Microdetermination of proteins with the arsenazo-DBN-Al(III) complex by Rayleigh light-scattering technique and application of the method

The determination of proteins with arsenazo-DBN and Al3+ by Rayleigh light-scattering (RLS) is described. The weak RLS of arsenazo-DBN and BSA can be enhanced greatly by addition of Al3+ in the pH range 5.3-7.0; this resulted in two enhanced RLS signals at 420-440 nm and 460-480 nm. The reaction between arsenazo-DBN, Al3+, and proteins was studied and a new method was developed for quantitative determination of proteins. This method is very sensitive (0.34-41.71 microg mL(-1) for bovine serum albumin, BSA, and 0.29-53.41 microg mL(-1) for human serum albumin, HSA), rapid (< 2 min), simple (one step), and tolerant of most interfering substances. The effects of different surfactants were also examined. When these proteins were determined in four human serum samples the maximum relative error was not more than 2% and the recovery was between 97 and 103%.     

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 with tetracarboxy manganese(II) phthalocyanine by resonance light scattering technique

A novel method for the determination of proteins by using tetracarboxy manganese(II) phthalocyanine (MnC4Pc) as a resonance light scattering (RLS) probe has been developed. At pH 3.0 Britton-Robinson (B-R) buffer solution, the RLS intensity of MnC4Pc at 385 nm is greatly enhanced in the presence of proteins. The effects of pH, reaction time, concentration of MnC4Pc and interfering substances on the enhanced RLS intensity are investigated, respectively. Under optimal conditions, the linear ranges of the calibration curves are 0-2.00 microg mL(-1) for bovine serum albumin (BSA) and human serum albumin (HSA), 0.0-1.75 microg mL(-1) for human-IgG and ovalbumin, with a detection limit of 16.37 ng mL(-1) BSA, 17.62 ng mL(-1) HSA, 19.41 ng mL(-1) human-IgG and 20.72 ng mL(-1) ovalbumin. The method has been applied to the determination of total proteins in human serum samples collected from a hospital and the results are in good agreement with those reported by the hospital.     

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。     

Study of the reaction of proteins with Beryllon II-AlIII by the Rayleigh light scattering technique and its application

The determination of proteins with tetrasodium 2-(3,6-disulfo-8-hydroxynaphthylazo)-1,8-dihydroxynaphthalene-3,6-disulfonate (Beryllon II) by Rayleigh light scattering (RLS) was studied. The weak RLS of the Beryllon II-bovine serum albumin (BSA) complex can be greatly enhanced by the addition of Al3+ in the pH range 5.6-7.2; there was a maximum RLS platform at 400-420 nm. Based on the reaction between Beryllon II, Al3+ and proteins, a new method for the determination of proteins was developed. This method is very sensitive [0.20-41.42 micrograms ml-1 for BSA and 0.18-48.15 micrograms ml-1 for human serum albumen (HSA)], rapid (< 2 min), simple (one step) and tolerant towards most interfering substances. The effects of different surfactants were also examined. Four samples of protein in human serum were determined; the maximum relative error was no more than 5% and the recovery was 96-105%.     

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 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.     

Resonance light scattering spectroscopy of beta-cyclodextrin-sodium dodecylsulfate-protein ternary system and its analytical applications.

A simple, highly sensitive and dye-less assay for proteins was reported using a resonance light-scattering (RLS) technique based on the enhanced RLS intensity of beta-cyclodextrin (beta-CD)-sodium dodecylsulfate (SDS)-protein system. Under the optimum conditions, the enhanced RLS intensity is in proportion to the concentration of proteins in the range of 0.01 to 2.3 microg ml(-1) for bovine serum albumin (BSA), 0.01 to 2.0 microg ml(-1) for human serum albumin (HSA), 0.015 to 5.0 microg ml(-1) for gamma-globulin (gamma-G), 0.02 to 3.5 microg ml(-1) for egg albumin (EA), 0.02 to 4.0 microg ml(-1) for pepsin (Pep), and 0.02 to 3.6 microg ml(-1) for alpha-chymotrypsin (Chy). Their detection limits (S/N = 3) are 1.1, 1.6, 2.4, 6.7, 5.4 and 4.2 ng ml(-1), respectively. Synthetic samples and human serum samples were determined satisfactorily, and the results were in reasonable agreement with those obtained by a documented spectrophotometric (Bradford) method.     

Determination of proteins by flow injection analysis coupled with the Rayleigh light scattering technique

A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of protein concentrations. This method is based on the weak intensity of RLS of p-nitrohenzene-azo-3,6 disulfo-1-amino-8-naphthol-7-azo-benzene disodium salt (Amide Black-10B) which can be enhanced by addition of protein in weakly acidic solution. It has proved that the application of this method to quantify the proteins by using human serum albumin was available in real samples. In addition, this method is very sensitive (the determination limits are 0.11 μg/mL for human serum albumen (HSA) and 0.85 μg/mL for bovine serum albumen, BSA), simple, rapid and tolerance of most interfering substances. The FIA-RLS method was more stabile than the general RLS method and the average R.S.D. value of FIA-RLS less than general RLS. The effects of different interfering substances will be also examined. The amount of proteins in human serum sample was determined and the maximum relative error was no more than 3.00% as well as the recovery was between 94.9 and 105.9%.     

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.     

A highly sensitive assay for protein with dibromochloro-arsenazo-Al(3+) using resonance light scattering technique and its application

A simple, sensitive and selective method has been developed for the determination of protein using resonance light scattering (RLS) technique. The method is based on the interaction of protein and arsenazo-DBC-Al(3+) in the pH range of 5.0-7.0, which causes a substantial enhancement of the resonance scattering signal of arsenazo-DBC-Al(3+) in the wavelength range of 300-550 nm with the maximum RLS platform at 405-420 nm. With this method, 2.50-50.00 mug ml(-1) of bovine serum albumin (BSA) and 2.50-60.00 mug ml(-1) of human serum albumin (HSA) can be determined, and the detection limits, calculated three times the standard deviation (S.D.) of six blank measurements, for BSA and HSA were 123.4 and 89.6 ng ml(-1), respectively. Moreover, the method is free from interference from many amino acids and metal ions. The method, with high sensitivity, selectivity and reproducibility, was satisfactorily applied to the determination of total protein in human serum samples. Mechanism studies indicated that arsenazo-DBC-Al(3+) could bind to BSA depending mainly on electrostatic forces, which results in enhanced RLS in the arsenazo-DBC-Al(3+)-protein system.     

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.     

Flow injection analysis: Rayleigh light scattering technique for total protein determination

A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of total protein concentrations. This method is based on the weak intensity of RLS of bromothymol blue (BB) (3',3"-dibromothymolsulfonephthalein) which can be enhanced by the addition of protein in weakly acidic solution. A common spectrofluorimeter was used as a detector. It was proved that the application of this method to quantify the total proteins in real samples by using bovine serum albumin was possible. The RLS signal was detected at lambda(ex)= lambda(em)=572 nm. The linear range was 7.0-70.0 microg mL(-1), the detection limit was 3.75 microg mL(-1), the reproducibility was 5.5% (n=7), and the sample throughput was 26 h(-1).     

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.     

A Novel Protein Assay With an Azo Dye Using Rayleigh Light Scattering Technioque

ABSTRACT

The interaction of SPADNS [3-(4-sulfophenylazo)-4, 5-dihydroxy-2, 7-naphthalene disulfonic acid] with proteins in acidic solution was studied by spectrophotometric and Rayleigh light scattering (RLS) methods. SPADNS reacts rapidly with albumin at room temperature, and the intensity of the weak RLS of SPADNS was enhanced remarkably and quantitatively by this reaction.

Based on this observation, a novel RLS method for serum albumin determination was developed. The linear range is 0.125-14.9 μg/ml for BSA. The relative standard deviation (n=10) for 5.0 μg/ml BSA was 3.94%. The method was applied to the determination of proteins in human plasma, and the results were compared with the traditional proteins assay (CBB method). There is almost no interference from amino acids, metal ions and other coexistent substances. The reaction mechanism was also discussed.     

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.     

Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with macromolecules nanoparticles of PS-AA

The polystyrene-acrylic acid (PS-AA) nanoparticles have been prepared by ultrasonic polymerization, characterized by FT-IR and TEM. It is the first report on the determination of proteins with macromolecules nanoparticles of PS-AA by resonance light-scattering (RLS). At pH 6.9, the RLS of macromolecules nanoparticles of PS-AA can be enhanced by proteins. Based on this, a novel quantitative assay of proteins at the nanogram levels has been proposed. At pH 6.9, the RLS signals of PS-AA were greatly enhanced by proteins in the region of 250-700 nm characterized by the peak at 342 nm. Under optimal conditions, the linear ranges of the calibration curves were 0.02-11.0 microgml-1, 0.04-10.0 microgml-1 and 0.03-10.0 microgml-1 for gamma-globulin (gamma-IgG), bovine serum albumin (BSA) and human serum albumin (HSA), respectively. The detection limits were 16.0 ngml-1, 19.0 ngml-1, and 15.0 ngml-1 for gamma-IgG, BSA and HSA, respectively. The method has been applied to the analysis of total proteins in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital, which indicates that the method presented here is not only sensitive, simple, but also reliable and suitable for practical application.     

Determination of proteins at nanogram levels based on their enhancement effects of Rayleigh light scattering on dibromomethylchlorophosphonazo

A new Rayleigh light scattering (RLS) assay of protein was conducted in this paper. At the optimum pH conditions, and in the presence of Tween-20, the weak RLS of dibromomethylchlorophosphonazo (DBM-CPA) can be enhanced greatly by the addition of proteins. Based on this, the reactions of DBM-CPA and proteins were studied. A new quantitative determination method for proteins has been developed. The method is simple, practical and relatively free from interference from coexisting substances, as well as much more sensitive (the dynamic ranges of 0.065-40.05 microg ml(-1) and detection limit of 30 ng ml(-1) for bovine serum albumin (BSA)) than most of the existing assays. The determination results of human body serum samples are identical to those by the CBB method, with relative S.D. of six determination of 0.5-2.2%.     

Fluorescence for the determination of protein with functionalized nano-ZnS

ZnS nanoparticles have been prepared and modified with sodium thioglycolate. The functionalized nanoparticles are water-soluble. They were used as fluorescence probes in the determination of proteins, which was proved to be a simple, rapid and specific method. In comparison with single organic fluorophores, these nanoparticle probes are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimum conditions, linear relationships were found between the enhanced intensity of fluorescence at 441 nm and the concentration of protein in the range 0.1-4.0 microg mL(-1) for human serum albumin (HSA), 0.2-3.0 microg mL(-1) for bovine serum albumin (BSA) and 0.1-4.5 microg mL(-1) for gamma-globulin (gamma-G). The limits of detection were 0.015 microg mL(-1) for HSA, 0.024 microg mL(-1) and 0.017 microg mL(-1) for BSA and gamma-G, respectively. The method has been applied to the analysis of human serum samples collected from the hospital and the results were in good agreement with those reported by a hospital, indicating that the method presented here is not only sensitive and simple, but also reliable and suitable for practical application.     

Determination of trace proteins with pyronine Y and SDS by resonance light scattering

A new resonance light scattering (RLS) probe for determining proteins is presented. The weak RLS of pyronine Y–SDS can be enhanced substantially by adding proteins in the presence of H₂SO₄, resulting in a strong and wide RLS band in the region 310–425 nm. The interaction of pyronine Y–SDS with proteins was studied on the basis of this behavior and a new quantitative method was developed for determining proteins. The enhanced RLS intensity is proportional to the concentration of proteins in the range 0.15–3.6 μg mL⁻¹ for bovine serum albumin (BSA) and 0.06–4.8 μg mL⁻¹ for human serum albumin (HSA), with detection limits of 21.0 and 12.0 ng mL⁻¹, respectively. This method is characterized by high sensitivity, rapidity of reaction, and simplicity. Four synthetic samples were determined satisfactorily and recovery was 99.5–101.5%. Results for human serum and urine samples were in agreement with those obtained by the Bradford method, with relative standard deviations (RSD) of 1.5–3.1%.