【作者】 刘健；

【导师】 刘绍璞；

【作者基本信息】 西南大学 ， 分析化学， 2010， 硕士

【摘要】 二十世纪九十年代发展起来的共振瑞利散射(RRS)和共振非线性散射(RNLS)因其高的灵敏度、简便快速的分析过程和简单价廉的设备而得到越来越多的关注和研究。目前该技术已经广泛地应用于药物分析、无机小分子之间的相互作用研究、小分子和生物大分子之间的相互作用以及某些物理-化学参数的测定等领域,但是对于大分子特别是生物大分子之间的相互作用、结合模式等研究甚少。因此,本文以蛋白质和多糖为研究对象,研究探讨了它们之间的相互作用机理、结合模式、结合位点以及结合作用力等,并发展了RRS和RNLS法测定蛋白质和多糖的新方法。主要研究内容如下：1.蛋白质作探针共振瑞利散射法及共振非线性散射法测定硫酸软骨素A在pH 2.5-4.0的Britton-Robinson(BR)缓冲溶液介质中,人血清白蛋白(HSA)、牛血清白蛋白(BSA)、糜蛋白酶(Chy)和α-淀粉酶(α-Amy)等蛋白质能通过静电引力、氢键作用力和疏水作用力与酸性多糖的硫酸软骨素A(CSA)形成结合产物。此时将引起共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射显著增强并产生新的散射光谱。在蛋白质过量时,三种散射增强(△IRRS、△ISOS和△IFDS)均在一定范围内与CSA的浓度成正比,方法具有高灵敏度。当用HSA、Chy、BSA和α-Amy作探针时,三种散射法对于CSA的检出限分别在1.1-2.0 ng／mL(RRS),1.9-2.9 ng／mL(SOS)和3.1-13.2 ng／mL(FDS)。其中以HSA-CSA体系的RRS法最灵敏(检出限1.1 ng／mL),可用于痕量CSA的测定。本文研究了反应体系的RRS、SOS和FDS的光谱特征、适宜的反应条件和影响因素,并以HSA-CSA体系为例,讨论了二者之间的反应机理和结合模式：认为静电引力、氢键作用和疏水作用是它们的主要作用力,而CSA的乙酰氨基与HSA中芳香氨基酸(主要是色氨酸和酪氨酸)的生色团和荧光团的相互作用是吸收光谱变化和荧光猝灭的重要原因,而分子体积增大、疏水界面的形成及荧光-散射共振能量转移作用是共振光散射增强的重要因素。2.用硫酸软骨素A作探针共振瑞利散射及共振非线性散射法测定蛋白质在pH 1.8-2.5的BR缓冲介质中,硫酸软骨素A(CSA)的硫酸酯基离解而以带多个负电荷的大阴离子存在,而HSA、BSA、Chy、α-Amy和溶菌酶(Lyso)等蛋白质处于其等电点(pI)之下,则是带多个正电荷的大阳离子,两者可借静电引力、氢键作用、疏水作用而结合形成复合物。此时将引起共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射(RNLS)的显著增强并出现新的散射光谱。3种散射的散射增强(△IRRS、△ISOS的△IFDS)均在一定范围内与蛋白质的浓度成正比,方法具有高灵敏度。三种方法对蛋白质的检出限分别为4.5-12.0 ng／mL(RRS法)、8.9-15.8 ng／mL(SOS法)和13.4-31.5 ng／mL(FDS法),其中以CSA-BSA体系灵敏度最高(检出限可达4.5 ng／mL)。本文研究了反应体系的RRS、SOS和FDS的光谱特征、适宜的反应条件和影响因素,讨论了反应机理、结合模式以及散射增强的原因。并以CSA-BSA体系为例考察了共存物质的影响,表明方法有良好的选择性。方法可用于正常人血清及尿样中蛋白质的测定。3.蛋白质与透明质酸钠相互作用的共振瑞利散射及共振非线性散射光谱研究及其分析应用在适当酸度的BR缓冲溶液介质中,透明质酸钠(Sodium Hyaluronate, SH)的D-葡萄醛酸单元上的羧酸离解而带上负电荷,而与在处于等电点以下带正电荷的HSA、BSA、Chy和a-Amy借助静电引力、疏水作用等结合而形成离子缔合物。此时将引起共振瑞利散射(RRS)显著增强,并产生新的RRS光谱,其最大散射波长位于300nm(Chy-SH)、290 nm(HSA-SH, BSA-SH);和305 nm(α-Amy-SH)处。散射增强(AIRRS)与SH浓度在一定范围内成正比,可用于SH的定量测定。HSA-SH,BSA-SH, Chy-SH以及α-Amy-SH体系的检出限(3σ)分别是：3.0,7.7,4.3和3.5ng／mL(RRS)；5.4,16.3,10.5和6.8 ng／mL(SOS)；8.9,21.0,17.2和8.8 ng／mL(FDS)。本文研究了适宜的反应条件,考察了共存物质的影响,表明方法有较好的选择性。基于此可以建立起一种灵敏度高、简便、快速测定透明质酸钠的新方法。4.用蛋白质作探针共振瑞利散射和共振非线性散射发测定痕量藻酸钠在适当酸度的BR缓冲溶液介质中,藻酸钠(Sodium Alginate, Alg)的甘露糖醛酸单元上的羧酸离解而带上负电荷,而与在此酸度介质中处于等电点(pI)以下的带正电荷的HSA、BSA、Chy和Lyso借助静电引力、疏水作用等结合而形成离子缔合物。此时将引起共振瑞利散射(RRS)、二级散射(SOS)和倍频散射(FDS)等共振非线性散射(RNLS)的显著增强,并产生新的散射光谱,其最大RRS散射波长分别位于304 nm(Chy-Alg)、300 nm(HSA-Alg,BSA-Alg)和301 nm(Lyso-Alg)处。散射增强(△IRRS、△ISOS和△IFDS)与藻酸钠浓度在一定范围内成正比,可用于痕量藻酸钠的定量测定。HSA-Mg,BSA-Alg,Chy-Alg以及Lyso-Alg体系的检出限(36)分别是：1.9,2.1,2.1和1.6 ng／mL(RRS)；2.6,2.9,3.0和2.6 ng／mL(SOS)；5.0,5.1,5.5和5.5 ng／mL(FDS)。本文研究了适宜的反应条件,考察了共存物质的影响,表明方法有较好的选择性。基于此可以建立起一种灵敏度高、简便、快速测定藻酸钠的新方法。5.糜蛋白酶作探针共振瑞利散射法和共振非线性散射法测定痕量的羧甲基纤维素钠在pH 4.4的HAc-NaAc缓冲介质中,羧甲基纤维素钠(CMC)由于分子链上的羧基钠电离而呈带多个负电荷的大阴离子,而此时处于等电点一下的糜蛋白酶则是带多个正电荷的大阳离子型体存在,二者可以通过静电引力和疏水作用力而相互结合形成复合物。此时将引起RRS和SOS、FDS等RNLS的显著增强,并出现新的散射峰。散射增强(△IRRS、△ISOS和△IFDS)的程度与一定浓度范围内的羧甲基纤维素钠成正比,且方法有高的灵敏度。三种方法(RRS法、SOS法和FDS法)对CMC的检出限(3σ)分别为2.3、4.8、4.3 ng／mL,其中以RRS法的灵敏度最高。本文研究了反应体系的RRS、SOS和FDS的光谱特征、适宜的反应条件和影响因素,并讨论了散射增强的原因,考察了共存物质的影响,表明方法有良好的选择性。方法可用于烟丝中CMC的测定。更多还原

【Abstract】 Resonance Rayleigh scattering (RRS) and resonance non-linear scattering (RNLS) catch people’s more attention because of their high sensitivity, rapid analysis speed and cheapness instrument. Now they have been widely used to pharmaceuticals analysis, the study of reactions between the micromolecules and biological macromolecules and reactions among inorganic molecules. But they are rarely applied to the study of reactions among biological macromolecules and their binding mode.In this work, the applications of RRS and RNLS to the analysis of proteins and polysaccharides were studied; the reation mechanism, binding mode, binding sites and the main interaction forces were investigated.1. Determination of chondroitin sulfate A by resonance Rayleigh scattering and resonance non-linear scattering using proteins as probesIn pH 2.5-4.0 Britton-Robinson (BR) buffer medium, some proteins such as human serum albumin (HSA), bovine serum albumin (BSA), Chymotrypsin (Chy) andα-Amylase (α-Amy) can react with chondroitin sulfate A (CSA) to form binding products by means of electrostatic, hydrogen bonding and hydrophobic forces. As a result, the resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS) were enhanced greatly and new scattering spectra appeared. The increments of scattering intensity (ΔI) were directly proportional to the concentrations of CSA in certain ranges. The detection limits (3σ) of CSA were 1.1-2.0 ng/mL (RRS),1.9-2.9 ng/mL (SOS) and 3.1-13.2 ng/mL (FDS), separately. Among them, the RRS method exhibited the highest sensitivity and the HSA-CSA system was more sensitive than other reaction systems (DL=1.1 ng/mL). The characteristic of the spectra and optimum conditions of RRS method were investigated. Taking RRS method of HSA-CSA system for example, the reaction mechanism and binding mode were discussed, which indicated that the main interaction forces were electrostatic attraction, hydrogen bonding and hydrophobic interaction. The interaction between acetylamino group of CS and chromophore and fluorophore of aromatic amino acids of HSA (mainly in tryptophan and tyrosine) was the important cause of the change of absorption spectra and the fluorescence quenching. But the increase of molecular volume, formation of hydrophobic interface and fluorescence-RRS resonance energy transfer were the main factors of scattering enhancements.2. Determination of proteins by resonance Rayleigh scattering and resonance non-linear scattering methods with chondroitin sulfate A as a probeIn pH 1.8-2.5 BR buffer medium, negatively charged chondroitin sulfate A (CSA) can react with positively charged proteins, such as HSA, BSA, Chy, a-Amy and lysozyme (Lyso), via electrostatic attraction, hydrogen bonding and hydrophobic interaction. This led to the enhancements of resonance Rayleigh scattering (RRS) and resonance non-linear scattering (RNLS), including second order scattering (SOS) and frequency doubling scattering (FDS), and the appearance of new spectra. The scattering intensities (ΔIRRs,ΔISOS andΔIFDs) were directly linear to the concentrations of proteins in certain ranges. Each method had high sensitivity and the detection limits (3σ) were 4.5-12.0 ng/mL (RRS),8.9-15.8 ng/mL (SOS) and 13.4-31.5 ng/mL (FDS) for proteins. The CSA-BSA system was the most sensitive in which the detection limit for BSA was 4.5 ng/mL. In this article, the spectral characteristics, optimum conditions and influencing factors were investigated. The reaction mechanism and binding mode were discussed. Taking RRS method of CSA-BSA system for example, the effects of coexisting substances on the reaction were investigated. The method can be applied to the determination of protein in serum and urine samples with satisfactory results.3. The study of resonance Rayleigh scattering and resonance non-linear scattering of proteins-hyaluronate systems and their analytical applicationsIn suitable acidity of BR buffer medium, sodium hyaluronate (SH) with negative charge can react with possitively charged proteins, such as HSA, BSA, Chy and a-Amy. via electrostatic attraction, hydrogen bonding and hydrophobic interaction. The intensities of RRS and RNLS were enhanced obviously and new spectra appeared. The enhanced scattering intensities (ΔIRRS,ΔISOS andΔIFDS) were directly proportional to the concentrations of hyaluronate in certain ranges. The detection limits (3σ) of hyaluronate for HSA-SH, BSA-SH. Chy-SH andα-Amy-SH were 3.0,7.7,4.3 and 3.5 ng/mL (RRS): 5.4,16.3,10.5 and 6.8 ng/mL (SOS);8.9,21.0,17.2 and 8.8 ng/mL (FDS), respectively. In this article, the spectral characteristics, optimum conditions of the reactions and influencing factors were investigated. Based on the above researches, a high sensitive, simple and fast method for the determination of hyaluronate has been established.4. Determination of sodium alginate by resonance Rayleigh scattering and resonacne non-linear scattering using proteins as probesIn suitable acidity of BR buffer medium, sodium alginate (Alg) can react with some proeins such as HSA, BSA, Chy and Lyso by means of electrostatic attraction, hydrogen bonding and hydrophobic interaction, which led to the enhancement of RRS and RNLS intensities such as SOS and FDS. The maximum scattering wavelenghs were 304 nm (Chy-Alg),300 nm (HSA-Alg, BSA-Alg) and 301 nm (Lyso-Alg) for RRS. The enhanced scattering intensities (ΔIRRS,ΔISOS andΔIFDS) were directly linear to the concentrations of Alg in certain ranges. The detection limits (3σ) of alginate for HSA-Alg, BSA-Alg, Chy-Alg and Lyso-Alg were 1.9,2.1,2.1 andl.6 ng/mL (RRS); 2.6,2.9,3.0 and 2.6 ng/mL (SOS); 5.0,5.1,5.5 and 5.5 ng/mL (FDS), respectively. The optimum conditions, influence factors and the effect of coexisting substances were investigated. Based on above reseacches, a high sensitive, simple and quick method is developed to determine the sodium alginate.5. Determination of sodium carboxymethylcellulose by resonance Rayleigh scattering and resonance non-linear scattering using chymotrypsin as a probeIn pH 4.4 Hac-NaAc buffer medium, sodium carboxymethylcellulose (CMC) can react with Chy by means of electrostatic attraction and hydrophobic interaction. The intensities of RRS, SOS and FDS were enhanced greatly and new scattering spectra appeared. The enhanced scattering intensities (ΔIRRS,ΔISOS andΔIFDS) were directly proportional to the concentrations of CMC in certain ranges. The detection limits (3σ) of three method were 2.3,4.8,4.3 ng/mL for RRS, SOS and FDS, respectively. The optimum conditions, influence factors and the effect of coexisting substances have been investigated. The method can be applied to determination of CMC in cut tobacco.更多还原