【作者】 童裳伦；

【导师】 刘维屏；

【作者基本信息】 浙江大学 ， 环境科学， 2005， 博士

【摘要】 近年来,离子探针方法及其应用越来越受到人们的重视,正逐步发展成为一种常规的测试手段,广泛应用于生物学、医学、化学等学科,特别是在生物大分子结构与功能研究中,有着广阔的应用前景。目前大多数DNA荧光探针试剂具有很强的毒性,可能为致突变剂,尤其是当其与DNA的结合为不可逆的。因此,对毒性低、选择性好、灵敏度高的DNA探针的研究是一个非常有前景的研究领域。本论文总体分为四个部分,具体的研究内容与取得的结果如下: 第一部分:结合自己在稀土离子荧光探针及其应用领域所做的工作基础上,比较系统地介绍了稀土离子荧光探针在药物与生化分析中的应用以及在生物大分子中的应用研究进展。在稀土荧光探针及其应用方面进行了如下研究:（1）人工合成的双酰代吡唑酮类螯合剂,1,6-双（1’-苯基-3’-甲基-5’-吡唑酮-4’-基）已二酮（BPMPHD）是一种新型的稀土离子荧光配体,具有优良的螯合性能。BPMPHD能与Dy³⁺离子和阳离子表面活性剂溴代十六烷基三甲胺（CTMAB）形成稳定的三元离子缔合物并发射出Dy³⁺的特征荧光峰,Gd³⁺离子的加入,能与该体系形成共发光效应,增强了体系的荧光强度,在详细研究该共发光体系的影响因素基础上,建立了灵敏测定痕量稀土Dy³⁺的分析方法,其测定线性范围为1.0×10^-7-1.3×10^-5mol/L,检出限为2.0×10^-8mol/L（S/N=3）。（2）实验发现稀土铽离子能与去甲肾上腺素发生络合反应,并发射出铽离子的特征荧光。在对铽离子与去甲肾上腺素的络合发光反应进行详细研究的基础上,提出了一种简便、快速、灵敏检测去甲肾上腺素的新方法,其检出限可达1.0ng/mL（S/N=3）。（3）实验发现,抗坏血酸在水溶液中能将无荧光的铈（Ⅳ）离子还原成能发射特征荧光的铈（Ⅲ）离子,加入三磷酸钠,可使体系的荧光强度大大增强,由此建立了间接灵敏测定抗坏血酸的方法,其检出限为1.0x10^-8mol/L（S/N=3）。（4）研究表明,在表面活性剂SDBS存在下,能使氟喹诺酮-铽体系荧光强度大大增强,由此建立了表面活性剂敏化的铽离子荧光探针灵敏测定氟喹诺酮类药物的分析方法,其检出限可达10^-9mol/L（S/N=3）,为临床检测与药动力学研究以及环境中氟喹诺酮类药物含量的监测提供灵敏,快速,简便的方法。更多还原

【Abstract】 In recent years, ions as luminescence probes and its application have attracted more and more attention, and will become a normal analytical method, which is widely used in the biology, medicine, chemistry and so on, especially in the study of the structure and function of the biological macromolecules. At present, many of these fluorescent DNA probes are highly toxic and can be mutagenic, especially if the union to DNA is irreversible. Therefore, the search for low toxic, selective and sensitive DNA probes is an active field of research. The dissertation consists of the following four parts.In the first part, the rare-earth luminescence probes and its application in the study of the pharmaceutical analysis, biological substances analysis and the structure and function of the biological macromolecules were systemically reviewed, which includes my research works in the above fields. The following study on the rare-earth luminescence probes and its application was developed. （1） The artificial synthetic derivative of diacylated pyrazolone, 1,6-bi（1’-phenyl-3’-methyl-5’-pyrazolone-4’-） hexanedione（BPMPHD） is a new fluorescent chelating agent for RE ions, which can form a large ion association with Dy³+ and the cation surfactant cetyltrimethylammonium bromide（CTMAB）, and can emit the intrinsic fluorescence of Dy3+. In this system, some rare earth ions such as Gd3+、 Y3+、 La3+ can exert an evident fluorescence enhancement effect, this is a newly found co-luminescence system. From this, a rapid, simple and sensitive method was developed for the determination of trace amounts of Dy3+ . The results indicated that the fluorescence intensity of the system was a linear relationship with the concentration of Dy3+ in the range of 1.0× 10^-7- 1.2×10^-5 mol/L, the detection limit（S/N=3） was 3.0×10^-8mol/L. （2） Experiments indicated that terbium（ III） ion could form complex with norepinephrine, then emitted its characteristic fluorescence. In this paper, the fluorescence reaction between terbium（III） ion and norepinephrine was studied in detail. Thus a new method for the determination of norepinephrine was put forward.The fluorescence intensity was measured in a 1 cm quartz cell with excitation and emission wavelengths of 300, 545nm, respectively. The result showed that the fluorescence intensity of the system presented a linear relationship with the concentration of norepinephrine in the range of 0.01-50 u g /mL, the detection limit（S/N=3） was 1.0 ng/mL. （3） Experiments indicated that cerium（IV） ion which could not emit fluorescence was deoxidized to cerium（III） ion which could emit characteristic fluorescence by ascorbic acid in water solution, while sodium triphosphate added could greatly enhance the fluorescence intensity of the system. From this, a indirect sensitive method for determining ascorbic acid was developed. The fluorescence intensity of the system was measured in a 1 cm quartz cell with excitation and emission wavelengths of 303 nm and 353 run, respectively. The results showed that the fluorescence intensity of the system presented a linear relationship with the concentration of ascorbic acid in the range of l.OxlO"7 -6.0x10"6 mol/L, the correlation coefficient r was 0.9989, the detection limit（S/N=3） was l.OxlO"8 mol/L. （4） The experiments indicated that terbium（III） ion could complex with the fluoroquinolone, then emitted the characteristic fluorescence of terbium（III） ion. While added the surfactant of SDBS, the fluorescence intensity of the system was greatly increased. From this, a sensitive method of determining the fluoroquinolone was set up. The fluorescence intensity was determined by a 1-cm quartz cell with the excitation wavelengths of 300nm and the emission wavelengths of 545nm. the detection limit could obtain 10"9 mol/L.In the second part, the fluorescence enhancement of the fluoroquinolone -Tb3+ complex by DNA was studied. A sensitive fluorescence method for the determination of DNA was developed. It was found that the fluorescence probe of the fluoroquinolone-Tb3+ complex not only had higher sensitivity for determining the single-stranded content of DNA at nanogram levels than most of the reported probes, but also could directly determine double-stranded content of nature calf thymus DNA （except NFLX-Tb3+）.The fluorescence probe of the fluoroquinolone-Tb3+complex can also be used to study the interaction between the contaminants and DNA. In addition, these DNA probes are environment friendly.更多还原