【作者】 杨传孝；

【导师】 黄承志；

【作者基本信息】 西南师范大学 ， 分析化学， 2003， 硕士

【摘要】 共振光散射技术是基于普通荧光分光光度计所开发出来表征超分子组装化学及其分析应用研究的新技术。应用该技术已建立了高灵敏的测定核酸、蛋白质、痕量金属离子、表面活性剂的分析方法，表征了分子聚集的光谱特征和核酸的超螺旋结构。本文在讨论Al³⁺-DNA、镁试剂Ⅰ阳离子表面活性剂、固红VR-蛋白质、丽春红G-蛋白质四个体系的RLS光谱特征的基础上，证明荧光分光光度计的仪器条件和样品的分子吸收等因素会影响共振光散射（RLS）光谱的形状，降低检测的灵敏度。在此基础上本文引入校正因子建立了RLS光谱校正理论，并在普通荧光光度计上引入吸收装置测定分子吸收，根据同一台仪器所测定的数据讨论了固红VR-蛋白质、丽春红G-蛋白质和meso-四-（对三甲氨基苯基）卟啉（TAPP）-DNA体系的RLS光谱的校正。 在pH 2.21的酸性介质中，Al³⁺与脱氧核糖核酸（DNA）发生静电作用产生以291.0nm为特征峰的共振光散射（RLS）增强光谱。Al³⁺没有生色基团，它与DNA作用的RLS光谱受光吸收的影响小，因而Al^<sub>3+用于DNA测定的灵敏度高，线性范围宽。在pH 6.09和离子强度为0.03mol／L的条件下，阳离子表面活性剂氯化十四烷基苄基二甲铵（2eph）、溴化十六烷基三铵（CTMAB）与镁试剂Ⅰ的相互作用使其共振光散射（RLS）增强，产生以470.0nm、485.0nm和495.0nm为特征峰的RLS信号。方法成功用于合成样和自来水样的测定。 上述试验表明散射光谱随体系的分子吸收和仪器条件的变化而发生变化，因此类似荧光光谱的校正那样，通过引入校正因子，校正仪器因素和体系中分子吸收的内滤效应。 在酸性介质中，偶氮染料固红VR（Fast red VR, FRV）和丽春红G（Ponceau G,PG）与牛血清白蛋白（BSA）、人血清白蛋白（HSA）、溶菌酶（Lys）、γ-球蛋白（γ-IgG）等蛋白质作用产生共振光散射（RLS）增强信号，最大散射峰分别位于287.0nm和288.0nm处。FRV和PG用于测定BSA、HSA、Lys、γ-IgG的检测限均在25.0ng／ml以下。方法成功的应用于合成样和人血清样品的测定，测定结果与考马斯亮蓝法一致。用吸收装置改变荧光分光光度计入射光的光路直接测得FRV-蛋白质和PG-蛋白质复合物的分子吸收，用校正因子校正FRV-蛋白质和PG-蛋白质体系的RLS强度。研究表明，校正由于分子对激发光和散射光的吸收对RLS光谱的影响后，校正共振光散射（CRLS）光谱灵敏度可提高2倍左右。z 共级光散射光沿的柱正及其分析应用研兜￥分别在卜2500和卜4500荧光 多光光度计上对meso－四－对三甲氨基苯基)卜l 啦汀APP)与DNA作用的RLS光谱进行校正。校正强度后，灵敏度有显著提高。￥引入散射系数蚓村，井成功的把表观吸收光谱中的散射和吸收成分分离。研究发g 现，从F－2500和 F－4500荧光分光光度计上分离出的TAPP与 DNA作用的散射g 成分，其形状基本一致。更多还原

【Abstract】 Resonance light scattering technique is a newely established technique for characterizing supramolecular assemblies and their analytical applications developed by a common spectrofluorometer. With this technique, we have proposed highly sensitive analytical methods of DNA, proteins, trace amount metallic ions and surfactants, characterized the characteristic spectrum of molecular aggregation, and the structure of the supramolecular double-stranded DNA. By examining RLS spectral features of four systems including Al3+-DNA, azoviolet-cationic surfactants, fast red VR-proteins, ponceau G-proteins in this thesis, and proved that the instrumental conditions of spectrofluorometer have strong effect on the shape of resonance light scattering (RLS) spectra. Molecular absorption of sample will lower the detection sensitivity of RLS method. Thus a correction theory was established by introducing a correction factor, which measured molecular absorption directly on a common spectrofluorometer by using an absorption cell holder. According to data that measured on the same instrument, the corrections of resonance light scattering spectra of the three systems including fast red VR-proteins, ponceau G-proteins and α,β,γ, δ-tetrakis[4-(trimethylammoniumyl)pheny]porphine(TAPP)-DNA have been discussed.In acidic medium of pH 2.21, the interaction of Al3+ with deoxyribonucleic acid (DNA) results in enhanced resonance light scattering spectra, characterized by the peak at 291nm. Al3+has no chromothore, and its light absorption has little effect on the RLS spectra of the interaction between Al3+ and DNA. So the sensitivity of determination of DNA with Al3+ is very high, and the linear range is wide. A simple assay of cationic surfactants in water samples was developed based on the measurements of enhanced resonance light scattering. On the conditions of pH 6.09 and ionic strength 0.03 mol/L, the interactions of azoviolet (AV) with cationic surfactants, including zephiramine (Zeph), and cetyl trimethyl ammonium bromide (CTMAB), result in enhanced RLS signals characterized by the peaks of 470.0 nm,485.0 nm and 495.0 nm. Determinations of cationic surfactants in synthetic and tap water samples were successfully made with the recovery of 90.5-108.2%.The experiments stated above have showed that RLS spectra vary with the molecular absorption of systems and the instrumental conditions. Just like the correction of fluorescence spectra, we have corrected the instrumental factors and the inner fittering of the molecular absorption in the systems by introduing a correction factor.In an acidic medium, the interaction of fast red VR (FRV) and Ponceau G (PG) with proteins including bovine serum albumin (BSA), human serum albumin (HSA), pepsin (Pep) a-chymotrypsin (Chy), and lysozyme (Lys) was characterized by measuring enhanced resonance light scattering signals. The maximum RLS peaks were 287.0 nm and 288.0 nm, respectively. The limits of determination for BSA, HSA, Lys, y-IgG were below 25.0 ng/ml. The present method is successfully applied to determine proteins in synthetic samples, and the total content of proteins in human blood plasma samples, respectively. The results for determination human blood serum by this method are consistent with those obtained by the Coomassie brilliant blue G-250 assay. By using an absorption cell holder to change the propagation direction of the incident light beam of a common spectrofluorometer, the molecular absorptions of FRV-porteins and PG-porteins systems were directly measured through a spectrofluorometer, and corrected the RLS intensities of FRV-porteins and PG-porteins with a correction factor. With measurements of the corrected resonance light scattering (CRLS) signals of the interaction of fast red VR (FRV) with proteins and PG with proteins, respectively, we have proved that the present correction for the RLS spectra in terms of the molecular absorption of excitation and scattering radiation could improve the detection sensitivity by about two fold.We corrected the RL更多还原