【作者】 薛蓓；

【导师】 胡之德；

【作者基本信息】 兰州大学 ， 分析化学， 2008， 硕士

【摘要】 共振光散射(RLS)光谱法二十世纪90年代发展起来的一种分析测试新技术。1995年,共振光散射技术作为一种研究生色团聚集的方法被Pasternack正式提出。随着共振光散射技术在理论和分析方法上的日趋成熟,共振光散射技术以其简单的操作,较高的灵敏度广泛应用于蛋白质、核酸、药物、细胞、金属离子、非金属离子、纳米粒子和表面活性剂等的分析。共振光散射法与一般的荧光法相比有更高的灵敏度,可以用于稀溶液的研究;检测更为方便,并可研究共振光散射光谱特征;可为研究分子结构和反应特征提供更丰富的信息;共振瑞利散射对于大分子非键作用,如缔合、聚集、偶极-偶极作用、长距离组装非常敏锐,这对于生物大分子的测定、表征及反应历程的研究极为有利。但是共振光技术还存在着不足之处,譬如抗干扰能力不强,其共振光散射信号易受到外来共存物质的干扰;重现性不好;油溶性试剂中使用不便等。本论文在总结前人工作基础上,对共振光散射法测定蛋白质进行了创新性的研究:1、将自行组装的流动注射装置与荧光光谱仪联用,确立了流动注射-共振光散射联用测定人血清样品中蛋白质含量的新方法,提高了共振光散射技术的重现性和稳定性。2、建立了聚乙二醇辛基苯基醚-蛋白质-亮丽春红5R三元体系检测人血清中蛋白质含量的新方法,提高了检测的灵敏度。论文共分为三章:第一章:简要介绍了共振光散射技术的原理和应用,以及共振光散射分析新方法的发展。并且对实验中用到的表面活性剂的在共振光散射检测中的应用进行了简单综述。第二章:用十二烷基硫酸钠作为探针,在流动注射-共振光散射联用系统中在线测定人血清中蛋白质的含量。该体系对人血清白蛋白检测的线性范围在3.0-28.0μg/mL之间,检测限为70ng/mL,检测结果的相对标准偏差不大于2.12%,样品回收率在97.83-106.09%之间。该方法克服了不稳定体系重现性较差的缺点,分析速度快,灵敏度高,实现了蛋白质的在线检测。第三章:基于在pH2.11的酸性溶液中,聚乙二醇辛基苯基醚(OP)对亮丽春红5R-蛋白质体系的共振光散射信号有强烈的增敏现象,建立了聚乙二醇辛基苯基醚-蛋白质-亮丽春红5R三元体系测定人血清中蛋白质浓度的新方法。该体系对人血清白蛋白检测的线性范围在0-10.0μg/mL之间,检测限为5ng/mL,检测实际样品的回收率在97.80-109.62%之间,该方法令人满意。更多还原

【Abstract】 Rayleigh light scattering (RLS) is a new technology developed in 1990s for trace analysis. It was founded by Pasternalk et al in 1993. There are more researches and applications of RLS recently. It is widely used in the research and determination of proteins, nucleic acids, carbohydrates, medication, non-particle, inorganic particles and surfactants and shows great foreground of use. Comparing with other methods, the RLS method has more acuminous sensitivity, more simple manipulation. And it is able to provide details for molecule’s configuration. However, the RLS method has disadvantage in reproducibility and stability.In this dissertation, on the basis of the previous research, novel Rayleigh light scattering methods were developed to investigate the quantification of proteins in human blood serum. The following major innovative works were carried out:1: The flow injection combined with Rayleigh light scattering technique was used to quantify the protein in human serum samples. The homemade instruments of flow injection were connected with spectrofluorophotometer. Protein in human serum samples were determined using the combination technique mentioned above, the results’ reproducibility and stability were elevated.2: A novel system of OP-HSA- Brilliant ponceau 5R was founded for the quantitative detection of protein in human serum samples by Rayleigh light scattering. The method had getten more acuminous sensitivity.This dissertation consists of three chapters: Chapter 1: The principles, up to date applications and novel development of Rayleigh light scattering were briefly described. And the surfactants used in this dissertation were mentioned.Chapter 2: A novel flow injection analysis (FIA) method coupled with Rayleigh light scattering (RLS) was developed for the determination of protein concentrations. This method is based on that the weak intensity of RLS signals of dodecane-1-sulfonic acid sodium salt can be enhanced by the addition of protein in weakly acidic solution. The determination limit was 70ng/mL for human serum albumin, the linear range was 3.0-28.0μg/mL, the maximum relative standard deviations (R.S.D.) was no more than 2.12% and the recovery of the samples was between 97.83-106.09%. The FIA-RLS method was more stabile, rapid, and reproducible than the general RLS method.Chapter 3: In the presence of OP and in the pH2.11 buffer, the Brilliant ponceau 5R combined proteins to form complex, causing an enhance of RLS signals. The sensitivity raised 2 times in the presence of OP. For human serum albumin, the linear range was 0-10.0μg/mL, and the determination limit was 5ng/mL. The recovery of the samples was between 97.80-109.62%. This method was more sensitive than the general RLS method and obtained satisfactory results.更多还原