Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with composite nanoparticles of CdS/PAA

This paper describes the development of composite nanoparticles. A novel composite nanoparticle has been prepared by an in situ polymerization method. The nano-CdS has been prepared, then the polymerization of acrylic acid (AA) was carried out by initiator potassium persulfate (KPS) under ultrasonic irradiation. The surface of the composite nanoparticles was covered with abundant carboxylic groups (-COOH). The nanoparticles are water-soluble, stable and biocompatible. Reaction of the composite nanoparticles with proteins results in an enhanced resonance light scattering (RLS) at 380 nm. Based on this, a new resonance light-scattering (RLS) method was developed for the determination of proteins including BSA, HSA and human gamma-IgG. Under the optimum conditions, the enhanced RLS intensity is linearly proportional to the concentration of proteins. The liner range is 0.1-15 microgmL(-1) for HSA, 0.2-20 microgmL(-1) for BSA and 0.1-50.0 microgmL(-1) for human gamma-IgG, respectively. The method has been applied to the determination of the total protein in human serum samples collected from the hospital and the results are in good agreement with those reported by the hospital. This method proved to be very sensitive, rapid, simple and tolerant of most interfering substances.     

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.     

Direct fluorimetric determination of gamma-globulin in human serum with organic nanoparticle biosensor

This paper describes the development of organic fluorescence nanoparticles. The nanoparticles have a narrow, tunable, symmetric emission spectrum and a broad, continuous excitation spectrum. The nanoparticles have high room-temperature fluorescence quantum yields and long fluorescence lifetime. They are also photochemically stable and water-soluble. They were used as fluorescence biosensor in the determination of proteins, which was proved to be a simple, rapid and specific method. In comparison with single organic fluorephores, these nanoparticles are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimal conditions, the linear ranges of the calibration curves were 0.1-4.5 microg ml(-1) for human serum albumin (HSA), 0.2-3.5 microg ml(-1) for bovine serum albumin (BSA) and 0.04-0.8 microg ml(-1) for gamma globulin (gamma-IgG), respectively. The detection limits were 0.062 microg ml(-1) for HSA, 0.036 microg ml(-1) for BSA and 0.022 microg ml(-1) for gamma-IgG, respectively. However, when the content of HSA is lower than 0.8 microg ml(-1), HSA makes little contribution to the fluorescence quenching. So, the method was applied to direct selective quantification of gamma-IgG in human blood serum without separation of HSA. The results were in good agreement with these reported by the hospital, indicating that the method presented here is not only sensitive, selective and simple, but also reliable and suitable for practical applications.     

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%.     

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.     

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 proteins at nanogram levels by synchronous fluorescence scan technique with a novel composite nanoparticle as a fluorescence probe

A novel composite nanoparticle has been prepared by an in situ polymerization method and applied as a protein fluorescence probe. The nano-CdS has been prepared, then the polymerization of acrylic acid (AA) was carried out by initiator potassium persulfate (KPS) under ultrasonic irradiation. The surface of the composite nanoparticles was covered with abundant carboxylic groups (--COOH). The nanoparticles are water-soluble, stable, and biocompatible. The synchronous fluorescence intensity of the composite nanoparticles is significantly increased in the presence of trace protein at pH 6.90. Based on this, a new synchronous fluorescence scan (SFS) analysis was developed for the determination of proteins including BSA, HSA, and human gamma-IgG. When Delta lambda = 280 nm, maximum synchronous fluorescence is produced at 290 nm. Under the optimum conditions, the response is linearly proportional to the concentration of proteins. The linear range is 0.1-10 microg ml(-1) for HSA, 0.09-8.0 microg ml(-1) for BSA, and 0.08-15 microg ml(-1) for human gamma-IgG, respectively. The method has been applied to the determination of the total protein in human serum samples collected from the hospital and the results are satisfactory.     

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%.     

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.     

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。     

Novel magnetic and fluorescent nanocomposite as a sensitive probe for the determination of proteins

The Fe(3)O(4)/(sodium oleic acid/ethyltrimethyl ammonium bromide)(n)/4-aminobenzoic acid (Fe(3)O(4)/(NaOL/CTAB)(n)/PABA) nanocomposites have been prepared by a layer-by-layer self-assembly approach. This kind of nanocomposites have fluorescent, magnetic and water-soluble properties. Taking advantage of the magnetic property of nanocomposites, we can separated them from solution easily by using a permanent magnet. By using their strong fluorescence, we can detect proteins. At pH 6.98, the fluorescence of Fe(3)O(4)/(NaOL/CTAB)(n)/PABA nanocomposites can be enhanced by the proteins. Under optimal conditions, the linear ranges of calibration curves were 0.2-20, 0.2-13, 0.2-10 microg mL(-1) for gamma-globulin (gamma-IgG), human serum albumin (HSA), and bovine serum albumin (BSA), respectively. The detection limits were 0.02, 0.01, 0.02 for gamma-IgG, HSA and BSA, respectively. The method has been applied to analyze the total proteins in human samples and the results were in good agreement with those reported by the hospital. This method is sensitive, simple and potential in many areas.     

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%.     

Determination of proteins at nanogram levels by their quenching effect on large particle scattering of colloidal silver chloride

A novel quantitative method for the determination of proteins in aqueous solutions has been based on the quenching of the resonance scattering light of colloidal silver chloride in the presence of proteins. The detection limits for eight kinds of proteins (BSA, HSA, egg albumin, human gamma-IgG,alpha-chymotrypsin, E. Coli. alpsase, myoglobin, alpha-casein) were at about 8 ng/mL; the linear ranges of the calibration curves were 10-400 ng/mL under optimal conditions,except for human gamma-IgG (20-400 ng/mL), myoglobin (10-300 ng/mL), and alpha-casein (10-300 ng/mL). Three wavelengths (398 nm, 475 nm, 499 nm) were all suitable for the determination and any acidity from pH 3.0 to pH 9.0 could be chosen. A few non-protein substances at high concentration levels interfered with this method, but this problem could simply be overcome by diluting the samples before the assay. Mechanism studies showed that the quenching effect of proteins on the scattering light of colloidal silver chloride was mainly due to the coagulation of AgCl particles retarded by protein. The method was employed for the determination of total protein in human serum with satisfactory results.     

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.     

Resonance light scattering technique for the determination of proteins with polymethacrylic acid (PMAA)

As a resonance light scattering (RLS) probe, the polyelectrolyte polymethacrylic acid (PMAA) was applied in this assay. The bovine serum albumin (BSA) and human serum albumin (HSA) were determined by the electrostatic interaction of PMAA and proteins. At pH 3.8 Na(2)HPO(4)-citric acid buffer solution, the RLS intensities of PMAA-BSA (HSA) system were greatly enhanced. The characteristic peaks were appeared at the wavelength 320, 546 and 594 nm. The optimization conditions of the reaction were also examined and selected. Under the selected conditions, the RLS intensities were proportional to the protein concentrations in the range of (0.0200-2.00) x 10(-6) mol/L for BSA and (0.0200-2.40) x 10(-6) mol/L for HSA. The influences of some foreign substances were also examined. The synthetic samples containing proteins and some real samples were analyzed and the results obtained were satisfactory.     

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 serum albumin in the presence of poly(diallyldimethylammonium chloride) by resonance light scattering technique

By means of the resonance light scattering (RLS) technique, a new method was developed to determine the bovine serum albumin (BSA) and human serum albumin (HSA) by the interaction of serum albumin with poly(diallyldimethylammonium chloride) (PDDA). At Tris-NaOH buffer solution, the RLS intensity of serum albumin at the wavelength 320, 550 and 590 nm was obviously enhanced in the presence of PDDA. The influences of some experimental factors, including incubation time, addition sequence of reagents, pH value, concentration of PDDA and foreign substances, on the enhancement of the RLS intensity were examined. The optimum conditions of the experiment were selected. Under the selected experimental condition, the enhanced RLS intensities were directly proportional to the concentrations in the range of (0.0250-2.75)x10(-6) mol/L for BSA and (0.0235-1.17)x10(-6) mol/L for HSA. The detection limits (S/N=3) were 8.40x10(-9) mol/L for BSA and 7.39x10(-9) mol/L for HSA. The synthetic samples were analysed and the results obtained were satisfactory.     

四羧基镍酞菁共振散射法测定蛋白质

建立了一种测定蛋白质的新方法．在pH3．6的Britton-Robinson（B-R）缓冲溶液中,蛋白质与四羧基镍酞菁NiPc（COOH）4发生相互作用,使体系在λ=388nm处的共振散射（RLS）增强,并且增强的散射强度（IRLS）与蛋白质的含量成比例,据此利用四羧基镍酞菁NiPc（COOH）4为光谱探针共振散射法测定人血清中的总蛋白质,同时优化了体系光散射检测的实验参数．在最佳的实验条件下,对牛血清白蛋白（BSA）、人血清白蛋白（HSA）、人血清总蛋白（TP）的线性范围分别为0．00～1．20mg／L、0．00～1．00mg／L、0．00～1．00mg／L,相应检测限分别为5．97×10^-4mg／L、2．90×10^-4mg／L、4．76×10^-4mg／L．将该方法应用于实际人血清样品中总蛋白的测定,结果与考马斯亮蓝法比较,令人满意．     

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 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.