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.     

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 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.     

异硫氰酸荧光素-人血清白蛋白标记物化学发光反应的研究

采用反相流动注射分析方法,研究了异硫氰酸荧光素（FITC）－人血清白蛋白（HSA）标记物的化学发光性能和反应的最佳条件,建立了化学发光测定人血清白蛋白的新方法。体系的化学发光强度与人血清白蛋白的含量在0．08－16μg/mL内呈线性关系,方法的检出限为0．04μg/mL。讨论了蛋白质标记物化学发光性增强的原因。     

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.     

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 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 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 γ-IgG,α-chymotrypsin, E. Coli. alpsase, myoglobin, α-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 γ-IgG (20–¶400 ng/mL), myoglobin (10–300 ng/mL), and α-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 sactifactory results.     

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 highly sensitive and rapid assay for protein determination in human urine and penicillin using a Rayleigh light-scattering technique with benzeneazo-8-acetylamino-1-naphthol-3,6-disulfonic acid sodium salt.

A simple and sensitive method was conducted for the determination of trace amounts of proteins with benzeneazo-8-acetylamino-1-naphthol-3,6-disulfonic acid sodium salt (azophloxine, AP) using a Rayleigh light-scattering (RLS) technique. At pH 2.60 and in the presence of an emulsifier OP microemulsion, the RLS of AP can be greatly enhanced by proteins, owing to the interaction between AP and protein. The enhanced intensity is proportional to the concentration of proteins. Four proteins, including bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lys) and gamma globulin (gamma-G) have been tested. For example, the linear range of BSA was 0 - 0.06 microg mL(-1) with detection limits of 2.38 ng mL(-1). The method was applied to the analysis of protein in human urine and penicillin samples with satisfactory results. The relative standard deviation was in all instances less than 4.0%, and the recovery was in the range of 97.5 - 104%.     

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%.     

[HgI4]2－-蛋白质离子缔合物体系共振瑞利散射和共振非线性散射光谱及其分析应用

在0.0035～0.0045 mol/L硫酸介质中, 牛血清白蛋白(BSA)、人血清白蛋白(HSA)、卵白蛋白(OVA)和血红蛋白(HGB)等蛋白质以带正电荷的阳离子存在. 它们能借助于静电引力和疏水作用力与配阴离子[HgI4]2－-反应形成结合产物, 此时将引起共振瑞利散射(RRS)、二级散射(SOS)和倍频散射(FDS)显著增强, 并且出现新的散射光谱. 其最大RRS, SOS和FDS波长分别位于390, 760和390 nm附近. 在一定范围内, 三种散射增强(ΔIRRS, ΔISOS和ΔIFDS)与蛋白质浓度成正比, 方法具有高灵敏度, 三种方法对于不同蛋白质的检出限分别在5.7～15.9 ng/mL (RRS), 8.2～15.4 ng/mL (SOS)和11.2～22.1 ng/mL (FDS)之间, 均可用于痕量蛋白质的测定. 本文研究了[HgI4]2－与蛋白质相互作用对RRS, SOS和FDS光谱特征和强度的影响, 考察了适宜的反应条件, 并以RRS为例考察了共存物质的影响, 表明方法有良好的选择性. 据此, 利用[HgI4]2－与蛋白质的相互作用发展了一种用共振光散射技术、灵敏度高、简便、快速测定蛋白质的新方法. 本方法可用于血清和人尿中总蛋白质的测定.     

A novel method for determination of protein in human serum utilizing the trihydroxylphenylfluorone-molybdenum(VI) complex as a probe by fluorescence spectrometry

The fluorescence intensity of the trihydroxyl-phenylfluorone-molybdenum(VI) [Mo(VI)] complex is quenched by protein. Based on this, a novel method for protein assay in aqueous solution was developed. With pH 3.75 acetic acid-sodium acetate buffer solution, in the presence of p-octyl polyethylene glycol phenyl ether microemulsion, the quenched fluorescence intensity is proportional to the concentration of bovine serum albumin (BSA) in the range of 0-7.00 microg/mL, and the detection limit of BSA is 5.65 ng/mL. There is no interference from amino acids and most metal ions. The method developed in this paper has been used for the successful determination of protein in human serum.     

Time-resolved chemiluminescence technique for the microdetermination of proteins based on their complexation with copper(II).

Based on the complexation between proteins and Cu(II) coupled with the time-resolved chemiluminescence (CL) technique, a highly sensitive and quantitative assay for measuring proteins in solution is described. The complexes of proteins with Cu(II) have a strongly catalytic effect on the luminol-H2O2 CL reaction. Because the CL emission produced by the complexes is much more long-lived than that by Cu(II), the CL signals originating from proteins can be easily identified and measured with a time-resolved technique. On this basis, bovine albumin fraction V (BAF V) can be quantitatively determined in the range of 0.01 - 5.0 microg/ml with a detection limit of 5.8 ng/ml. The results show that the proposed assay exhibits a small variation in the response values for the same amount of different proteins, as compared to the Lowry as well as Bradford assays. The CL assay has also been studied for the detection of immobilized proteins.     

Microdetermination of proteins using m-carboxychlorophosphonazo as detection probe by enhanced resonance light scattering spectroscopy

Proteins can be determined using a common spectrofluorometer to detect the intensity of resonance light scattering (RLS). Under acidic conditions, the reaction between m-carboxychlorophosphonazo (CPA-mK) and proteins enhances the weak light scattering of CPA-mK drastically. This enhanced intensity is proportional to the concentration of proteins. The linear ranges for human serum albumin are 0.5-35.0 microg/mL, with detection limits of 0.104 microg/mL. The method yields results comparable to those of the calorimetric method using Coomassie Brilliant Blue G-250 (CBB) with relative standard deviations of 0.72-2.10% (n = 10). There is almost no interference by amino acids and most of the metal ions.     

Determination of bisphenol A and 10 alkylphenols in serum using SDS micelle capillary electrophoresis with gamma-cyclodextrin.

A new micelle capillary electrophoresis based on cyclodextrin micellar electrokinetic chromatography (MEKC) for the determination of bisphenol A and 10 alkylphenols in rat serum is reported. Several surfactants and dextrins were studied. Bisphenol A and alkylphenols were separated using a 50 microm x 50 cm capillary with 20 mM borate phosphate buffer (pH 8.0) containing 20 mM sodium dodecylsulfate and 5 mM gamma-cyclodextrin as carrier. The method could determine 0.6-2000 microg/mL of phenols in 100 microL serum by photometric detection at 214 nm. Using 2.0 mL serum, 1.0 ng/mL of phenols could be determined. The relative standards deviations were 6.3-7.7% at 10 microg/mL in serum. The recoveries were 91.8-93.0% with 10 microg/mL serum samples.     

A simple HPLC method for the quantification of mizoribine in human serum: pharmacokinetic applications

A simple high-performance liquid chromatographic (HPLC) method was developed and validated for the quantification of mizoribine in human serum. After the addition of 70% perchloric acid and 3-methylxanthine (50 microg/mL, internal standard) to human serum, the samples were mixed and centrifuged at 12,000 rpm (1432 g) for 10 min. The supernatant was injected onto a C(18) column eluted with a mobile phase of 20 mm Na2HPO4 and methanol (93:7, v/v, pH 3) containing 0.04% octanesulfonic acid and detected utilizing an ultraviolet detector at 275 nm. The linear calibration curve was obtained in the concentration range of 0.1-4.0 microg/mL and the lower limit of quantification was 0.1 microg/mL. This method was validated with selectivity, linearity, precision and accuracy. In addition, the method was successfully applied to estimate the pharmacokinetic parameters of mizoribine in Korean subjects following an oral administration of 100 mg mizoribine (two Bredinine 50 mg tablets). The maximum serum concentration (C(max)) of 2.30 +/- 0.83 microg/mL was reached 2.27 +/- 0.66 h after an oral dose. The mean AUC(0-12 h) and the elimination half-life (t(1/2)) were 13.2 +/- 4.79 microg h/mL and 3.10 +/- 0.74 h, respectively.     

化学发光底物分辨双组分免疫分析法测定胶质瘤标志物NSE和CA15-3

本文建立了一种基于辣根过氧化物酶(HRP)和碱性磷酸酯酶(ALP)化学发光底物分辨的双组分免疫分析新技术,用以检测人胶质瘤血清标志物神经元特异性烯醇化酶(NSE)和糖链抗原15-3(CA15-3).实验详细考察了捕获抗体、检测抗体、HRP和ALP酶标记物的用量,结果发现NSE和CA15-3的线性范围分别为0.5～20ng/mL(R20.99)和0.5～20U/mL(R20.99),最低检出限分别为0.2ng/mL和0.2U/mL;高、中和低三个浓度的血清加样回收率良好;天内和天间相对标准偏差均小于10%;且一份血清,两组分同时检测无交叉干扰.综合而言:本法能够一次实验,高灵敏、高特异地同时检测两种疾病标志物,具有血清用量少、检测时间短、操作简单、结果可靠的优点,有望为胶质瘤的临床早期诊断提供坚实的支持.     

Study on the chemiluminescence behavior of bovine serum albumin with luminol and its analytical application

In this paper, the luminescence behavior of bovine serum albumin (BSA) and luminol was first studied by flow injection chemiluminescence (CL). It was found that the hyperchromic effect of luminol in the presence of BSA led to the acceleration of the electrons transferring rate of excited 3-aminophthalate, which greatly enhanced the CL intensity of luminol/dissolved oxygen reaction. The increments of CL intensity were proportional to the concentrations of BSA with a linear range from 0.01 to 7 nmol L(-1). It was also found that azithromycin could inhibit the CL intensity of luminol/BSA reaction. The decrements of CL intensity were logarithm over the concentrations of azithromycin ranging from 0.1 to 700 ng mL(-1). At a flow rate of 2.0 mL min(-1), a complete analytical process, which included sampling and washing, could be performed within 30s with relative standard deviations of less than 3.1%. This proposed method was successfully applied in assaying azithromycin in pharmaceutical and human serum samples with recoveries from 91.0 to 104.3%. The possible luminescence mechanism of luminol/BSA/azithromycin reaction was discussed in detail by CL, UV and fluorescence methods.     

流动注射-抑制化学发光法测定岩白菜素

本文基于岩白菜素对鲁米诺-牛血清白蛋白体系化学发光信号显著的抑制作用,建立了快速灵敏测定岩白菜素的流动注射-化学发光分析新方法。实验发现,化学发光强度的降低值与岩白菜素质量浓度对数值呈良好的线性关系,方法的线性范围为3.0~5.0×10~5 pg/mL,检出限(3σ)为1.0pg/mL。当溶液流速为2.0mL/min时,完成一次分析过程仅需30s,采样频率120/h。本法用于片剂、人血清和尿液中岩白菜素的含量测定,回收率为98.1%~102.7%,相对标准偏差小于2.0%(n=7)。同时对化学发光反应机理进行了探讨。