Estimation and in-situ detection of thorium in human liver cell culture by arsenazo-III based colorimetric assay.

To understand the biological effects of Thorium-232 (Th) in human cells and animal models as well as to assess mitigation strategies for its detoxification, there is a need to develop a sensitive, specific, high-throughput and easily-implementable assay for detection and estimation of Th in biological samples. Here, we have optimized arsenazo-III dye based colorimetric assay to detect Th in biological samples. The concentration of arsenazo-III (i.e. 50 µM) was optimized, which can reliably estimate Th in the concentration range of 2.5 to 40 µM. The optimized assay can specifically detect Th without interference from other metal ions (La, Ce, U, Fe, Ca, Cu, Zn and Mn). A significant correlation (R2 = 0.999) was found between arsenazo-III-based detection of Th and total reflection X-ray fluorescence. The conditions of present assay successfully estimated Th in cell culture medium, cell harvesting (trypsin-EDTA) solution and cell lysate obtained from human liver cell culture. Moreover, for the first time, we detected Th in-situ in adherent liver cells in culture after staining with arsenazo-III. This study confirms that Th can be specifically determined in biological samples using arsenazo-III with the sensitivity, which is relevant to thorium toxicity research.

Synthesis and evaluation of N-allylthiourea-modified chitosan for adsorptive removal of arsenazo III dye from aqueous solutions.

N-allylthiourea chitosan (ATUCS), a chelating material, was prepared, characterized, and studied for the removal of arsenazo III (As (III)) dye from aqueous solution. Scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and 1H- and 13C-nuclear magnetic resonance (NMR) were used to characterize the prepared adsorbent and to investigate the adsorption mechanism. Furthermore, the adsorption behavior of chitosan (CS) and ATUCS were studied under various conditions. The equilibrium adsorbed amount of As (III) onto ATUCS was found to be 116.3 mg/g, compared to 87.3 mg/g with respect to CS. The regeneration of the loaded CS and ATUCS were studied using 1:1 solution of H2O2-H2SO4 and reused with certain change in efficiency after the third cycle. The adsorption process was found to fit well with pseudo-second-order kinetic model. The equilibrium data were better described with the Freundlich isotherm. The monolayer adsorption capacity was found to be 204.08 and 90.90 mg/g for the As (III)/ATUCS and As (III)/CS systems, respectively, at 25 °C. The pH of the higher uptake of As (III) onto ATUCS and CS was 4-5 and 8.0, respectively. The results demonstrated improved adsorption of As (III) using ATUCS as compared to the CS.

Selectivity enhancement of Arsenazo(III) reagent towards heavier lanthanides using polyaminocarboxylic acids: a spectrophotometric study.

A new study has been conducted to quantify lanthanide(III) ions using Arsenazo III-polyaminocarboxylic acid (PACA) system. The study disclosed two different analytically important information: (i) λmax of lanthanide-Arsenazo III complexes for lighter lanthanides like Ce(III) and Nd(III) did not shift from its original position on addition of PACA and (ii) for heavier lanthanides like Dy(III), Tm(III) and Lu(III) a new λmax at 538 nm was observed, while wavelengths at 610 nm and 654 nm were disappeared in presence of ethylenediaminetertracetic acid (EDTA) and trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid (DCTA), further the intensity of peak decreased with increase in lanthanide(III) ion concentration. Effect of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and N-(2-hydroxyethyl) ethylenediamine-N,N',N'-triacetic acid (EDTA-OH) on Arsenzo(III)-Ln(III) complex is very weak and there is no analytically importance of such interaction. Moreover, this work confirms that Nd(III) and heavy lanthanides can be successfully determined with high accuracy in the working range of concentration of these metal ions.

Influence of gamma irradiation on uranium determination by Arsenazo III in the presence of Fe(II)/Fe(III).

Arsenazo III is a widely used reagent for the concentration measurement of uranium and other actinides in aqueous samples. This study indicates that, for routine aqueous samples, due to the strong complexing ability with Arsenazo III, Fe(III) can significantly decrease the UV-Vis absorbance of the U(VI)-Arsenazo III complex, whereas the influence of Fe(II) on the absorbance is negligible. However, when Fe(II) is present in a gamma-irradiated U(VI) aqueous sample, it can give rise to the Fenton reaction, which produces oxidizing radicals that decompose the subsequently added Arsenazo III, leading to a sharp decrease in the absorbance of the U(VI)-Arsenazo III complex. The decrease in absorbance depends on the iron content and irradiation dose. Furthermore, the oxidizing radicals from the Fenton reaction induced by gamma irradiation can be continually produced. Even if the irradiated solution has been aged for more than one month in the absence of light at room temperature and without the exclusion of oxygen, the reactivity of the radicals did not decrease toward the subsequently added Arsenazo III. This finding demonstrates that the presence of Fe(II) in gamma-irradiated U(VI) aqueous samples can lead to incorrect U(VI) measurement using the Arsenazo III method, and a new method needs to be developed for the quantitative determination of U(VI) in the presence of gamma radiation and ferrous iron.

Optimization of the arsenazo-III method for the determination of uranium in water and plant samples.

This work reports a reliable, fast and optimized photometric technique based on the specific chemical complexation of uranyl ion with arsenazo-III. In the case of solid samples (plant samples), for which mineralization under acidic and oxidative conditions was used, addition of ascorbic acid led to stabilization of the arsenazo-uranyl complex over time. The results, in total agreement with data obtained from α and γ spectrometries, demonstrate that the present technique is able to precisely quantify uranium in water as well as in plant samples, within the µg/L and mg/g ranges respectively.

Flow injection online spectrophotometric determination of uranium after preconcentration on XAD-4 resin impregnated with nalidixic acid.

In this work, spectrophotometer was used as a detector for the determination of uranium from water, biological, and ore samples with a flow injection system coupled with solid phase extraction. In order to promote the online preconcentration of uranium, a minicolumn packed with XAD-4 resin impregnated with nalidixic acid was utilized. The system operation was based on U(VI) ion retention at pH 6 in the minicolumn at flow rate of 15.2 mL min(-1). The uranium complex was removed from the resin by 0.1 mol dm(-3) HCl at flow rate of 3.2 mL min(-1) and was mixed with arsenazo III solution (0.05 % solution in 0.1 mol dm(-3) HCl, 3.2 mL min(-1)) and driven to flow through cell of spectrophotometer where its absorbance was measured at 651 nm. The influence of chemical (pH and HCl (as eluent and reagent medium) concentration) and flow (sample and eluent flow rate and preconcentration time) parameters that could affect the performance of the system as well as the possible interferents was investigated. At the optimum conditions for 60 s preconcentration time (15.2 mL of sample volume), the method presented a detection limit of 1.1 µg L(-1), a relative standard deviation (RSD) of 0.8 % at 100 µg L(-1), enrichment factor of 30, and a sample throughput of 42 h(-1), whereas for 300 s of the preconcentration time (76 mL of sample volume), a detection limit of 0.22 µg L(-1), a RSD of 1.32 % at 10 µg L(-1), enrichment factor of 150, and a sampling frequency of 11 h(-1) were reported.

A simplified suite of methods to evaluate chelator conjugation of antibodies: effects on hydrodynamic radius and biodistribution.

INTRODUCTION: Antibodies covalently conjugated with chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) are required for radioimmunoscintigraphy and radioimmunotherapy, which are of growing importance in cancer medicine. METHOD: Here, we report a suite of simple methods that provide a preclinical assessment package for evaluating the effects of DOTA conjugation on the in vitro and in vivo performance of monoclonal antibodies. We exemplify the use of these methods by investigating the effects of DOTA conjugation on the biochemical properties of the DAB4 clone of the La/SSB-specific murine monoclonal autoantibody, APOMAB, which is a novel malignant cell death ligand. RESULTS: We have developed a 96-well microtiter-plate assay to measure directly the concentration of DOTA and other chelators in antibody-chelator conjugate solutions. Coupled with a commercial assay for measuring protein concentration, the dual microtiter-plate method can rapidly determine chelator/antibody ratios in the same plate. The biochemical properties of DAB4 immunoconjugates were altered as the DOTA/Ab ratio increased so that: (i) mass/charge ratio decreased; (ii) hydrodynamic radius increased; (iii) antibody immunoactivity decreased; (iv) rate of chelation of metal ions and specific radioactivity both increased and in vivo, (v) tumor uptake decreased as nonspecific uptake by liver and spleen increased. CONCLUSION: This simplified suite of methods readily identifies biochemical characteristics of the DOTA-immunoconjugates such as hydrodynamic diameter and decreased mass/charge ratio associated with compromised immunotargeting efficiency and, thus, may prove useful for optimizing conjugation procedures in order to maximize immunoconjugate-mediated radioimmunoscintigraphy and radioimmunotherapy.

Kinetic-spectrophotometric determination of trace amounts of vanadium(V) based on its catalytic effect on the reaction of DBM-arsenazo and potassium bromate.

A simple and sensitive kinetic-spectrophotometric method is developed for the determination of trace vanadium(V), based on the catalytic effect of vanadium(V) on the oxidation of DBM-arsenazo by potassium bromate in 0.0138 moll(-1) phosphoric acid medium and at 100 degrees C in the presence of citric acid as activator. The absorbance is measured at 528 nm with the fixed-time method. The optimization of the operating conditions regarding concentrations of the reagents, temperature and interferences are also investigated. The working curve is linear over the concentration range 0-20 ngml(-1) of vanadium(V) with good precision and accuracy and the detection limit was down to 3.44 ngl(-1). The relative standard deviation for a standard solution of 14 ngml(-1) is 0.28% (n=11). The apparent activity energies of the catalytic reaction and the non-catalytic reaction are 73.48, 113.5 kJ/mol, respectively. The proposed method proved highly sensitive, selective and relatively rapid for the assay of vanadium at low-level range of 0-20 ngml(-1) without any pre-concentration step. Thw method was applied to the determination of vanadium(V) in steels, rice, flour, cabbage, potato, fish, shrimp and tea samples with satisfactory results. The obtained results for the steel samples were excellent agreement with the standard reference values. The analytical results of the rice, flour, cabbage, potato, fish, shrimp and tea samples were excellent agreement with those of atomic absorption spectrometry. The recovery experiments have been made for the rice, flour, cabbage, potato, fish, shrimp and tea samples except the steels; excellent results were obtained. The relative standard deviations were over the range of 0.18-2.60% and the recoveries were over the range of 98.00-102.4%, respectively. The analytical results obtained were satisfactory.

Quantification of bifunctional diethylenetriaminepentaacetic acid derivative conjugation to monoclonal antibodies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The results of the characterization of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based method that was developed to establish the stoichiometry of CHX-A''-diethylenetriaminepentaacetic acid (DTPA) or benzyl-DTPA conjugated to a recombinant immunoglobulin G (IgG) are reported. This simple method does not require an accurate measurement of the sample protein concentration to accurately quantify the number of DTPA conjugated. It is also not necessary to thoroughly remove nonconjugated DTPA from the sample. The average number of moles of DTPA attached per mole of IgG was calculated from the difference in the observed masses of DTPA-IgG and nonconjugated IgG divided by the molecular weight of the DTPA derivative. As more DTPA is attached, the [M+H](+) peak of DTPA-IgG becomes broader and noisier. Also, the signal intensity in the mass spectrum decreases, apparently due to the increase in the heterogeneity in the number of DTPA attached to each molecule of IgG. The standard deviation of the measured mass and that of the stoichiometry of the DTPA attached per IgG increased as more DTPA was attached. The standard deviation, expressed as coefficient of variation for samples with 2 to 4 mol of DTPA attached per mole of IgG, was 8 to 9%.

Surface functionalization of magnetoliposomes in view of improving iron oxide-based magnetic resonance imaging contrast agents: anchoring of gadolinium ions to a lipophilic chelate.

Small unilamellar phospholipid vesicles containing the phosphatidylethanolamine-diethylene triamine pentaacetic acid (PE-DTPA) conjugate as one of the building stones were constructed. The ability of these colloids to complex gadolinium(III) ions at the surface of both the inner and outer bilayer shells was verified using a colorimetric method with Arsenazo III as a dye indicator. On incubation of these functionalized vesicles with magnetoliposomes (MLs, nanometer-sized magnetite cores encapsulated in a phospholipid bilayer), PE-DTPA percolates into the ML coat. The PE-DTPA content could be fine-tuned by varying the conjugate concentration in the donor vesicles. In the experimental conditions applied, up to 500 Gd(3+) ions were immobilized per ML colloid. The resulting ML-Gd(3+) complexes might have great potential, for example, as a novel magnetic resonance imaging contrast agent.

Galvanic cell without liquid junction for potentiometric determination of copper.

This paper describes potentiometric measurements in an integrated galvanic cell with both indicator and reference electrodes. Both electrodes are conducting polymer-based. The copper-sensitive indicator electrode is made by using poly(3,4-ethylenedioxythiophene) (PEDOT) doped with 2-(o-arsenophenylazo)-1,8-dihydroxynaphthalene-3,6-disulphonic sodium salt (Arsenazo-I) as the electroactive substance in the film, while the reference electrode is based on PEDOT doped by 2-morpholineoethanesulfonic acid (MES). It is shown that the galvanic cell can be used for determination of copper both in non-aqueous media (where all PVC-based membranes failed) and in the presence of chloride ions, which disturb the signal of conventional copper ion-selective electrodes with solid-state membranes. It is further shown that the titration of copper ions can be successfully monitored using the described electrochemical cell.

A microchip sensor for calcium determination.

A newly designed glass-PDMS microchip-based sensor for use in the determination of Ca(2+) ions has been developed, utilizing reflectance measurements from arsenazo III (1,8-dihydroxynaphthalene-3,6-disulfonic acid-2,7-bis[(azo-2)-phenyl arsenic acid]) immobilized on the surface of polymer beads. The beads, produced from cross-linked poly(p-chloromethylstyrene) (PCMS), were covalently modified with polyethylenimine (PEI) to which the Arsenazo III could be adsorbed. The maximum amount of Arsenazo III which could be immobilized onto the PEI-attached PCMS beads was found to be 373.71 mg g(-1) polymer at pH 1. Once fabricated, the beads were utilized at the detection point of the microfluidic sensor device with a fiber optic assembly for reflectance measurements. Samples were mobilized past the detection point in the sensor where they interact with the immobilized dye. The sensor could be regenerated and re-used by rinsing with HCl solution. The pH, voltage, linear range, and the effect of interfering ions were evaluated for Ca(2+) determination using this microchip sensor. At the optimum potential, 0.8 kV, and pH 9.0, the linear range of the microchip sensor was 3.57 x 10(-5) - 5.71 x 10(-4) M Ca(2+), with a limit of detection (LOD) of 2.68 x 10(-5) M. The microchip biosensor was then applied for clinical analysis of calcium ions in serum with good results.

Application of arsenazo III in the preparation and characterization of an albumin-linked, gadolinium-based macromolecular magnetic resonance contrast agent.

A macromolecular magnetic resonance contrast agent (MMCA) was prepared by linking bovine serum albumin (BSA) to gadolinium (Gd) via a chelating agent, diethylenetriaminepentaacetic acid (DTPA). Colorimetric testing with 2,7-bis(o-arsenophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid (arsenazo III) was performed to check for the appearance of free gadolinium during preparation and to quantify the Gd content in the final product. The complex was purified by dialysis, concentrated by lyophilyzation and characterized by magnetic resonance (MR) proton relaxation times. The resultant product had a molecular weight of about 90 kDa, Gd:BSA ratio of 14:1, and T1 and T2 relaxation times of 128.3 and 48.9 ms, respectively, at a field strength of 7Tesla (T) and at 20% concentration. Contrast enhancement of Gadomer-17 (a dendritic MMCA) and Gd-linked to BSA (Gd-BSA) was sequentially evaluated in a rat brain gliosarcoma model (n = 5) by MR imaging (MRI). Following intravenous injection, the blood concentration of Gadomer-17 fell rapidly, whereas that of Gd-BSA was almost constant for the duration of imaging. The areas of enhancement of both MMCAs were comparable. The spatial distribution of Gd-BSA showed good agreement with Evans blue-tagged albumin. Treatment with dexamethasone decreased Gd-BSA enhancement in the tumor. These results suggest that the arsenazo III method is applicable in preparing Gd-BSA to image brain tumors and their response to treatment. This simple method may also be useful for preparing other gadolinium-linked MMCAs.

Study on the binding of Arsenazo-TB to human serum albumin by Rayleigh light scattering technique and FT-IR.

The interaction between Arsenazo-TB and human serum albumin (HSA) was studied by Rayleigh light scattering (RLS) technique and Fourier transformed IR (FT-IR). The binding parameters of Arsenazo-TB with HSA were studied at different temperature of 288, 298, 308, 318 K under the optimum conditions. It is indicated by the Scatchard plots that the binding constant K decreased from 5.03 x 10(7) to 7.13 x 10(6) and the maximum binding number N reduced from 53 to 36 with the increasing of the temperature. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction. The free energy change deltaG0, the enthalpy change deltaH0 and the entropy change deltaS0 of 288 K were calculated to be -42.46 kJ/mol, -49.17 kJ/mol and 318.15 J/mol K, respectively. The alterations of protein secondary structure in the presence of Arsenazo-TB in aqueous solution were quantitatively calculated from FT-IR spectroscopy with reductions of alpha-helix from 57% to 40% and with increases of beta-sheet from 36% to 39%, beta-turn from 7% to 21%.

Spectrophotometric determination of uranium with arsenazo-III in perchloric acid.

A short, sensitive and reliable spectrophotometric method, which has advantages over all known "wet chemistry" methods for uranium determination with regard to tolerance to common interferences, has been developed for the determination of uranium. Selectivity, molar absorptivity and the determination range of uranium have been enhanced by using 0.07% arsenazo-III as a chromogenic reagent. The use of 3 mol dm(-3) perchloric acid as a medium of determination was found to be excellent in terms of good solvent compatibility on dilution, destruction of organic contamination and simplicity of operation. The uranium-arsenazo-III complex formed instantly, and was found to be stable for more than 3 weeks with constant absorbance. Beer's law was obeyed up to a uranium concentration of 16 microg g(-1), with a molar absorptivity at 651 nm of 1.45x10(5) mol(-1) dm(3) cm(-1) at 24+/-2 degrees C. Only phosphate and citrate at 70-fold excess over uranium interfere seriously, whereas other anions studied could be tolerated up to a 70-fold excess over uranium. Of the cations studied, only Mn(II), Co(II), Ni(II), Cu(II) and Cr(III) decreased the normal absorbance of the complex. Iron(III), Ce(III) and Y(III) enhanced the absorbance. Other cations studied did not affect the absorbance up to a 50-fold excess. The accuracy was checked by determining uranium from standard solutions in the range 10-50 microg g(-1). It was found to be accurate with a 96.0-98.6% recovery rate. The method has been successfully applied to standard reference materials and ore samples at microg g(-1) levels.

An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction.

Alizarin red S (ARS) staining has been used for decades to evaluate calcium-rich deposits by cells in culture. It is particularly versatile in that the dye can be extracted from the stained monolayer and assayed. This study describes a sensitive method for the recovery and semiquantification of ARS in a stained monolayer by acetic acid extraction and neutralization with ammonium hydroxide followed by colorimetric detection at 405 nm. This method was three times more sensitive than an older method involving cetylpyridinium chloride (CPC) extraction and resulted in a better signal to noise ratio, especially for weakly stained monolayers. The assay facilitates detailed inspection of mineralization by phase microscopy and semiquantification of the entire monolayer by extraction and quantification. The sensitivity of the assay is improved by the extraction of the calcified mineral at low pH and, since the mineral is already stained in a quantitative manner, there is no requirement for an additional colorimetric quantification step. Furthermore, the linear range is much wider than those of conventional assays for calcium, making dilutions of mineral extracts prior to measurement unnecessary. It has a wide range of potential uses including tumor characterization, mesenchymal stem cell evaluation, and osteogenic compound screening. Although more labor intensive than CPC extraction, the protocol is more sensitive and yields more reliable results for weakly mineralizing samples.

Determination of Mn(II) and Co(II) with Arsenazo III.

Complex formation between Arsenazo III and Mn2+ and Co2+ at equilibrium has been investigated at pH 7.2, and the stoichiometry and stability of the complexes have been determined. The data indicate that Arsenazo III is suitable for determination of Mn2+ and Co2+ on the micromolar scale. The dissociation constants of the phosphate complexes of Mn2+ and Co2+ at pH 7.2 were estimated with Arsenazo III as 3.6 and 10 mM, respectively.

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%.

The high-sensitivity determination of protein concentrations by the enhancement of Rayleigh light scattering of Arsenazo-DBN.

A new Rayleigh light scattering (RLS) assay of protein is presented in this paper. At the optimum pH 4.10, the weak RLS of Arsenazo-DBN can be greatly enhanced by the addition of proteins due to the interaction between protein and Arsenazo-DBN. Based on this, the reactions of Arsenazo-DBN and proteins, including bovine serum albumin, human serum album, gamma-globulin, egg albumin, lysozyme and trypsin, were studied. A new quantitative determination method for proteins has been developed. The linear range for human serum albumin, for example, is 0.085-34.62 micrograms mL-1 with a detection limit of 44.8 ng mL-1. Besides high sensitivity, the method is characterized by good reproducibility, rapidity of reaction, good stability, and few interfering substances. The determination of the proteins in human serum and urine samples by this method give results very close to those obtained using Coomassie Brilliant Blue G-250 colorimetry, with relative standard deviations of 0.7-2.5%.