共振瑞利散射法灵敏测定蛋白质
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摘要
共振瑞利散射(Resonance Rayleigh scattering, RRS)法是一种20世纪90年代后发展起来的新的定量分析技术。近年来,RRS法由于操作简单、灵敏度高而受到人们的广泛关注。目前,RRS法不仅应用到生物大分子、无机离子、医药和阳离子表面活性剂的测定,而且在胶束浓度和包容常数等方面的测定也被广泛应用。
蛋白质在生物体中担负着各种重要的生理功能。它是构成细胞内原生质的主要组成成分,是组成自然界中各类生命的最重要的物质,可以这样说,没有蛋白质就没有生命。因此,蛋白质的定量测定对于生命科学和生物化学研究具有重要意义,是临床检验中的重要指标,也是许多生化物质和药物分离提纯中质量检验及食品检验中常见的分析项目。蛋白质的测定方法主要有：紫外吸收法、荧光光度法和共振光散射法等。共振瑞利散射法是近几年才发展起来的新技术。其特点是灵敏度高和简便实用,在蛋白质及核酸等生物大分子的分析测定中优势明显。
本论文基于共振瑞利散射法,开展了蛋白质的检测工作,主要包括两个方面内容：
1、在pH 4.2的BR(H3PO4,H3BO3和CH3COOH与0.2mol/L NaOH溶液按一定比例混合)缓冲溶液中,甲基蓝与免疫球蛋白相互作用生成复合物,使溶液的350nm处的共振瑞利散射光谱显著增强,增强程度与免疫球蛋白的浓度成正比关系。据此建立了测定免疫球蛋白的新方法,方法的线性范围为0.3-67.2μg/mL,检出限为0.03μg/mL。方法稳定性好、灵敏度高,用于人血清样品测定,结果令人满意。
2、以巯基乙酸为稳定剂,合成了CdSe量子点,用该量子点作探针共振瑞利散射法测定人血清蛋白。在pH 7.4的Tris-HCl缓冲溶液中,巯基乙酸包被的CdSe量子点通过共价偶联标记了人血清蛋白,生成的复合物导致溶液的共振瑞利散射光谱和二级散射光谱(second-order scattering, SOS)显著增强,散射光强度的增加与人血清蛋白的浓度在0.4-48μmol/L的范围内成正比关系,检出限分别为0.1μmol/L (RRS)和0.25μmol/L (SOS)。实验结果表明,RRS法的灵敏度比SOS高。该方法具有简单、灵敏度高和选择性好等优点。将所建立的方法用于尿样中HSA的测定,结果令人满意。
Resonance Rayleigh scattering(RRS) method is a new quantitative analytical technique which was developed in 1990s. In recent years, More attention has been paid to RRS methods, since it is very sensitive and simple. The RRS technique can not only be applied to the determination of biological macromolecules, inorganic ions, pharmaceuticals, and cationic surfactant, but also can be used to the determination of critical micelle concentration and inclusion constant.
Protein has many physiological functions. Intracellular proteins that form the main components of protoplasm, which occupy an important place in vivo, are composed of various types of natural life in the most important material. It can be said that without the protein there is no life. Quantitative analysis of protein is often involved in biochemistry and other biological disciplines. It has been an important indicator of clinical testing. Quantitative analysis of protein is also the means of purifying and separation in many biochemicals. Many methods have been developed for protein determination, such as UV absorption spectrophotometry, fluorescence spectrometry, resonance light scattering techniques, etc. Resonance Rayleigh scattering method is a new technology. The main advantage of this technique is sensitive and simple.
The main research content and innovation points are as follows:
1. In pH 4.2 BR buffer, the binding reaction of methyl blue with proteins enhanced remarkably the resonance Rayleigh scattering (RRS) signal at maximum emission wavelength of 350 nm. It was found that the increase value of RRS intensities were in proportion to the concentrations of IgG under optimum conditions. Based on this, a new method for the determination of IgG using methyl blue as indicator has been developed. The linear ranges of the calibration curves were 0.3-67.2μg/mL, and the detection limit was 0.3μg/mL. The method has the advantages of simplicity and high sensitivity. It has been used to determine IgG in human serum samples. The results were satisfactory.
2. CdSe quantum dots were synthesized in aqueous solution by using thioglycolic acid as the stabilizer. The interaction between CdSe quantum dots and human serum albumin was investigated by resonance Rayleigh scattering and second-order scattering spectra. In pH 7.4 Tris buffer solution, the CdSe quantum dots (CdSe-QDs) capped thioglycolic acid (TGA) were labeling with HSA by covalence coupled action, resulting in the great enhancements of intensities of RRS and second-order scattering (SOS), respectively. The enhancements of scattering intensity were directly proportional to the concentration of HSA in a certain range. The detection limits were 0.1μmol/L for RRS method and 0.25μmol/L for SOS. The results showed that the method was simple and sensitive. In addition, the reaction mechanism was discussed. The proposed method was successfully applied to the determination of HSA in uric samples with satisfactory.
蛋白质在生物体中担负着各种重要的生理功能。它是构成细胞内原生质的主要组成成分,是组成自然界中各类生命的最重要的物质,可以这样说,没有蛋白质就没有生命。因此,蛋白质的定量测定对于生命科学和生物化学研究具有重要意义,是临床检验中的重要指标,也是许多生化物质和药物分离提纯中质量检验及食品检验中常见的分析项目。蛋白质的测定方法主要有：紫外吸收法、荧光光度法和共振光散射法等。共振瑞利散射法是近几年才发展起来的新技术。其特点是灵敏度高和简便实用,在蛋白质及核酸等生物大分子的分析测定中优势明显。
本论文基于共振瑞利散射法,开展了蛋白质的检测工作,主要包括两个方面内容：
1、在pH 4.2的BR(H3PO4,H3BO3和CH3COOH与0.2mol/L NaOH溶液按一定比例混合)缓冲溶液中,甲基蓝与免疫球蛋白相互作用生成复合物,使溶液的350nm处的共振瑞利散射光谱显著增强,增强程度与免疫球蛋白的浓度成正比关系。据此建立了测定免疫球蛋白的新方法,方法的线性范围为0.3-67.2μg/mL,检出限为0.03μg/mL。方法稳定性好、灵敏度高,用于人血清样品测定,结果令人满意。
2、以巯基乙酸为稳定剂,合成了CdSe量子点,用该量子点作探针共振瑞利散射法测定人血清蛋白。在pH 7.4的Tris-HCl缓冲溶液中,巯基乙酸包被的CdSe量子点通过共价偶联标记了人血清蛋白,生成的复合物导致溶液的共振瑞利散射光谱和二级散射光谱(second-order scattering, SOS)显著增强,散射光强度的增加与人血清蛋白的浓度在0.4-48μmol/L的范围内成正比关系,检出限分别为0.1μmol/L (RRS)和0.25μmol/L (SOS)。实验结果表明,RRS法的灵敏度比SOS高。该方法具有简单、灵敏度高和选择性好等优点。将所建立的方法用于尿样中HSA的测定,结果令人满意。
Resonance Rayleigh scattering(RRS) method is a new quantitative analytical technique which was developed in 1990s. In recent years, More attention has been paid to RRS methods, since it is very sensitive and simple. The RRS technique can not only be applied to the determination of biological macromolecules, inorganic ions, pharmaceuticals, and cationic surfactant, but also can be used to the determination of critical micelle concentration and inclusion constant.
Protein has many physiological functions. Intracellular proteins that form the main components of protoplasm, which occupy an important place in vivo, are composed of various types of natural life in the most important material. It can be said that without the protein there is no life. Quantitative analysis of protein is often involved in biochemistry and other biological disciplines. It has been an important indicator of clinical testing. Quantitative analysis of protein is also the means of purifying and separation in many biochemicals. Many methods have been developed for protein determination, such as UV absorption spectrophotometry, fluorescence spectrometry, resonance light scattering techniques, etc. Resonance Rayleigh scattering method is a new technology. The main advantage of this technique is sensitive and simple.
The main research content and innovation points are as follows:
1. In pH 4.2 BR buffer, the binding reaction of methyl blue with proteins enhanced remarkably the resonance Rayleigh scattering (RRS) signal at maximum emission wavelength of 350 nm. It was found that the increase value of RRS intensities were in proportion to the concentrations of IgG under optimum conditions. Based on this, a new method for the determination of IgG using methyl blue as indicator has been developed. The linear ranges of the calibration curves were 0.3-67.2μg/mL, and the detection limit was 0.3μg/mL. The method has the advantages of simplicity and high sensitivity. It has been used to determine IgG in human serum samples. The results were satisfactory.
2. CdSe quantum dots were synthesized in aqueous solution by using thioglycolic acid as the stabilizer. The interaction between CdSe quantum dots and human serum albumin was investigated by resonance Rayleigh scattering and second-order scattering spectra. In pH 7.4 Tris buffer solution, the CdSe quantum dots (CdSe-QDs) capped thioglycolic acid (TGA) were labeling with HSA by covalence coupled action, resulting in the great enhancements of intensities of RRS and second-order scattering (SOS), respectively. The enhancements of scattering intensity were directly proportional to the concentration of HSA in a certain range. The detection limits were 0.1μmol/L for RRS method and 0.25μmol/L for SOS. The results showed that the method was simple and sensitive. In addition, the reaction mechanism was discussed. The proposed method was successfully applied to the determination of HSA in uric samples with satisfactory.
引文
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