【作者】 陆巍；

【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】 Pasternack等于20世纪90年代初提出的共振光散射(resonance light scattering,RLS)技术是一项借助普通荧光分光光度计灵敏检测聚集体光散射信号的分析技术。RLS技术因其操作简便并有力克服了传统光散射技术灵敏度低和仪器条件要求严格的缺点而迅速引起了诸多研究者的浓厚兴趣和广泛关注。从1996年到现在短短的7年间，RLS技术已经广泛和成功地应用于生物大分子和药物的分析研究领域。这些研究发现，与测定生物大分子和药物常常采用的吸光光度法和荧光光度法相比，RLS技术除了能获得更高灵敏度的优点外，还具有探针范围更广泛的优势。 本文分别以与蛋白质作用后既无明显颜色又无显著荧光性质变化的阴离子表面活性剂十二烷基苯磺酸钠(sodium dodecyl benzene sulfonate,SDBS)和酸性三苯甲烷染料水溶性苯胺蓝(water blue,WB)为例，成功建立了能简单、快速和灵敏地分析蛋白质的RLS方法，从而进一步证明了RLS技术在拓展分析探针方面的独特优势。 在pH1.98，离子强度为0.001时，十二烷基苯磺酸钠(sodium dodecyl benzene sulfonate,SDBS)的RLS信号能被蛋白质强烈增强，其最大散射峰位于470.0nm。增强的RLS信号与蛋白质浓度在一定范围内成正比，该方法分析蛋白质的检测限小于58.1ng／ml。该方法成功地应用于合成样和尿样中蛋白质的分析。 在pH2.09，离子强度为0.001时，水溶性苯胺蓝(water blue,WB)的RLS信号能被蛋白质强烈增强，其最大散射峰位于346.0nm。增强的RLS信号与蛋白质浓度在一定范围内成正比，该方法分析蛋白质的检测限小于1.5ng／ml。该方法成功地应用于合成样和尿样中蛋白质的分析。 虽然RLS技术具有上述的优点，但依然存在着一些不足之处。例如，RLS技术还不能把待分析物的光散射信号与其共存物质的光散射信号区分开来。这就在很大程度上限制了RLS技术选择性的进一步提高。为了弥补RLS技术的不足，近来，我们把RLS技术与液／液界面(liquid／liquid interface)上的全内反射光(total internal reflection light)相结合，成功地开发出了全内反射共振光散射(total internal reflected resonance light scattering,TIR-RLS)技术：一种通过检测液／液界面上的RLS西南师范大学硕士论文 界面光散射分析技术的开发及其在生化和药物分析中的应用信号的变化进行待分析物检测的界面光散射技术（interfacial h吵t scatteringtechnique卜在该技术中，待分析物通过与其它试剂结合形成双亲复合物（alnphiphilicspecies）而吸附在液／液界面上。这样，在由入射光于液／液界面全反射时在界面上形成的渐消失波一vanesceflt wave）的激发下，该双亲复合物能产生显著增强的RLS信号。由于待分析物通过形成双亲复合物而吸附在液／液界面上，故很好达到了与其共存物质相分离的目的。因此，TIR－gys技术能进一步提高RLS技术的选择性。此外，在此过程中，待分析物也在液／液界面上发生了富集，故与RLS技术相比，TIR－ffes技术能获得更高的灵敏度。 本文根据液／液界面反应机理（liquid／liqui inte血e reaction mechanls叫、协同吸附机理（synerglstlc adsorption mechanism）和共吸附机理（co刎sorptlon mecharusm），分别采用 Eu（Ill）三辛基氧化磷（trioctylphosphine oxide，TOPO）、罗丹明 BfrhodamineB，RhB）一演化十六烷基三甲按（cetyltnmethylanunonium bromide，CTMAB）和叮咬橙（3cnndin orange，AO卜漠化十y烷基。甲按ketylgmethyl。omm bromide，CTMAB）与核酸作用以及分别采用阴离子表面活性剂十二烷基苯磺酸钠kodiumdodecyl benzene sulfonate，SDBS）、十二烷基硫酸钠（sodium dodecylsulfonate，SDS）和十二烷基磺酸钠kodium lauryl sulfate，SLS）与维生素 B；…itamin BI，VBI）作用，在水／四氯化碳（HZO／CCIO界面上进行核酸和VB；的测定。 在 pH 8石9，离于强度为 0刀08时，核酸一EZ（lll和 TOPO在 HZO／CCb界面上发生络合作用形成三元双亲复合物核酸E（Ill）1OPO。在此过程中，核酸被吸附和富集在HZO／CCly界面上，引起强烈增强的TIR－RLS信号，其最大散射峰位于348刀urn。由此可以灵敏地测定核酸。该方法对干扰物质具有良好的选择性。 在 PH 5．72，离子强度为 0．006 M的条件下，在水相中核酸通过与具有双亲性的Aim和CTMAB相互作用形成三元双亲复合物RhB一核酸．CTMAB，而被M和CTMAB协同吸附在H。O／CC八界面上，引起显著增强的TIR－RLS信号，其最大散射峰位于367刀 urn。由此可以灵敏地测定核酸。该方法对干扰物质具有良好的选择性。 在PH 7．56，离子强度为 0刀07 M的条件下，核酸通过与具有双亲性的AO和CTMAB相互作用形成三元双亲复合物AO一核酸一CTMAB，而被AO和CTMAB协同吸附在HZO／CC14界面上，引起显著增强的TIR－RLS信号，其最大散射峰位于368刀 urn。由此可以灵敏地测定核酸。该方法对干扰物质具有良好的选择性。 在 pH 3．29，离子强度为 0刀03时，通过静电作用力 VB；能分别与 SDBS、SDS和 SLS相互作用形成双亲复合物VB；?更多还原

【Abstract】 Resonance light scattering (RLS) technique, proposed by Pasternack et al in the beginning of 1990’s, is a sensitive means to detect light scattering signals of aggregates with a conventional fluorescence spectrophotometer. After that, RLS technique attracted many researchers’ much interest and extensive concern not only for its simple manipulation but also for its high sensitivity and convenient experimental conditions, which cannot be achieved by traditional light scattering technique. From the 1996 year to now, the short period of 7 years, RLS technique has been widely and successfully applied to determine biomacromolecules and Pharmaceuticals. During these investigations, it was found that RLS technique has the advantage of possessing more extensive area of probe reagents as well as much better sensitivity, than spectrophotometry and spectrofluorimetry, which are often used as a routine way to assay biomacromolecules and pharmaceuticals. In this contribution, based on the significantly enhanced RLS signals of proteins in the presence of sodium dodecyl benzene sulfonate (SDBS) and water blue (WB), respectively, we established simple, rapid and sensitive assays of proteins although neither obvious color nor fluorescence change occurs in these interactions, further displaying the particular advantage of RLS technique in expanding the area of probe reagents.At pH 1.98 and ionic strength 0.001 M, the interactions of proteins with sodium dodecyl benzene sulfonate (SDBS) give rise to greatly enhanced RLS spectra with the maximum light scattering peak located at 470.0 nm. The enhanced RLS signals are in good proportional to the concentrations of proteins in the range of microgramme levels. With this method, proteins, including bovine serum albumin (BSA), human serumalbumin (HSA),α-chymotrypsin (Chy), lysozyme (Lys), can be determined with the limit of detection below 58.1 ng ml"’. Artificial samples and human urine samples were analyzed satisfactorily.At pH 2.09 and ionic strength 0.001, strongly enhanced RLS spectra of the mixture of proteins and WB with the maximum light scattering peak at 346.0 nm can be observed and the enhanced RLS signals at 346.0 nm are in good proportion to the concentrations of proteins in the range of microgramme levels. With the enhanced RLS signals at 346.0 nm, proteins, including bovine serum albumin (BSA), human serum albumin (HSA), γ-globulin (γ-IgG), a-chymotrypsin (Chy), lysozyme (Lys) and cellulose (Cel), can be determined with the limit of detection below 1.5 ng ml-1. Synthetic samples and human urine samples were analyzed satisfactorily.However, a regretting case is that RLS technique still suffers from several disadvantages, such as failing to distinguish light scattering signals of analytes with those of their coexisting foreign substances, which are not beneficial to improve further selectivity and sensitivity. One way to solve this problem, in our opinion, is to introduce an oil/water system. In an oil/water system, amphiphilic species, involving both hydrophilic moieties and hydrophobic moieties, are repelled from both the water and oil phases, but can be well adsorbed at the oil/water interface. As a result, an analyte can be well separated from its coexisting substances and be enriched to the oil/water interface by means of developing an amphiphilic species with appropriate reagents, and thus determination of the analyte with better selectivity and sensitivity can be available. Lately, we successfully combined RLS method with total internal reflected (TIR) light to propose a total internal reflected resonance light scattering (TIR-RLS) method to analyze amphiphilic species at an oil/water interface by simply constructing a TIR optical arrangement in the sample compartment of a conventional fluorescence spectrophotometer. In the present method, RLS signals are obtained from the amphiphilic species in the oil/water interfacial region with the excitation of an evanescent field coupled with total internal reflection of light at the interface. Since the inte更多还原