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 μg/mL, with detection limits of 0.104 μg/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 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.     

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).     

Resonance Rayleigh scattering for detection of proteins in HPLC

An HPLC-resonance Rayleigh scattering (RRS) (HPLC-RRS) detection system is described for separation and detection of proteins. This system is based on the modification of a commercial HPLC instrument involving the addition of a pump and a T-shaped interface, and a common fluorescence detector was used for detection. The detection principle is based on the change of RRS intensity of the ion-association complex formed from biebrich scarlet (BS) and protein. The RRS signal was detected at lambdaex=lambdaem=376 nm. The utility of the presented method was demonstrated by the separation and determination of four proteins involving cytochrome (Cyt-c), lysozyme (Lys), HSA, and gamma-globulin (gamma-Glo). An LOD of 0.2-1.0 microg/mL was reached and a linear range was found between peak area and concentration in the range of 0.20-3.0 microg/mL for Cyt-c, 0.25-2.5 microg/mL for Lys, 1.5-10 microg/mL for HSA, and 2.0-15 microg/mL for gamma-Glo, with linear regression coefficients all above 0.99. The method presented has been applied to determine HSA and gamma-Glo in human serum samples synchronously.     

High-performance liquid chromatography of lactose with evaporative light scattering detection, applied to determine fine particle dose of carrier in dry powder inhalation products

A method for quantification of the fine particle dose of lactose is described, using a hydrophilic interaction chromatography (HILIC) method and evaporative light scattering detection. The HILIC method used an aminopropyl column and a mobile phase consisting of acetonitril/water (80/20, v/v) for isocratic elution. Sensitive chromatography was obtained using a low concentration of water in the extraction solvent. The detection limit (RSD<10%) at an injection volume of 10 microL is 10 microg/mL. Linearity was obtained in the range of 10-80 microg/mL (R(2)>0.99). A relative standard deviation (RSD) of 0.5% (N=6) demonstrated good precision of the optimized method.     

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 Rayleigh method for the determination of proteins with Orange G.

In pH 0.6 - 2.0 HCl-sodium acetate buffer solution, proteins react with an acidic monoazo dye such as Orange G, Methyl Orange, Methyl Red and Orange IV to form a combination product. This results in a significant enhancement of resonance Rayleigh scattering (RRS) and a new RRS spectrum appears. Owing to the fact that Orange G-protein system is the most sensitive, it was taken as an example to study. The RRS spectral characteristics of its combination product and the optimum condition for the reaction were investigated. The intensity of RRS is directly proportional to the concentration of protein in the range of 0 - 5.0 microg/mL. The method has high sensitivity; its detection limits are 2.6 ng/mL for BSA, 3.4 ng/mL for HAS and 7.1 ng/mL for alpha-chymotrypsin, respectively. A new method for the determination of trace amounts of proteins on the basis of RRS spectra has been developed.     

Microdetermination of proteins by enhanced Rayleigh light scattering spectroscopy with morin

Under acidic conditions, the reaction between 3,5,7,2′,4′-pentahydroxyflavone (morin) and proteins enhances the weak light scattering of morin drastically. The enhanced light scattering intensity is proportional to the content of proteins. This fact is the basis of a new method for the quantitative analysis of proteins. The linear range is 0.45–7.15 and 0.46–11.14 μg/mL for BSA and HSA, respectively. The relative standard deviation is 3.76% (n = 12) for BSA in the middle of the linear range. The results of this assay for human serum samples were comparable with those from the traditional method (CBB method). There is almost no interfere from amino acids and most of the metal ions. The scattering spectrum of morin was also discussed.     

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%.     

The interaction of poly(ethylenimine) with nucleic acids and its use in determination of nucleic acids based on light scattering

For the first time, poly(ethylenimine) (PEI) was used to determine nucleic acids with a light scattering technique using a common spectrofluorometer. The interaction of PEI with DNA results in greatly enhanced intensity of light scattering at 300 nm, which is caused by the formation of the big particles between DNA and PEI. Based on this, a new quantitative method for nucleic acid determination in aqueous solutions has been developed. Under the optimum conditions, the enhanced intensity of light scattering is proportional to the concentration of nucleic acid in the range of 0.01-10.0 microg ml(-1) for herring sperm DNA (hsDNA), 0.02-10.0 microg ml(-1) for calf thymus DNA (ctDNA), 0.02-20.0 microg ml(-1) for yeast RNA (yRNA). The detection limits are 5.3, 9.9, and 13.7 ng ml(-1), respectively. Synthetic samples were determined satisfactorily. At the same time, the light scattering technique has been successfully used to obtain the information on the effects of pH and ionic strength on the formation and the stability of the DNA/PEI complex, which is important in some fields such as genetic engineering and gene transfer. Using ethidium bromide (EB) as a fluorescent probe, the binding of PEI with hsDNA was studied. Both the binding constant of EB with DNA and the number of binding sites per nucleotide decrease with increasing concentration of PEI, indicating noncompetitive inhibition of EB binding to DNA in the presence of PEI. And the association constant of PEI to DNA obtained is 1.2 x 10(5) M(-1). IR-spectra show that PEI interacts with DNA through both the phosphate groups and the bases of DNA and the formation of DNA/PEI complex may cause the change of the conformation of the DNA secondary structure, which is also proved by UV-spectra.     

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.     

Microdetermination of proteins by enhanced Rayleigh light scattering spectroscopy with morin

Under acidic conditions, the reaction between 3,5,7,2′,4′-pentahydroxyflavone (morin) and proteins enhances the weak light scattering of morin drastically. The enhanced light scattering intensity is proportional to the content of proteins. This fact is the basis of a new method for the quantitative analysis of proteins. The linear range is 0.45–7.15 and 0.46–11.14 μg/mL for BSA and HSA, respectively. The relative standard deviation is 3.76% (n = 12) for BSA in the middle of the linear range. The results of this assay for human serum samples were comparable with those from the traditional method (CBB method). There is almost no interfere from amino acids and most of the metal ions. The scattering spectrum of morin was also discussed. Received: 11 October 1998 / Revised: 18 February 1999 / Accepted: 24 February 1999     

Spectroscopic study on the interaction of acridine yellow with adenosine disodium triphosphate and its analytical application

The interaction of adenosine disodium triphosphate (ATP) with acridine yellow and its analytical application have been studied. In an alkalescent medium, adenosine disodium triphosphate react with acridine yellow to form an ion-association by virtue of electrostatic attraction and hydrophobic interaction, resulting in a remarkable enhancement of resonance light scattering (RLS) intensity of acridine yellow. The maximum scattering wavelength is at 325 nm. The spectral characteristics of the ion-associates, the effective factors and the optimum conditions have been investigated. The enhanced RLS intensity is directly proportional to the concentration of ATP in the range of 0.80-20.0 microg ml(-1) with the detection limit 0.086 microg ml(-1). The method has been successfully applied to the quick determination of ATP in tablet and injection samples. The results of the present method are in agreement with those obtained by the method in the Chinese Pharmacopoeia.     

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.     

Development of a novel method based on liquid chromatography-evaporative light scattering detection for the direct determination of streptomycin and dihydrostreptomycin in raw materials, pharmaceutical formulations, culture media and plasma

A novel method for the non-derivatization liquid chromatographic determination of streptomycin (STR) and dihydrostreptomycin (DHSTR) was developed and validated based on evaporative light scattering detection (ELSD). Utilizing a ThermoHypersil BetaBasic C18 analytical column, evaporation temperature of 50 degrees C and pressure of nebulizing gas (nitrogen) of 3.5 bar, the optimized mobile phase was 1.25 mL L(-1) TFA aqueous solution, in an isocratic mode at a rate of 1.0 mL min(-1). STR was eluted at 5.6 min and DHSTR at 7.8 min with a resolution of 4.4. Linear calibration curves were obtained from 2 to 120 microg mL(-1) (r > 0.9990) for STR and 2-75 microg mL(-1) (r > 0.9994) for DHSTR, with a LOD equal to 0.7 and 0.5 microg mL(-1), respectively. The developed method was applied for the assay of STR and DHSTR (sulfate) in pharmaceutical raw materials and formulations, while the simultaneous direct determination of sulfate was feasible (tR = 2.5 min, LOD = 1.4 microg mL(-1), double logarithmic calibration curve in the range of 4-50 microg mL(-1), r > 0.9998). Modified isocratic mobile phase (H2O-ACN, 90:10, v/v, containing 1.25 mL L(-1) TFA), was used for the determination of streptomycin B impurity in STR sulfate raw material and a gradient mobile phase (H2O-ACN containing TFA) was used for the determination of DHSTR in the presence of penicillinG procaine. The developed method was also applied for the assay of commercial formulations (STR powder and DHSTR injection solution and suspension) (%recovery 98-102, %RSD < 1.3, n = 3 x 3), for the determination of STR in bacteria culture medium (%recovery 99.6, %RSD = 0.8, n = 3 x 3), and for the determination of DHSTR in human plasma (2.0-23.0 microg mL(-1)) after solid phase extraction using carboxylate cartridges (%recovery 98.4-101.8, %RSD = 3.2, n = 3 x 3).     

Determination of trace chlorine dioxide based on the plasmon resonance scattering of silver nanoparticles

A plasmon resonance scattering (PRS) method for chlorine dioxide is reported based on the oxidization of silver nanoparticles (NPs) by it, in pH 9.1 ammonia-ammonium nitrate buffer solutions. Silver NPs exhibit strong PRS signals at 470nm, and can be oxidized by ClO(2), which results in PRS quenching at 470nm. It was found that the PRS quenching intensity is proportional to the concentration of chlorine dioxide over the range of 0.0011-0.185microg/mL, with a detection limit (3sigma) of 0.00050microg/mL and the correlation coefficient of 0.9995. The method is simple, rapid and cost effective. It was applied to the determination of chlorine dioxide in drinking water, with satisfactory results.     

花菁-脱氧核糖核酸的共振光散射光谱及其分析应用

研究了一种苯并噻唑阳离子花菁与脱氧核糖核酸(DNA)作用的共振光散射光谱,在pH 6.0的六次甲基四胺-HCl缓冲介质中,痕量DNA的加入使花菁在590nm的共振光散射强度显著增强。在最佳实验条件下,增强的共振光散射强度与DNA浓度具有良好的线性关系,据此建立了一种测定DNA的共振光散射光谱法。方法的线性范围为:小牛胸腺DNA(CT DNA),0～20μg/mL,鱼精子DNA(FS DNA),0～15μg/mL;检出限分别为0.005μg/mL和0.008μg/mL。该方法已用于合成样品中DNA的测定。     

Resonance Rayleigh scattering measurement of aminoglycoside antibiotics with Evans Blue.

In a weak acid medium, some aminoglycoside antibiotics, such as kanamycin (KANA), gentamicin (GEN), tobramycin (TOB) and neomycin (NEO), or acid bisazo dye Evans Blue (EB) can only produce very weak resonance Rayleigh scattering (RRS) signals. However, when two agents react with each other to form ion-association complexes, the RRS intensity can be greatly enhanced and a new RRS spectrum with a significant enhancement of the RRS intensity in the wavelength range from 350 nm to 600 nm can be observed. The maximum scattering peak is at 570 nm. There is a linear relationship between the RRS intensity and the antibiotic concentration in the range of 0.01-6.0 microg mL(-1) at 570 nm. This RRS method for the determination of aminoglycoside antibiotics at trace-amount levels has been developed. The detection limits (3sigma) of the four antibiotics, whose order of sensitivity from high to low ranks as KANA > NEO > TOB > GEN, are 5.2-6.9 ng mL(-1). This method has good selectivity and has been successfully applied to the quick determination of antibiotics not only for injections and ear drops, but for clinic serum samples as well. In addition, the reaction mechanism by using a quantum chemistry method and the influencing factors of the RRS spectra and the enhancement reasons of RRS have been discussed.     

Determination of vertilmicin sulfate and its related substances by HPLC-ELSD and HPLC-MS2

A new and simple high-performance liquid chromatography (HPLC)-evaporative light scattering detection (ELSD) method for the determination of vertilmicin sulfate and its related substances is developed. The column is an Agilent SB-C(18) (250 x 4.6 mm, 5 microm). The mobile phase is 0.05 mol/L trifluoroacetic acid-methanol (85:15). Good separation of vertilmicin from the main related substances is achieved. The standard curve is rectilinear in the range of 270-1350 microg/mL (r = 0.9998). The average recovery is 99.8%. The limit of detection is 10 microg/mL. The HPLC-mass spectrometry-mass spectrometry (MS(2)) method is used to characterize the structures of vertilmicin sulfate and its related substances. In positive mode, vertilmicin sulfate and its related substances are elucidated by use of electrospray ion trap MS in the multi-stage MS full scan mode. The possible structure of an unknown impurity in vertilmicin is deduced based on the HPLC-MS(2) data.     

Determination of proteins at nanogram levels by their quenching effect on the chemiluminscence reaction between luminol and hydrogen peroxide with manganese-tetrasulfonatophthalocyanine as a new catalyst

The manganese-tetrasulfonatophthalocyanine (MnTSPc) catalyzed luminol-hydrogen peroxide chemiluminescence (CL) systems can be quenched in the presence of proteins. A highly sensitive CL quenching method has been developed for the determination of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.1-20 microg/mL for human serum albumin (HSA), 0.2-20 microg/mL for human gamma-IgG, and 0.5-50 microg/mL for the bovine serum albumin (BSA) with the corresponding detection limits were 1.9 ng/mL, 2.7 ng/mL, and 3.4 ng/mL. The method has been applied to the analysis of total proteins in human serum samples and the results were in good agreement with clinical data provided.