后向光散射分析技术开发及其在生化分析中的应用
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摘要
共振光散射技术(RLS)自其建立以来，因操作简便和灵敏度高已广泛应用于研究分子的聚集行为及生物大分子和药物分析等领域，但存在选择性差的缺点。本文在普通RLS技术的基础上，设计了后向光散射(Backward resonance lightscattering，BRLS)信号检测装置，通过检测后向的共振光散射信号，实现了对生物大分子的高灵敏及高选择性分析。
在pH 3.50的酸性介质中，萘铬绿与蛋白质发生静电作用产生以342.0nm为特征峰的共振光散射(RLS)增强光谱。在此波长下，蛋白质的浓度与增强共振光散射强度(ΔI_(RLS))呈线性关系，据此建立了痕量蛋白质的共振光散射分析法，方法成功地应用于尿样中总蛋白质分析。
在pH 2.90，离子强度为0.003 M时，在阳离子表面活性剂溴化十六烷基三甲铵(CTMAB)存在下，QT-蛋白质相互作用产生的粒子富集在H_2O／CCl_4界面上时，会在376nm产生BRLS特征峰，增强的BRLS信号，与HSA和BSA浓度成正比例关系，据此建立了灵敏测定蛋白质的分析方法。
在pH 3.00，铝离子和水相中DNAs的结合能够产生增强的共振光散射信号，Al(Ⅲ)-DNAs这种二元复合物还可以结合CCl_4溶液中的tetraphenylporphyrin(TPP)，形成新的三元复合物TPP-Al(Ⅲ)-DNAs并富集在界面上。该复合物469nm产生强烈增强的BRLS信号，并且I_(BRLS)正比于ctDNA和fsDNA的浓度，可用于灵敏的测定核酸，方法的检出限在皮克级。
在pH 6.80，三辛基氧膦(TOPO)的存在下，Cr(Ⅲ)的水解物和核酸、肝素、黄原胶等生物大分子在H_2O／CCl_4界面上发生共吸附，形成较大的散射颗粒，增强的O_(BRLS)正比于生物大分子的浓度，据此建立了生物大分子的分析方法，发展了新类型的散射分析探针。
Since resonance light scattering (RLS) technique is developed, for its remarkable characteristics of simple operation and high sensitivity, this technique has been extensively applied in the investigations of molecular aggregation behavior and the analyses of biological supermolecular and Pharmaceuticals and so on. In this contribution, based on the common RLS method, we established a new backward resonance light scattering (BRLS) technique. A highly sensitive and selective assay method of biological macromolecules is proposed based on the measurements of BRLS signals at water/tetrachloromethane (H2O/CCl4) interface.
At pH 3.50, the static interaction of Naphthochrome Green (NG) with proteins occurs and results in greatly enhanced resonance light scattering (RLS) signals characterized by a peak at 277.0nm and 342.0nm. It was found that the enhanced RLS intensity (IRLS) at 342.0 nm is proportional to the concentration of proteins, and a new method for trace proteins was established accordingly. The results of determinations for urine samples were in agreement with the desired values.
At pH 3.00, the interaction of aluminum (III) with the phosphate group of DNAs in aqueous solution can result in enhanced resonance light scattering (RLS) signals. The binary complex of Al(III)-DNAs then interacts with tetraphenylporphyrin (TPP) in tetrachloromethane, forming a ternary complex of TPP-Al(III)-DNAs. It was observed that greatly enhanced backward resonance light scattering (BRLS) signals occurs with maximum peak at 469 nm when the ternary complex of TPP-Al(III)-DNAs were absorbed to the liquid/liquid interface. It was found that the enhanced backward resonance light scattering intensity (Ibrls) is in proportion to the concentration of calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA). The limits of detection (3a) are in the
picogram level.
At pH 2.90 and ionic strength 0.003M, in the presence of cationic surfactant, we study the interaction of proteins with quercetin (QT). The resulted species of the interaction, when concentrated at H2O/CCl4 interface, generate enhanced BRLS signals characterized at 376.0 nm which were found to be proportional to human serum albumin (HSA) and bovine serum albumin (BSA) in the range of 0.8-1250 ng/mL and 1.8-1250 ng/mL, respectively. Limit of determination (3d) of 74.6 and 184 pg/mL are available for the two proteins.
At pH 6.80, in the presence of TOPO, the interaction of hydrolyzed Cr(III) with the calf thymus DNA, heparin, xanthan gum in aqueous solution can result in enhanced backward resonance light scattering (BRLS) signals. When the ternary complex were absorbed to the liquid/liquid interface, it was observed that greatly enhanced BRLS signals occurs with maximum peak at 378 nm. We found that the enhanced /brls is in proportion to the concentration of biological macromolecules. The limits of determination (3d) are 0.85 ng ml-1, 1.65 ng ml-1, and 2.90 ng ml'1 for ctDNA, heparin sodium, and xanthan gum, correspondingly. We established new assay method and develop new RLS probe.
在pH 3.50的酸性介质中，萘铬绿与蛋白质发生静电作用产生以342.0nm为特征峰的共振光散射(RLS)增强光谱。在此波长下，蛋白质的浓度与增强共振光散射强度(ΔI_(RLS))呈线性关系，据此建立了痕量蛋白质的共振光散射分析法，方法成功地应用于尿样中总蛋白质分析。
在pH 2.90，离子强度为0.003 M时，在阳离子表面活性剂溴化十六烷基三甲铵(CTMAB)存在下，QT-蛋白质相互作用产生的粒子富集在H_2O／CCl_4界面上时，会在376nm产生BRLS特征峰，增强的BRLS信号，与HSA和BSA浓度成正比例关系，据此建立了灵敏测定蛋白质的分析方法。
在pH 3.00，铝离子和水相中DNAs的结合能够产生增强的共振光散射信号，Al(Ⅲ)-DNAs这种二元复合物还可以结合CCl_4溶液中的tetraphenylporphyrin(TPP)，形成新的三元复合物TPP-Al(Ⅲ)-DNAs并富集在界面上。该复合物469nm产生强烈增强的BRLS信号，并且I_(BRLS)正比于ctDNA和fsDNA的浓度，可用于灵敏的测定核酸，方法的检出限在皮克级。
在pH 6.80，三辛基氧膦(TOPO)的存在下，Cr(Ⅲ)的水解物和核酸、肝素、黄原胶等生物大分子在H_2O／CCl_4界面上发生共吸附，形成较大的散射颗粒，增强的O_(BRLS)正比于生物大分子的浓度，据此建立了生物大分子的分析方法，发展了新类型的散射分析探针。
Since resonance light scattering (RLS) technique is developed, for its remarkable characteristics of simple operation and high sensitivity, this technique has been extensively applied in the investigations of molecular aggregation behavior and the analyses of biological supermolecular and Pharmaceuticals and so on. In this contribution, based on the common RLS method, we established a new backward resonance light scattering (BRLS) technique. A highly sensitive and selective assay method of biological macromolecules is proposed based on the measurements of BRLS signals at water/tetrachloromethane (H2O/CCl4) interface.
At pH 3.50, the static interaction of Naphthochrome Green (NG) with proteins occurs and results in greatly enhanced resonance light scattering (RLS) signals characterized by a peak at 277.0nm and 342.0nm. It was found that the enhanced RLS intensity (IRLS) at 342.0 nm is proportional to the concentration of proteins, and a new method for trace proteins was established accordingly. The results of determinations for urine samples were in agreement with the desired values.
At pH 3.00, the interaction of aluminum (III) with the phosphate group of DNAs in aqueous solution can result in enhanced resonance light scattering (RLS) signals. The binary complex of Al(III)-DNAs then interacts with tetraphenylporphyrin (TPP) in tetrachloromethane, forming a ternary complex of TPP-Al(III)-DNAs. It was observed that greatly enhanced backward resonance light scattering (BRLS) signals occurs with maximum peak at 469 nm when the ternary complex of TPP-Al(III)-DNAs were absorbed to the liquid/liquid interface. It was found that the enhanced backward resonance light scattering intensity (Ibrls) is in proportion to the concentration of calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA). The limits of detection (3a) are in the
picogram level.
At pH 2.90 and ionic strength 0.003M, in the presence of cationic surfactant, we study the interaction of proteins with quercetin (QT). The resulted species of the interaction, when concentrated at H2O/CCl4 interface, generate enhanced BRLS signals characterized at 376.0 nm which were found to be proportional to human serum albumin (HSA) and bovine serum albumin (BSA) in the range of 0.8-1250 ng/mL and 1.8-1250 ng/mL, respectively. Limit of determination (3d) of 74.6 and 184 pg/mL are available for the two proteins.
At pH 6.80, in the presence of TOPO, the interaction of hydrolyzed Cr(III) with the calf thymus DNA, heparin, xanthan gum in aqueous solution can result in enhanced backward resonance light scattering (BRLS) signals. When the ternary complex were absorbed to the liquid/liquid interface, it was observed that greatly enhanced BRLS signals occurs with maximum peak at 378 nm. We found that the enhanced /brls is in proportion to the concentration of biological macromolecules. The limits of determination (3d) are 0.85 ng ml-1, 1.65 ng ml-1, and 2.90 ng ml'1 for ctDNA, heparin sodium, and xanthan gum, correspondingly. We established new assay method and develop new RLS probe.
引文
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