【作者】 郭宏平；

【导师】 黄承志；

【作者基本信息】 西南师范大学 ， 分析化学， 2005， 硕士

【摘要】 基于光散射粒子高的光发射能力,在临床检验和生物分析、超灵敏免疫检测和DNA探针分析中进行光散射微粒标记显微成像分析已引起人们广泛关注。实验证明应用共振光散射（RLS）粒子作为标记比荧光标记的灵敏度能够得到显著提高。本文在普通RLS技术的基础上,建立了一种共振光散射成像技术,可以观察到单个聚集粒子的光散射性质,通过对单个散射粒子的光散射信号检测,实现了生物大分子及药物分子的分析测定。研究内容包括下列四个方面: （1）研究了铬水解聚合物的光谱性质,并且运用共振光散射（RLS）光谱研究其与核酸的相互作用。铬聚合物在415nm和580 nm处具有特征吸收,在350 nm处具有最大荧光发射。在酸性条件下,铬聚合物与核酸作用产生强烈增强的RLS信号,其特征峰位于299.0 nm,据此讨论无机聚合物试剂与生物大分子之间的作用,并建立了核酸的分析方法。 （2）在酸性介质中,当蛋白质存在时,四-（对磺基苯基）卟啉（TPPS₄）和四-（5-磺基噻吩）卟啉[T（5-ST）P],可以发生J-聚集,从而导致增强的RLS信号,且其特征峰分别位于490.0 nm与469.0 nm处。运用488 nm氩离子激光光源激发溶液中的聚集粒子,在与激光光束呈直角的方向,我们可以通过普通的显微镜观察到单个聚集粒子的散射光,并且能够用一个冷却型电荷耦合装置（CCD）进行成像。对这些数字化图像进行数据分析的结果表明,在焦平面上检测到的聚集粒子个数与溶液中的蛋白质浓度成正比,据此建立了灵敏测定蛋白质的分析方法,并且成功地用于人血清样品中蛋白质的测定。 （3）在中性介质中,四（四-氨基苯基）卟啉（TAPP）与肝素通过静电和疏水作用相互结合,在430.0nm处产生RLS特征峰。在441.6 nm He-Cd激光线的激发下,单个TAPP-肝素聚集粒子的光散射信号可以通过普通的显微镜观察到,并且能够用CCD对其成像进行表征。数据分析表明,焦平面一定面积内的成像粒更多还原

【Abstract】 Based on the high light-scattering power of resonance light scattering (RLS) particles, the applications of RLS particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications, ultrasensitive immuno- and DNA probe assays combined with microscopy and imaging techniques have being addressed more and more interests. It has proved that the use of RLS particles as labels can obtain the significant increase in sensitivity over fluorescent labels. In this contribution, based on the common RLS method, we established a new resonance light scattering imaging technique and can observe the features of individual scatterers. The method has been applied to sensitive assay of biological macromolecules and medicine molecules by imaging and detecting the single light scattering species. The paper consists of four sections.(1) The spectral properties of hydrolyzed Cr(III) oligomers were investigated and their interaction with nucleic acids was also investigated by measurement of enhancement of resonance light scattering (RLS) signals. Hydrolyzed Cr(III) oligomers have characteristic absorption peaks of 415 nm and 580 nm. It was observed that Cr(III) oligomers displayed a fluorescent property with maximum excitation and emission wavelengths at 225 nm and 350 nm, respectively. In the acid medium, the hydrolyzed Cr(III) oligomers can interact with DNA, resulting in strong enhanced RLS signals characterized at 299.0 nm. Based on this, the interaction of inorganic polymer reagent with biological macromolecules was discussed, and the sensitive method of nucleic acids was developed.(2) In acidic medium, J-aggregation of α.β.γ.δ-tetrakis(p-sulfophenyl)porphine(TPPS4) and α,β,γ,δ-tetrakis(5-sulfothienyl)prophine [T(5-ST)P] in the presence of proteins occurs, resulting in strong RLS signals characterized at 490 nm and 469.0 nm, respectively. Under the excitation of 488 nm light beam of argon ion laser source, RLS signals of single protein-induced .J-aggregation species could be observed at the right angle to the incident beam with a common microscope and the images could be digitally imaged by a cooled charge-coupled device (CCD). Data analysis showed that the counts of the aggregation species imaged in detection focus plane are proportional to the concentration of proteins. The sensitive method of proteins was established accordingly. The results of determination for three human blood serum samples were identical to the reference values according to the method of Coomassie Brilliant Blue (CBB G- 250).(3) In neutral medium, the combination of heparin with α,β,γ,δ-tetra(4-trimethyl aminonium phenyl)prophine (TAPP) by electrostatic and hydrophobic interaction force can result in strong RLS signals at 430 nm. The single aggregation species of heparin with TAPP could be illuminated by a 441.6 nm laser source, and the light scattering signals of single species could be observed with a common microscope. Data analysis for the images of aggregation species by a digitalized CCD camera showed that the counts of aggregate species in the detection focus plane are proportional to the concentration of heparin. The method was successfully applied to detect heparin content in heparin sodium injection samples.(4) A light scattering imaging method combined with microfluidic channel device was developed and applied to the determination of trace amount of berberine based on the high light scattering power of single light scattering particles. In neutral medium, a single aggregation species of berberine with a, p, y, 8-tetrakis(p-sulfophenyl)prophine (TPPS4) inside a microflow channel can be illuminated by a 488 nm laser beam, which is guided through a prism to incident the aggregation species. The light scattering signals of single species could be observed with a common microscope, and the images could be captured by a digital CCD更多还原