Determination of proteins by their enhancement of resonance light scattering by fuchsine acid

Based on the measurement of the enhancement of resonance light scattering (RLS) of fuchsine acid (FSA) by proteins, a novel sensitive assay of proteins in body fluid samples has been developed. Proteins, including bovine serum albumin (BSA), human serum albumin (HSA), pepsin (Pep), alpha-chymotrypsin (Chy), lysozyme (Lys), and cellulase (Cel), can bind to fuchsine acid (FSA), resulting in enhanced RLS signals at 277.0 nm. Linear relationships between the enhanced RLS intensity and the protein concentration were measured at different concentration of FSA, and the limits of detection for BSA, HSA, and Lys were found to lie in the nanogram range.     

A resonance light-scattering determination of proteins with fast green FCF.

The interaction of Fast Green FCF (FCF) with proteins (including bovine serum albumin (BSA), human serum albumin (HSA), pepsin (Pep) and alpha-chymotrypsin (Chy), and lysozyme (Lys)) was characterized by enhanced resonance light-scattering (RLS) measurements using a common spectrofluorometer. The enhanced RLS signals of FCF by proteins at 279.0 nm were obtained, and the mechanism of the RLS enhancement was considered in terms of the effects of the pH and ionic strength on the interaction. It was found that the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of nanogram levels, displaying that the present assay is much more sensitive than the reported RLS methods, with the limits of determination being 4.54, 0.6, 22.8, 4.32 and 1.75 ng/ml for BSA, HSA, Pep, Chy, and Lys. respectively.     

全氟辛烷磺酸与蛋白质体系的共振光散射光谱及其分析应用

研究了牛血清白蛋白(BSA)与全氟辛烷磺酸(PFOS)相互作用的共振光散射(RLS)光谱,建立了PFOS的共振光散射分析方法。 在pH值为4.1的BR缓冲溶液中,全氟辛烷磺酸根阴离子与质子化的BSA通过静电引力和疏水作用形成离子缔合物,引起共振光散射强度(IRLS)显著增强,最大散射波长位于285.0 nm处,增强的散射信号强度与PFOS浓度在0.2～25.0 μmol/L范围内呈线性关系,据此建立了测定PFOS的光散射分析方法,检出限为20.0 nmol/L。 讨论了体系的最佳反应条件及外来物质的干扰,并探讨了反应机理。 建立的共振光散射法用于环境水样中PFOS的测定,RSD≤4.4%。     

A flow injection sampling resonance light scattering system for total protein determination in human serum

A novel flow injection method with resonance light scattering detection was developed for the determination of total protein concentrations. This method is based on the enhancement of RLS signals from Methyl Blue (MB) by protein. The enhanced RLS intensities at 333 nm, in a pH 4.1 acidic aqueous solution, were proportional to the protein concentration over the range 2.0-37.3 and 1.0-36.0 microg ml-1 for human serum albumin (HSA) and bovine serum albumin (BSA), respectively. The corresponding limits of detection (3sigma) of 45 ng ml-1 for HSA and 80 ng ml-1 for BSA were attained. The method was successfully applied to the quantification of total proteins in human serum samples, the maximum relative error is less than 1% and the recovery is between 98% and 102%. The sample throughput was 60 h-1.     

Determination of proteins at nanogram levels with Bordeaux red based on the enhancement of resonance light scattering

A simple, sensitive and selective method was proposed for the determination of proteins by using a resonance light scattering technique. The weak resonance light scattering (RLS) of Bordeaux red (BR) can be enhanced greatly in the pH range 3.87-3.96 by the addition of micro amounts of proteins, resulting in four characteristic peaks in the wavelength range 250-600 nm. At the maximal wavelength of 363 nm, the enhanced RLS is proportional to the concentration of proteins in the range 0.12-10.8 microg ml-1 for bovine serum albumin (BSA) and 0.24-18.0 microg ml-1 for human serum albumin (HSA). The detection limits were 40.0 and 52.9 ng ml-1 for BSA and HSA, respectively. The present method has been applied to the determination of total proteins in human serum, urine and saliva samples. The obtained results are in good agreement with those obtained by the Bradford method with relative standard deviations (R.S.D.) of 0.9-2.3%.     

Resonance light scattering technique for the determination of proteins with resorcinol yellow and OP

A new resonance light scattering (RLS) assay of proteins is presented. In the citric acid-NaOH (pH 2.35) buffer, the RLS of Resorcinol yellow (RY)-protein system can be greatly enhanced by addition of nonionic surfactant OP, owing to the interaction between OP and RY-protein. The enhanced RLS is in proportion to the concentration of proteins in the range 0.02-4.0 mug ml(-1) for both bovine serum albumin (BSA) and bovine hemoglobin (HEM), the detection limits were 10.4 ng ml(-1) (S/N=3) for BSA and 11.4 ng ml(-1) (S/N=3) for HEM. Samples were determined satisfactorily.     

Interactions of Janus Green B with double stranded DNA and the determination of DNA based on the measurement of enhanced resonance light scattering

A novel assay of DNA with a sensitivity at the nanogram level is proposed based on the measurement of enhanced resonance light scattering (RLS) signals resulting from the interaction of Janus Green B (JGB) with DNA. At pH 6.37 and ionic strength < 0.20, the RLS signals of JGB were greatly enhanced by DNA in the region of 300-650 nm characterized by three peaks at 416.0, 452.0 and 469.2 nm. The binding properties were examined using a Scatchard plot based on the measurement of the enhanced RLS data at 416.0 nm at a high JGB: DNA molar ratio (R > 2.22), and an aggregation mechanism of JGB in the presence of DNA at the nanogram level is proposed. Linear relationships can be established between the enhanced RLS intensity and DNA concentration in the range 0-3.5 micrograms ml-1 for both calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA) if 2.0 x 10(-5) M JGB is employed. The limits of determination were 8.7 ng ml-1 for ctDNA and 9.9 ng ml-1 for fsDNA, respectively. Synthetic samples were analysed satisfactorily.     

Determination of Proteins by Measuring Total Internal-reflected Resonance Light Scattering Signals on Water／Tetrachloromethane Interface with Evans Blue and Cetyltrimethylammonium Bromide

A sensitive and selective assay of proteins is proposed based on measuring the total internal-reflected resonance light scattering(TIR-RLS) signals produced on the water/tetrachloromethane( H2O/CCl4 ) interface. In an aqueous medium with pH value in the range of 3. 29-3. 78, electrostatic attraction occurs between the negatively charged Evans Blue(EB) and positively charged proteins, forming hydrophobic ion associates and resulting in EB-protein adsorption on H20/CC14 interface. The presence of cetyhrimethylammonium bromide prompts this adsorption, resulting in strongly enhanced TIR-RLS signals. The intensity of the enhanced TIR-RLS at 360-370 nm was found to be proportional to the concentration of proteins. For bovine serum albumin and human serum albumin, the linear range of detection is 0. 07-1.2μLg/mL and the limits of detection are 6. 68 and 6. 30 ng/mL(3σ) , respectively, while for lysozyme, the linear range of detection is 0. 06-1.0μg/mL and the limit of detection is 6. 0 ng/mL(3σ). The content of the total albumin in a human urine samplc could be directly determiined by using the standard addition method with a percent recovery of 97.6%-104. 1% , and the RSD ranging from 1. 9% to 4. 2%.     

Determination of Proteins Based on Their Resonance Light Scattering Enhancement Effect on Manganese-Tetrasulfonatophthalocyanine

This is the first report on the determination of proteins with manganese-tetrasulfonatophthalocyanine (MnTSPc) by resonance light scattering (RLS). At pH 3.6, the interactions of MnTSPc with the enhanced RLS signals at 344nm, proteins, including bovine serum albumin (BSA), human serum albumin (HSA), human -IgG, and ovalbumin, can be determined with a limit of detection below 20ngmL^–1. The method has been applied to the analysis of total protein in human serum samples collected from a hospital, and the results were in good agreement with those reported by the hospital.     

Determination of nanograms of proteins based on the amplified resonance light scattering signals of Tichromine

A new high-sensitivity detection of protein assay at the nanogram level is developed based on the amplified resonance light scattering signals (RLS) of Tichromine (TCM). In Walpole (NaAc–HCl) buffer (pH 4.05), TCM reacts with proteins to form large particles, which results in remarkable enhanced RLS signals characterized by three peaks at 293 nm, 399 nm and 553 nm. Mechanistic studies showed that the enhanced RLS stems from a large complex of TCM–BSA formed for the electrostatic effect between TCM and BSA. With the enhanced RLS signals at the three wavelengths, the enhanced RLS intensity is proportional to the concentration of proteins in an appropriate range. The lowest limit of determination was 7.4 ng mL^?1. The proposed method was successfully applied to determine total protein in human serum samples.     

Determination of cationic surfactants in water samples by their enhanced resonance light scattering with azoviolet

A simple assay of cationic surfactants in water samples was developed based on the measurements of enhanced resonance light scattering (RLS). At pH 6.09 and ionic strength 0.03 M, the interactions of azoviolet (AV) with cationic surfactants, including zephiramine (Zeph) and cetyl trimethyl ammonium bromide (CTMAB), result in enhanced RLS signals characterized by the peaks of 470.0, 485.0 and 495.0 nm. The enhanced RLS intensity is proportional to the concentration of cationic surfactant of Zeph in the range of 0.2~6.0x10(-6) M, and to that of CTMAB in the range of 0.4~4.8x10(-6 )M. The limit of determination (3 sigma) is 2.1x10(-8) M and 3.8x10(-8) M for the two surfactants, respectively. Determinations of cationic surfactants in synthetic and tap water samples were successfully made with a recovery of 90.5~108.6%.     

硅钨酸与盐酸巴马汀相互作用光谱研究及其分析应用

在pH 为0.91 的HCl-NaOAc 缓冲介质中, 盐酸巴马汀与硅钨酸通过静电相互作用导致共振光散射信号显著增强,最大散射波长为297 nm, 增强的散射信号强度与盐酸巴马汀浓度在0.09～3.2 μg/mL 范围内呈线性关系, 据此建立了盐酸巴马汀的共振光散射分析方法, 检测限为9.1 ng/mL. 实验考察了pH、离子强度对体系的影响, 研究了体系紫外吸收光谱及荧光光谱, 讨论了共振光散射增强机理. 该方法用于黄藤素片、黄藤素胶囊及血清样品中盐酸巴马汀含量测定,RSD≤4.2%.     

盐酸奎宁与全氟辛烷磺酸体系的共振光散射光谱研究及其分析应用

研究了盐酸奎宁(Quinine dihydrochloride,简称Quinine)与全氟辛烷磺酸(perfluorooctane sulfonate,简称PFOS)相互作用的共振光散射(resonance light scattering,RLS)光谱,并建立了PFOS的共振光散射分析方法.在pH值为2.87的Britton-Robinson(BR)缓冲溶液中,全氟辛烷磺酸根阴离子与质子化的盐酸奎宁通过静电引力和疏水作用形成2:1的离子缔合物,引起共振光散射强度(IRLS)显著增强,最大散射波长位于283nm处,增强的散射信号强度与PFOS浓度在0.10～50.0μmol/L范围内呈线性关系,据此建立了测定PFOS的散射分析方法,检测限为9.88nmol/L.讨论了体系的最佳反应条件及外来物质的干扰,同时研究了体系的吸收光谱及荧光光谱,并探讨了反应机理.本方法用于水样及人体血清样品中PFOS的测定,RSD≤4.2%.     

同多酸和染料探针共振光散射法测定蛋白质

在酸性条件下,铬黑T、钼酸铵与蛋白质形成聚合物,使体系的共振光散射明显增强。据此建立了利用共振光散射技术测定总蛋白含量的新方法。在最佳条件下,体系的最大散射峰位于555nm处。共振光散射增强的程度与蛋白质的浓度呈良好的线性关系。牛血清白蛋白和人血清白蛋白的线性范围分别为0.20～10.0μg/mL和0.10～8.0μg/mL,检出限为0.050μg/mL和0.039μg/mL。方法已用于人血清样品的分析,并与考马斯亮蓝的测定结果进行了比较,两者无显著性差异。     

A resonance light scattering ratiometry applied for binding study of organic small molecules with biopolymer

Resonance light scattering (RLS) technique is a creative application of light scattering signals detected by using a common spectrofluorometer, but it has drawbacks such as the fluctuation of signals caused by poorly quantified or variable factors. Herein we develop a RLS ratiometry to overcome the drawbacks of the technique and apply to measure the binding nature of organic small molecules (OSM) with biopolymer using the binding of cation porphyrins with heparin (HP) as an example. In near neutral solution, cationic porphyrins meso-tetrakis [(trimethylammoniumyl) phenyl] porphyrin (TAPP) and meso-tetra (4-methylpyridy) porphyrin (TMPyP-4) interact with heparin, resulting in hypochromatic effect, and enhanced RLS signals. Linear relationship could be established between the ratio of enhanced RLS signals at two wavelengths, where the maximum and minimum are available in the ratio curve of UV-vis spectrum of porphyrin to that of heparin-porphyrin complex, and the logarithm of heparin concentration, and thus a wide dynamic range detection method of biopolymers could be developed. In comparison with RLS method, this RLS ratiometric one is less affected by environmental conditions such as pH, ionic strength. The mechanism of these interactions was investigated based on the charge density distribution of the two porphyrin molecules and it could be concluded that the enhanced RLS intensity is proportionally promoted by the charge capacity of components in the complex. Additionally, the binding number and binding constant were measured scientifically by Scatchard plot.     

Resonance Light Scattering Method for Microdetermination of Proteins Based on Aggregation of Au Nanoparticles

By using a resonance light scattering (RLS) technique, a highly sensitive method for protein determination based on the aggregation of Au nanoparticles on protein template is described. For the Au nanoparticles of 15 nm, the detection limit of bovine serum albumin was 5.0 ng/mL and the linear range was 10–300 ng/mL. The experimental results indicated that various metal ions do not interfere with this assay. The proposed RLS assay exhibited lower variation in response signals for the same weight of different proteins and showed satisfactory results when it was used for determination of proteins in human serum.     

Study of the interaction of proteins with curcumin and SDS and its analytical application

It is found that protein and sodium dodecyl sulphonate (SDS) can enhance resonance light scattering (RLS) of curcumin (CU). Based on this phenomenon, a new quantitative method for protein in aqueous solution has been developed. In the BR (pH 3.5) buffer, the RLS intensity of CU-SDS system is greatly enhanced by protein. The enhanced RLS is proportional to the concentration of protein in the range of 0.00020-20.0 microgml(-1) for bovine serum albumin (BSA) and 0.00040-1.0 microgml(-1) for human serum albumin (HSA) and their detection limits are 0.16 and 0.041 ngml(-1), respectively. An actual sample is satisfactorily determined. In addition, the interaction mechanism between protein and CU-SDS is also studied by using multi-techniques such as RLS, absorption spectroscopy and fluorescence, zeta potential assay measurement.     

Resonance light scattering technique for the determination of protein with rutin and cetylpyridine bromide system

A new resonance light scattering (RLS) assay of protein is presented. In Tris-NaOH (pH = 10.93) buffer, the RLS of rutin-cetylpyridine bromide (CPB) system can be greatly enhanced by protein, including bovine serum albumin (BSA) and human serum albumin (HSA). The enhanced RLS intensities are in proportion to the concentration of proteins in the range of 5 x 10(-9) to 2.5 x 10(-6) g ml(-1) for BSA and 2.5 x 10(-8) to 3.5 x 10(-6) g ml(-1) for HSA. The detection limits (S/N = 3) are 3.0 ng ml(-1) for BSA and 10.0 ng ml(-1) for HSA. Samples are determined satisfactorily.     

Determination of protein by its enhancement effect on the Rayleigh light scattering of carboxyarsenazo

This is the first report on the determination of proteins based on the interaction with carboxyarsenazo (CAA) by Rayleigh light scattering (RLS). At pH 4, the weak RLS of CAA can be enhanced greatly by the addition of proteins, resulting in three characteristic peaks. Based on this, the interactions of CAA with nine kinds of proteins were studied and a new quantitative determination method for proteins has been developed. This method is very sensitive (0.10-15.3 mug ml(-1) for bovine serum albumin (BSA)), rapid (<2 min), simple (one step), tolerant of most interfering substances, and gives a close value to that of the Coomassie brilliant blue (CBB) method in the determination of proteins in human serum. Thus, the CAA assay can be useful for routine analytical purposes and may overcome some of the limitations of other currently employed methods. Mechanism studies show that the three RLS peaks correspond to the absorption valleys of the CAA-protein complex.     

藉L-半胱氨酸包覆的ZnS纳米粒子的共振光散射定量分析蛋白质

合成和表征了功能性L-半胱氨酸包覆的ZnS纳米粒子。在pH5．12的NaAc-HAc溶液介质中,L-半胱氨酸包覆ZnS纳米粒子于波长308．0nm处出现共振光散射峰。一定量蛋白质的加入能明显增强体系的共振光散射,且峰强度增加值与蛋白质浓度间存在良好线性关系,据此建立了一种灵敏的测定微量蛋白质的方法。用L-半胱氨酸包覆ZnS纳米粒子作为探针,不仅克服了有机染料可能出现的光漂白等缺点,而且本身不具毒性。该法用于人血清试样中总蛋白的测定,其结果与临床数据一致。