Fluorescence enhancement effect of the morin-Al3+ -sodium dodecyl benzene sulphonate-protein system and the determination of proteins.

The fluorescence intensity of the morin-Al(3+) complex was greatly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this, a new fluorimetric method for the determination of protein was developed. Under optimum conditions, the enhanced intensity of fluorescence was in proportion to the concentration of proteins in the range 1.0 x 10(-8)-1.3 x 10(-5) g/mL for bovine serum albumin (BSA), 4.0 x 10(-8)-1.2 x 10(-5) g/mL for egg albumin (EA) and 5.0 x 10(-8)-1.2 x 10(-5) g/mL for human serum albumin (HSA). Their detection limits (S:N = 3) were 5.0 x 10(-9), 1.8 x 10(-8) and 1.6 x 10(-8) g/mL, respectively. The interaction mechanism was also studied.     

Resonance light scattering technique for the determination of proteins with Congo red and Triton X-100.

Resonance light scattering (RLS) of Congo red (CR) was greatly enhanced by BSA (HSA) in the presence of Triton X-100 (TX-100). In sodium citrate-HCl buffer (pH 2.7-3.0), the enhanced intensity of resonance light scattering at 360 nm was in proportion to the concentration of proteins [corrected] The linear relationship was obtained between the resonance light scattering intensity and proteins in the range 5.0 x 10(-8)-8.0 x 10(-6) g/mL and 1.0 x 10(-9)-6.0 x 10(-6) g/mL for BSA and HSA, respectively. Their detection limits were 1.4 x 10(-8) g/mL and 2.8 x 10(-10) g/mL (S:N = 3), respectively. Synthetic and actual samples were analysed satisfactorily.     

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples.     

Protein analysis with terbium (III) and sodium dodecyl sulphonate by a second-order scattering technique.

This is the first report of terbium(III) as a probe of second-order scattering (SOS) for the determination of proteins in human serum at nanogram levels. A sensitive method has been developed using light scattering, based on the fact that the weak SOS of proteins can be enhanced in the presence of terbium(III) and sodium dodecyl sulphonate (SDS). With this method, 7.0 x 10(-9)-1.0 x 10(-5) g/mL human serum albumin (HSA) and 5.0 x 10(-9)-5.0 x 10(-6) g/mL gamma-globulin can be determined; the detection limits were 4.4 ng/mL for HSA and 3.1 ng/mL for gamma-globulin. The method has been applied to the detection of total proteins in human serum samples, and the results are consistent with those obtained by the Coomassie brilliant blue (CBB) G-250 assay.     

Improvement of the acridine orange-protein-surfactant system for protein estimation based on aromatic ring stacking effect of sodium dodecyl benzene sulphonate.

The fluorescence of acridine orange (AO) is greatly quenched by the anionic surfactant sodium dodecyl benzene sulphonate (SDBS), but when protein is added into the AO-SDBS system, the fluorescence intensity of the latter is enhanced again. Based on this, a new fluorimetric method of determination of protein was developed. Under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of protein, such as bovine serum albumin (BSA), human serum albumin (HSA) and egg albumin (EA), over a wide range with detection limits at the 10(-9) g/mL level. This method has been satisfactorily used for the determination of protein in samples. We compared results using 280 nm and 490 nm excitation wavelengths and the mechanism of the assay.     

Homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin employing Ru(bpy)(2)(dcbpy)NHS and carrier protein.

A highly sensitive homogeneous electrogenerated chemiluminescence (ECL) immunoassay for the determination of anti-digoxin antibody and digoxin hapten was developed employing Ru(bpy)(2)(dcbpy)NHS (bpy = 2,2'-bipyridyl; dcbpy = 2,2'-bipyridine-4,4'-dicarboxylic acid; NHS = N-hydroxysuccinimide ester) as an electrochemiluminescent label and bovine serum albumin (BSA) as a carrier protein. A digoxin hapten was indirectly heavily labelled with Ru(bpy)(2)(dcbpy)NHS through BSA to form Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate. The ECL intensity of the immunocomplex of the conjugate with anti-digoxin antibody markedly decreased when the immunoreaction between Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate and anti-digoxin antibody took place. Two formats, direct homogeneous immunoassay for anti-digoxin antibody and competitive immunoassay for digoxin, were developed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration in the range 7.6 x 10(-8)-7.6 x 10(-6) g/mL was determined by direct homogeneous format. Digoxin hapten was determined throughout the range 4.0 x 10(-10)-1.0 x 10(-7) g/mL with a detection limit of 1.0 x 10(-10) g/mL by competitive format. The relative standard derivation for 6.0 x 10(-9) g/mL was 4.3%. The method has been applied to assaying digoxin in control human serum.     

Interaction of loratadine with serum albumins studied by fluorescence quenching method

The interactions between loratadine and bovine serum albumin (BSA) and human serum albumin (HSA) were studied using tryptophan fluorescence quenching method. The fluorescence intensity of the two serum albumins could be quenched 70% at the molar ratio [loratadine]:[BSA (or HSA)]=10:1. In the linear range (0-50 micromol L(-1)) quenching constants were calculated using Stern-Volmer equation. Temperature in the range 298 K-310 K had a significant effect (p<0.05) on the two serum albumins through ANOVA analysis and t-test. Furthermore the conformation changes in the interactions were studied using FTIR spectroscopy.     

Flow-injection resonance light scattering detection of proteins at the nanogram level.

A resonance light scattering (RLS) detection method for protein was developed, using a flow-injection system based on the enhancement of RLS signals from Biebrich scarlet (BS) by protein. The enhanced RLS intensities at 286.0 nm, in acidic aqueous medium, were proportional to the protein concentration over the range 0.005-18 microg/mL and 0.008-16 microg/mL for human serum albumin (HSA) and bovine serum albumin (BSA), respectively, with corresponding limits of detection (3sigma) of 5.00 ng/mL for HSA, and 7.80 ng/mL for BSA. The method was successfully applied to the quantification of total proteins in human serum samples.     

The sensitive determination of nucleic acids using a fluorescence-quenching method.

Experiments indicated that nucleic acids can quench the fluorescence of the Eu3+ -2-thenoyltrifluoroacetone (TTA)-1,10-phenanthroline (Phen) system. Based on this, a sensitive method for the determination of nucleic acids was proposed. The experiments indicated that under the optimum conditions, the quenched fluorescence intensity was in proportion to the concentration of nucleic acids in the range 1.0 x 10(-11)-1.0 x 10(-6) g/mL for yeast RNA (yRNA), 5.0 x 10(-11)-5.0 x 10(-7) g/mL for fish sperm (fsDNA) and 1.0 x 10(-10)-1.5 x 10(-6) g/mL for calf thymus DNA (ctDNA). Their detection limits were 3.0 x 10(-12), 4.0 x 10(-12) and 5.0 x 10(-11) g/mL, respectively. Therefore, the proposed method is one of the most sensitive methods available. The interaction between nucleic acids and Eu3+ -TTA-Phen is also discussed.     

Fluorimetric determination of proteins using 4‐chloro‐(2′‐hydroxylophenylazo)rhodanine–Ti(IV) complex as a spectral probe

A novel method for the determination of proteins was developed, based on the enhancement of fluorescence with 4‐chloro‐(2′‐hydroxylophenylazo)rhodanine–Ti(IV) [ClHARP–Ti(IV)] complex as a fluorescence probe. The excitation and emission wavelengths of the system were 335 nm and 376 nm, respectively. The presence of bis(2‐ethylhexyl)sulphosuccinate sodium salt (AOT) microemulsion greatly increased the sensitivity of the system. Under optimal conditions, four kinds of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), egg albumin (Ova), and γ‐globin (γ‐G) were studied. The detection limits were 0.182 µg/mL for BSA, 0.0788 µg/mL for HSA, 0.216 µg/mL for Ova and 0.484 µg/mL for γ‐G. The linear ranges of the calibration were 0–12.0, 0–10.0, 0–18.0 and 0–18.0 µg/mL, respectively. The method possessed high sensitivity, good selectivity and was applied to the analysis of protein in milk powder and cornmeal with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

Dye-binding protein assay using a long-wave-absorbing cyanine probe

A simple and fast protein assay that involves the binding of water-soluble sulfonate heptamethylene cyanine to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 778 to 904 nm, and the increase in absorption at 904 nm is monitored. This assay is very reproducible, of good color stability for at least 80 min, and sensitive at the 100 ng/mL level of human serum albumin (HSA) when a spectrophotometer with near-infrared wavelength is used to measure absorbance. Few chemicals except ionic surfactants such as cetyltrimethylammonium bromide and sodium dodecyl sulfonate interfere with the assay. Purified proteins have different capacities to interact with the dye; under the experimental conditions, the linear ranges of bovine serum albumin (BSA), HSA and gamma-IgG were 200-2000, 100-2400, and 200-3000 ng/mL, respectively. The relative standard deviation for the five replicate determinations of 1200 ng/mL BSA is 2.1%.     

Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application.

In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.     

Spectrofluorimetric determination of 5-hydroxytryptamine using chlorosulphonylthenoyltrifluoroacetone-europium probe.

A new spectrofluorimetric method was developed for the determination of trace amounts of 5-hydroxytryptamine (5-HT). Using chlorosulphonylthenoyltrifluoroacetone (CTTA)-europium (Eu(3+)) ion as a fluorescent probe in a buffer solution at pH 11.0, 5-HT can remarkably enhance the fluorescence intensity of the CTTA-Eu(3+) complex at lambda = 612 nm; the enhanced fluorescence intensity of Eu(3+) is proportional to the concentration of 5-HT. Optimum conditions for the determination of 5-HT were also investigated. The linear range and detection limit for the determination of 5-HT were 1.0 x 10(-7)-1.2 x 10(-5) mol/L and 8.5 x 10(-8) mol/L, respectively. This method is simple, practical and relatively free of interference from coexisting substances, and can be applied to assess 5-HT in urine samples.     

Effects of homogeneous media, binary mixtures and microheterogeneous media on the fluorescence and fluorescence probe properties of some benzo[b][1,8]naphthyridiens with HSA and BSA

A rapid and efficient method for the synthesis of various poly‐substituted benzo[b][1,8]naphthyridines in high yield has been developed via the Friedländer condensation of 2‐aminoquinoline‐3‐carbaldehyde 1 with various alicyclic ketones in a base catalyst (aq. potassium hydroxide). A series of benzo[b][1,8]naphthyridines branched with various side‐chains and substituents were prepared with the aim of being investigated as a fluorescent agents. Electronic absorption and fluorescence properties of some representative benzonaphthyridines (3d, 5b and 21f) in homogeneous organic solvents, dioxane–water binary mixtures and in the microheterogeneous media (sodium dodecyl sulphate (SDS), cetyl trimethyl ammonium bromide (CTAB) and Triton‐X100 micelles) have been examined. A linear correlation between solvent polarity and fluorescence properties was observed. Further, the interaction of these benzonaphthyridines (3d, 5b and 21f) with human serum albumin (HSA) and bovine serum albumin (BSA) in phosphate buffer have been examined by UV‐vis absorption and fluorescence spectroscopy. The fluorescence intensity of 3d, 5b and 21f increases with the increasing HSA and BSA concentration. These benzonaphthyridines also quench the 345 nm fluorescence of BSA in phosphate buffer (λ_ex 280 nm). These compounds have potential for use as neutral and hydrophobic fluorescence probes for examining the microenvironments in proteins, polymers, micelles, etc. Copyright © 2011 John Wiley & Sons, Ltd.     

A flow-injection chemiluminescence method for the determination of human serum albumin, using the reaction of fluorescein-human serum albumin-sodium hypochlorite by the enhancement effect of the cationic surfactant cetyltrimethylammonium bromide.

The interaction between surfactant and fluorescein was studied, using a fluorescence spectroscopy and flow-injection (FI) chemiluminescence (CL) method. It was found that the cationic surfactant cetyltrimethylammonium bromide (CTAB) could cause the structural transformation of fluorescein from the quinone to the spirolactone form, and greatly enhance the CL intensity of the fluorescein-human serum albumin (HSA) complex. Based on this finding, a rapid and sensitive FI-CL method was developed for the determination of HSA. Under the optimum conditions, the proposed method has a linear range of 0.05-24.0 microg/mL, with a detection limit of 0.03 microg/mL for HSA (3sigma). The relative standard deviation (RSD) of 1.2 microg/mL HSA (n = 8) is 0.8%. The method was applied to the determination of protein content in urine samples, with satisfactory results. Density functional theory was used to study the mechanism of surfactant-enhanced CL intensity of the fluorescein-HSA complex.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

Flow injection analysis-Rayleigh light scattering detection for online determination of protein in human serum sample

A flow injection analysis (FIA) system combined with Rayleigh light scattering (RLS) detection is developed for the sensitive and rapid determination of protein concentration in human serum sample. This method is based on the weak intensity of RLS of Eriochrome Black T (EBT, 2-hydroxy-1-(1-hydroxy-2-naphthylazo)-6-nitronaphthalene-4-sulfonic acid sodium salt), which can be enhanced by the addition of protein in weakly acidic solution. The effects of pH and interfering species on the determination of protein were examined. Calibrations for protein, based on RLS intensity, were linear in the concentration ranges of 7-36 microg/ml for human serum album (HSA) and 8-44 microg/ml for bovine serum album (BSA). The detection limits of the method were found to be 0.882 and 2.507 microg/ml for HSA and BSA, respectively. A relative standard deviation of 0.76% (n=5) was obtained with 20 microg/ml HSA standard solution. The FIA-RLS method was more stable than the general RLS method, and the average RSD value of FIA-RLS was less than that of the general RLS. The sample rate was determined to be 90 samples per hour.     

Interaction of a protein, BSA, and a fluorescent probe, Mag-Indo-1, influence of EDTA and calcium on the equilibrium.

Recent findings indicate that ion-chelator probes with tetracarboxylate structure bind proteins. It was suggested that these fluorescent probes are valuable tools to gain information on protein structure through the energy transfer from tryptophans to the bound probe. Here, the binding of the fluorescent probe Mag-Indo-1 to bovine serum albumin (BSA) was investigated. Mag-Indo-1 was reported previously to serve as a probe for magnesium cations (Kd = 2.8 x 10(-4) M for zero ionic strength) which can also interact with calcium cations (Kd = 7.5 x 10(-7) M). Probe complexation with protein results in a shift of the emission fluorescence spectrum of the probe from 480 to 457 nm. We used emission fluorescence techniques to monitor this interaction. Computational resolution of the complex fluorescence spectra and a new software to test the theoretical model were developed in our laboratory. This enabled us to calculate the number of interacting sites and the dissociation constants. The fluorescent probe Mag-Indo-1 binds at a singular site with high affinity (Kd = 1.8 x 10(-7) M) to bovine serum albumin (BSA). Since proteins are known to bind several compounds unspecifically, we have studied the influence of EDTA as a competitor of the probe. Our findings suggest that the BSA binding site is identical for both Mag-Indo-1 and EDTA. We found that EDTA binds the protein with Kd = 0.4 x 10(-3) M. We studied the influence of calcium and found that Mag-Indo-1 does not bind the calcium free Apo-protein anymore.     

A fluorimetric method for the determination of nucleic acids using Ce(IV) and sodium triphosphate.

A novel and stable fluorimetric method was established for the determination of nucleic acids. The proposed method is based on the reduction by nucleic acids of Ce(IV) to fluorescent Ce(III). The fluorescence intensity can be greatly increased by sodium triphosphate. The enhanced fluorescence intensity is proportional to the concentration of nucleic acids in the range 4.2 x 10(-8)-4.2 x 10(-6) g/mL for fish sperm DNA and 5.0 x 10(-8)-6.5 x 10(-6) g/mL for yeast RNA, and the detection limits (S/N = 3) are 13.5 ng/mL and 45 ng/mL, respectively. The reaction mechanism of the hydrolytic scission of nucleic acids by Ce(IV) is discussed.     

Use of sodium lauroyl sarcosinate in a high-sensitivity protein assay by resonance light scattering technique

A simple and high-sensitivity method has been developed for the determination of proteins in aqueous solutions by resonance light scattering (RLS) technique. At pH 3.4 and ionic strength 1.2 x 10(-3), the weak RLS intensity of sodium lauroyl sarcosinate was greatly enhanced by the addition of proteins with the maximum peak located at 391 nm. Under the optimum conditions, the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.04 to 2.1 microg/mL for lysozyme, 0.0025 to 1.2 microg/mL for bovine serum albumin, 0.0075 to 0.9 microg/mL for human serum albumin, 0.02 to 1.4 microg/mL for gamma-globulin, 0.02 to 0.8 microg/mL for egg albumin, and 0.01 to 0.6 microg/mL for hemoglobin. Low detection limits ranging from 0.8 ng/mL to 4.3 ng/mL depending on the kind of proteins that have been achieved. The protein concentrations in synthetic samples and real biochemical samples were determined with satisfactory results. This method presented here is not only sensitive and simple but also reliable and suitable for practical bioassay applications.