励外商投资的高新技术产品。它是将化学物质(待测物质)通过分子设计组成的敏感器件,定量和高选择性地转化为可监测的光电信号,并由高集成的电子仪器进行信息分析、处理,得到相关环境的化学物质的有关信息。化学传感器由于其具有灵敏度非常高,选择性好,携带方便,易微型化,能用于现场分析和监控等特点,在矿山开发、石油化工、生物医学及日常生活中越来越多的被用来作为易燃,易爆,有毒,有害气体的检测预报和自动控制装置,或用来测定多种含量极低的物质,甚至可以测量细胞中的离子浓度。医学上采用化学传感器作病情诊断及治疗过程的自动控制。生产和生活各个领域对化学传感器的广泛要求,使得化学传感器的研究和开发一直十分活跃,并表现出非常广阔的应用前景。本文第一章对腐殖酸的定义、分类、元素组成、官能团和结构、性质和作用等进行了比较全面的综述。第二章简要介绍了我国两种典型土壤——红壤和黑土的分布情况、特征及成土过程。第三章采用NaOH碱提法提取和纯化了我国两种典型土壤腐殖酸,并用元素分析、扫描电子显微镜、紫外光谱、傅立叶变换红外光谱、三维荧光光谱等方法对提取的腐殖酸进行了表征。结果表明,提取的腐殖酸由C、H、O、N元素组成,红壤腐殖酸含有较多的芳香族不饱和物质和较多的多糖或类多糖物质,而黑土腐殖酸则含有相对较多的芳香烃或多酚类官能团。两种腐殖酸均为片状、有序排列的无定形聚合体,黑土腐殖酸表面光滑而红壤腐殖酸表面粗糙且伴有少量的颗粒状结构。从三维荧光光谱分析可知,土壤腐殖酸在低浓度时只有一个明显的荧光峰,高浓度时出现几个荧光峰,并且发生了Ex/Em红移现象。离子强度对腐殖酸的3DEEM影响很小,而pH值对黑土腐殖酸的荧光强度影响较大,在相同条件下,红壤腐殖酸的荧光量子产率和荧光指数均大于黑土腐殖酸,据此可以区分这两种典型土壤腐殖酸。第四章研究了红壤腐殖酸和黑土腐殖酸与重金属离子(Cu2+和Cd2+)作用的荧光特性。研究发现,离子强度对红壤腐殖酸和黑土腐殖酸的三维荧光光谱影响很小；而溶液的pH值对其与金属离子所形成的络合物的3DEEM影响较离子强度大。在相同条件下,红壤腐殖酸和黑土腐殖酸对Cu2+的络合能力均比Cd2+强。红壤腐殖酸与Cu2+、Cd2+所形成的络合物的平均络合比分别为1：1.22和3.2：1,络合平衡常数分别为1.6×106和1.5×106；黑土腐殖酸与Cu2+、Cd2+所形成的络合物的平均络合比分别为1：1.1和1：1.5,络合平衡常数分别为1.25×105和3.5×103说明两种腐殖酸对土壤中的Cu2+有良好的调节作用。第五章采用自组装方法,将对巯基苯胺修饰到金电极表面,并通过偶联剂将腐殖酸与对巯基苯胺相连制得腐殖酸单层膜,再通过浸泡和相转移将细胞色素c固定在腐殖酸膜表面。采用场发射扫描电子显微镜、紫外可见光谱、电化学交流阻抗谱、拉曼光谱、循环伏安法等方法对电极的表面形态和电化学性质进行了比较详细的讨论。结果表明,腐殖酸对细胞色素c在金电极上的电化学响应具有良好的促进作用,细胞色素c有一对可逆的氧化还原峰,其表观电位为-0.101 V,且不随扫描速度的改变而改变。细胞色素c在金电极上呈单层,平均表面覆盖率为5.28 pmol/cm2,电子转移速度常数为2.14 s-1,该修饰电极吸附2,4-二氯苯酚后,有一对明显的新的氧化还原峰出现,可望制成一种检测2,4-二氯苯酚的新的传感器。第六章利用对巯基苯胺、双链DNA作为细胞色素c在金电极上电子转移的促进剂。并采用场发射电子显微镜、紫外可见光谱、电化学阻抗谱和循环伏安法,对该修饰电极进行了比较详细的研究。PATP-dsDNA-Cyt c修饰电极在pH为7.0的BR缓冲溶液中有一对稳定的、可逆的氧化还原峰,其标准电位为-0.106 V(相对饱和甘汞电极)。固定在修饰电极表面的细胞色素c的平均表面覆盖率为3.70pmol/cm2,电子转移速率常数为2.0 s-1,说明双链DNA可以稳定、有效地促进细胞色素c的快速电子转移,该修饰电极可望对特定序列的DNA进行分析以及对DNA链中的碱基突变进行检测。第七章研究了在低温下,通过混合石墨粉、日本血吸虫抗原、溶胶-凝胶,制备了一种基于溶胶-凝胶技术的可更新的安培分析免疫传感器。该传感器坚固、多孔,拥有一个可更新的表面。在辣根过氧化物酶标记的日本血吸虫抗体的协助下,通过竞争性的免疫分析来确定日本血吸虫抗体的含量。并对分析过程中的一些参数,如培育液中酶标日本血吸虫抗体的含量、培育时间、温度以及溶液的pH值进行了优化。以邻-氨基酚作为底物,-250 mV作为工作电位,响应电流与日本血吸虫抗原的浓度在0.32～19.00μg／ml范围内呈线性关系,检测限为0.18μg／ml。在此线性范围内,对兔血清样品中日本血吸虫抗体进行了定量测量。结果与临床诊断用的ELISA方法相比较,令人满意。第八章研制了基于新型分子导线化合物2-(2-(4-(2-(4-(2-(2,5-二庚基-4-(2-(噻吩-2-基)乙炔基)苯基)乙炔基)苯基)乙炔基)-2,5-二庚苯基)乙炔基)噻吩(简称FDB)测定Cr(VI)的光化学敏感膜。研究表明：Cr(Ⅵ)离子对固定于聚氯乙烯膜中的FDB有可逆荧光猝灭作用。敏感膜最佳组成为：55.0 mg聚氯乙烯、105.0 mg邻苯二甲酸二异辛酯和5.0 mg四苯硼钠。选用最大激发／发射波长为371 nm/407 nm,在0.60 mol/L的H2SO4溶液中,测定Cr(VI)离子的线性响应范围为3.78×10-6～1.26×10-3mol/L,检测限为5.16×10-7mol/L,响应时间小于1 min。敏感膜具有良好的重现性、可逆性和选择性,其它常见金属离子不干扰测定。应用于环境水中Cr(Ⅵ)离子含量的测定,结果满意。更多还原

【Abstract】 Soil is not only the loose surface in which material cycle and energy exchange occurs between the biological and environmental materials those have a life activity, but also the biogeochemical materials that be able to grow complex plants. Soil organic matter are the basic materials with biological properties and structures, not only the conditions but also the products of life activity. Humic substances (HS), a complex mixture of partially "decomposed" and otherwise transformed organic materials, are widely distributed in soil, natural water (e.g. lake, river, stream, sea and groundwater) and sediments, and contain a lot of functional groups such as carboxylic, phenolic, and methoxy groups. The size, chemical composition, structure, functional groups, and polyelectrolytic characteristics of HS may vary greatly, depending on the origin and age of the material.According to the solubility in acid and alkaline solution, HS can generally be divided into three main components:(1) Humic acid, HA, also known as brown rot acid, is soluble in dilute alkali solution, but flocculating in acidified alkaline extract. (2) Fulvic acid, FA, can dissolve in acid and alkaline solution. (3) Humin, Hu, the humus components, can not be extracted from soil samples in alkali and acid.HA, an important member of HS, plays a critical role in the fate and transportation of many toxic organic or inorganic chemicals and in nutrient cycling throughout the environment, which also has a great effect on the transport and transformation of both heavy metals and organic contaminants. The study on structural characteristic of HA will be helpful for us to know these reaction and processes due to the fact that the formation process of HA is quite complex. A better understanding of the structural and functional properties of HA may further improve our understanding of the intrinsic mechanisms of its complexation, reduction, mobilization or immobilization with heavy metals, radionuclides, pesticides, and other toxic chemicals. This, in turn, may improve our predictive capabilities of the behavior of HA and environmental pollutants in natural ecosystems.No single analytical tool, however, can provide definitive structural or functional information about HA because of its heterogeneous and ill-defined nature. The knowledge of the chemistry and reactions of HS may be possibly obtained only by the combination of a variety of spectroscopic and wet-chemical techniques. Many instrumental methods have been used to study the characteristics of HA, such as the nuclear magnetic resonance (NMR), Fourier-transform infrared spectrum (FTIR), surface enhanced Raman spectroscopy (SERS), Ultra Violet/Visible Spectrophotometry (UV-Vis), electron paramagnetic resonance (EPR), immobilized metal ion affinity chromatography (IMAC), High-pressure size exclusion chromatography (HPSEC), etc. Among analytical characterization methods, nondestructive spectroscopic techniques appear to be most useful, and they are directly applicable to both solid and liquid sample analysis. These methods have a number of attractive advantages including:(a) they are nondestructive; (b) only small amounts of samples are needed; (c) they are experimentally simple and do not require special manipulative sample preparation; and, perhaps most importantly, (d) they provide valuable information on molecular structure and chemical or functional properties of HA.At present, various kinds of fluorescence spectrum technology such as fluorescence excitation spectrum, fluorescence emission spectrum, synchronous fluorescence spectrum (SFS) and three-dimensional fluorescence spectrum (or three-dimensional excitation emission matrix fluorescence spectroscopy,3DEEM) are being used for determining the nature and physical chemical characteristic of HA. 3DEEM is a very useful spectrum fingerprint technology, because it can provide information of fluorescence intensity about HA when the excitation and emission wavelength are changed at the same time. By using of this technology and other related technologies, the more detailed properties about red soil humic acid and black soil humic acid were researched in this paper, and the similarities and differences between them were given. Meanwhile, in order to study the interaction between humic acid and heavy ions in soils and environment or biological molecules conveniently, photoeletrochemical sensors were prepared for detecting the heavy ions in soil and bio-molecules.Chemical sensor, combining the e-science, chemical sciences and materials science in one, is the high-tech products which belongs to the State 95 scientific and technological research and encouraged foreign investment projects. It can transform the sensitive device composed of chemical substance atfer molecular design, with quantitative and high selectivity, into monitorable electro-optical signals which then be analyzed and processed for environmental information about the chemical substances.Chemical sensors, because of its very high sensitivity, good selectivity and easy to carry, easy miniaturization, on-site analysis and monitoring, can be more and more used as prediction and automatic control devices for flammable, explosive, toxic and harmful gas detection in mining, petrochemical, bio-medicine and everyday lives. Chemical sensors are also used for determining a variety of substances with very low concentration, and even for ions concentration in cells. In medicine, people use the chemical sensors for diagnosis and treatment process automation. The widespread demand for chemical sensors in all areas lead to chemical sensor’s research and development be very active and which has shown a very broad application prospects.This first chapter in this paper gives a overview about the definition, classification, elemental composition, functional groups and structure, nature and role of humic acids. Chapter 2 briefly describes the distribution, features and soil formation processes of two typical soils(red and black soils) in China.In chapter 3, Humic Acids (HAs) were extracted from two typical soils in China including red soil and black soil. The HAs were characterized jointly with elemental analysis (EA), scanning electron microscopy (SEM), ultra voilet (UV) spectroscopy, FTIR and 3DEEM. The results showed that HAs extracted from different soils were composed of C, H,O and N, and they have almost the same chemical groups as carboxyl, hydroxyl and much aromatic unsaturated substances, but the content of these groups is varied. The content of aromatic unsaturated substances and polysaccharides or polysaccharide-like substances of HAs from red soil is relatively high, while the content of aromatic or polyphenolic functional groups of HAs from black soil is larger. The SEM images indicate that two HAs are sheet-like regular amorphous aggregates, the surface of HAs extracted from black soil is smooth, while those of HAs from red soil is rough and many small particles can be observed on it. The 3DEEM showed that the concentration of soil HAs affected the numbers of fluorescence peak, and arose the red shift of the Ex/Em maximum wavelength. The effect of ionic strength on the 3DEEM of soil HA was neglectable. The effect of pH on the fluorescence intensity of black soil humic acid (BHA) was greater than that of red soil humic acid (RHA). The fluorescence quantum yields and fluorescence index of RHA were bigger than that of BHA at the same condition, which can be used to distinguish BHA and RHA.In chapter 4, the fluorescence characteristics of the complexes of both RHA and BHA with heavy metals were studied. The results indicate that ionic strength over the range from 0 to 0.05 mol/L NaNO3 did not affect the 3DEEMs of RHA and BHA. The concentration of HAs and pH of the test solution had obvious effects on the 3DEEM. The complexation abilities of the Cu-RHA and Cu-BHA are all stronger. The average complex ratio of Cu-RHA is 1:1.22, the complex constant is 1.6×106, and the average complex ratio of Cd-RHA is 3.2:1, the complex constant is 1.5×107. The average complex ratios of Cu-BHA and Cd-BHA are 1:1.1,1:1.5, and the complex constants of Cu-BHA and Cd-BHA are 1.25×105 and 3.5×103, respectively.In chapter 5,p-aminothiophenol (PATP) and humic acids (HA or HAs) were applied jointly as the electron transfer accelerants of redox reactions of cytochrome c (Cyt c) on Au electrodes. The electrochemical properties of the modified electrodes were studied carefully by field emission scanning electron microscope (FESEM), UV-Vis, electrochemical impedance spectroscopy (EIS), Raman spectroscopy (RS) and cyclic voltammetry (CV). The immobilized Cyt c displayed a couple of stable and well-defined redox peaks with a formal potential of -0.101 V (vs. SCE) in pH 7.0 c). Cyt c adsorption is of a monolayer with average surface coverage of 5.28 pmol/cm2. The electron transfer rate constant was calculated to be 2.14 s-1. It indicated that the HA film acted as a good adsorption matrix for Cyt c and an excellent accelerant for the redox of Cyt c.In chapter 6, PATP and dsDNA were applied jointly as the electron transfer accelerants of redox reactions of cytochrome c (Cyt c) on Au electrodes. The electrochemical properties of the modified electrodes were studied carefully by field emission scanning electron microscope (FESEM), ultraviolet-visible spectroscopy (UV-Vis), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry. The immobilized Cyt c displayed a couple of stable and well-defined redox peaks with a formal potential of -0.106 V (vs. SCE) in pH 7.0 phosphate buffer solution (PBS). Cyt c adsorption is of a monolayer with average surface coverage of 3.70 pmol cm-2. The electron transfer rate constant was calculated to be 2 s-1. It indicated that the ssDNA film acted as a good adsorption matrix for Cyt c and an excellent accelerant for the redox of Cyt c after hybridization with the target sequence DNA. The PATP-ssDNA-Cyt c modified gold electrode will probably be an alternative method of detecting hybridization of target DNA.In chapter 7, an amperometric immunosensor was constructed by dispersing graphite, schistosoma-japonicum antigen (SjAg) and silica sol-gel at low temperature. The performance characteristics of the prepared immunosensor. were examined in the buffer solution of o-aminophenol (o-AP) used as a substrate. It exhibited excellent physical and electrochemical stability with a renewable external surface. A competitive binding assay was employed to determine schistosoma-japonicum antibody (SjAb) with the aid of horseradish peroxidase labeled SjAb (HRP-SjAb). The experimental parameters for SjAb assay were optimized, including the amount of labeled SjAb in incubation solution, incubation time, temperature and the pH of solution. The use of o-AP substrate and amperometric detection at-250 mV (vs. SCE) results in a determination limit of 0.32μg/ml and a linear range extending up to 0.18μg/ml. The results of SjAb assay in serum samples demonstrate the feasibility of using the proposed immunosensor for clinical analysis.In chapter 8, an optical chemical sensor has been developed for determination of chromium(VI) based on reversible fluorescence quenching of a conductor wire compound 2-(2-(4-(2-(2,5-diheptyl-4-(2-thiophenyl-2-)ethinyl)phenyl)ethinyl)phenyl) ethinyl)2,5-diheptophenyl)ethinyl)thiophene (FDB) immobilized in a PVC membrane. The sensing membrane consisted of 55.0 mg PVC,105.0 mg diisooctyl (o-)phathalate (DIOP) and 5.0 mg NaTPB. The maximum response of the sensing membrane for chromium(VI) was obtained in 0.6 mol/L sulfuric acid when the maximum EX/EM wavelength is 371/407 nm respectively. Under optimum conditions the method showed good linearly for 3.78×10-6～1.26×10-3 mol/L Cr(Ⅵ) with detection limit of 5.16×10-7 mol/L. The response time was less than 1 min. Common cations and anions did not interfere the measurements of Cr(Ⅵ). The method was verified by the determination of trace Cr(Ⅵ) in environmental water with satisfactory results.更多还原