Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb3+ complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.
This paper reports a highly sensitive fluorescent immunoassay for the detection of human immunoglobulin G based on PbS nanoparticles and DNAzyme. A sandwich immunoassay format was performed on a microtiter plate. Goat anti-human IgG was coated onto the polystyrene microtiter plate. The human IgG analyte was first captured by the goat anti-human IgG, and then sandwiched by a goat anti-human IgG antibody labeled with PbS nanoparticles. After being dissolved with HNO3, the released Pb2+ made the substrate chain of the DNAzyme labeled with the fluorophore dissociate from the enzyme strand of the DNAzyme labeled with the quencher, which resulted in fluorescence recovery. Then, the human IgG could be detected indirectly from the fluorescent signals. Under the optimized conditions, the linear range of the developed immunosensor was from 1 ng mL−1 to 10 μg mL−1 with a detection limit of 0.8 ng mL−1. This immunosensor could be used to detect the amount of human IgG in human serum samples.
The L-ascorbic acid concentration in beverages was measured after separation by silica gel thin layer chromatography (TLC) by visually determining the time in autocatalytic reaction for the L-ascorbic acid spot to turn the same yellow color of the background and disappear (the end time of the induction period) after spraying the slide with a 3,6-dihydroxyxanthane solution. There was a good linear relationship between the end time of the induction period and the concentration of L-ascorbic acid for concentrations in the range of 5.0 – 20 mM (r2 = 0.9944). In addition, there was a good relationship expressed by a quadratic equation in the concentration range of 0.1 – 5.0 mM (r2 = 0.9975). The relative standard deviations of the L-ascorbic acid values for 3 beverages (2.2 – 8.6 mM) were less than 5% (n = 5), and the recovery of 5.0 mM L-ascorbic acid from 4 beverages (0.7 – 7 mM) was 97 – 110%. A good correlation was also observed between the L-ascorbic acid values of 23 beverages (0 – 86 mM) determined by the proposed TLC method and the colorimetric method contained in a commercially available kit for L-ascorbic acid (r2 = 0.9945).
Several methods for quantifying the purine content in food and drink have been described using high-performance liquid chromatography (HPLC). We have developed an improved HPLC method that is based on a method reported by Kaneko et al. and that is more sensitive yet simple, and suitable for determining the purine content of beer and beer-like alcoholic beverages. Quantitative HPLC separation was performed on a Shodex Asahi Pak GS-320HQ column with an isocratic elution of 150 mmol/L sodium phosphate buffer (H3PO4/NaH2PO4 = 20:100 (v/v)). The retention times for the four analytes, namely, adenine, guanine, hypoxanthine and xanthine, were 19.9, 25.0, 29.3 and 43.0 min, respectively. The resolution was good, and there was no excessive interference from the other compounds in the beverages at these retention times. Furthermore, the detection limit for all the analytes was improved to less than 0.0075 mg/L, and all the calibration curves showed good linearity (r2 > 0.999) between 0.013 and 10 mg/L for adenine and guanine, and between 0.025 and 10 mg/L for hypoxanthine and xanthine. The pretreatment was simplified by removing some procedures and optimizing the perchloric acid hydrolysis and the enzymatic peak-shift assay. We reduced the sample dilution rate by almost 50%, and the time spent on pretreatment from 4 days to only 180 min. The recovery of the analytes from spiked samples was 94.8 – 103.8%. This method may be useful for evaluating quantitative and qualitative differences in the purine content of beer and beer-like alcoholic beverages.
A rapid on-site air sampling technique was developed with a miniaturized needle-type sample preparation device for a systematic evaluation of the indoor air environments in school facilities. With the in-needle extraction device packed with a polymer particle of divinylbenzene and activated carbon particles, various types of volatile organic compounds (VOCs) were successfully extracted. For evaluating the indoor air qualities in school facilities, air samples in renovated rooms using organic solvent as a thinner of the paint were analyzed along with measurements of several VOCs in indoor air samples taken in newly built primary schools mainly using low-VOCs materials. After periodical renovation/maintenance, the time-variation profile of typical VOCs found in the school facilities has also been monitored. From the results, it could be observed that the VOCs in most of the rooms in these primary schools were at a quite low level; however, a relatively higher concentration of VOCs was found in some specially designed rooms, such as music rooms. In addition, some non-regulated compounds, including benzyl alcohol and branched alkanes, were detected in these primary schools. The results showed a good applicability of the needle device to indoor air analysis in schools, suggesting a wide range of future employment of the needle device, especially for indoor air analysis in other types of facilities and rooms including hospitals and hotels.
A carbon-based nanoporous sorbent was first used for microextraction in a packed syringe (MEPS) before HPLC/UV analysis of some biophenols in rat plasma. A laboratory-made programmable apparatus was designed and used for automation of the extraction procedure. The MEPS syringe was packed with 2 mg of CMK-3 sorbent, between the barrel and the injection needle, and mounted on an apparatus for programming of the conditioning, sampling, washing, elution and cleaning steps. All steps of the microextraction procedure were carefully optimized on the system. For optimization of important factors, such as the number of adsorption and elution cycles, elution volume and pH, a multivariate central composite design method was used. The highest recoveries were obtained for 24 and 10 times of adsorption and elution cycles, respectively, using 100 μL of acetonitrile as the eluent and a sample pH of 2. Good results were obtained in terms of the precision (RSD 1.6, 2.5 and 2.3%) and detection limit (0.7, 4.7 and 0.25 μM) for caffeic acid, tyrsol and oleuropein, respectively. The method was simple, efficient and appropriate for sample clean up before analysis by HPLC, and was successfully applied to the determination of biophenols in the plasma of several rats that received an olive leaves extract either by a gavage or an intraperitoneal injection method. A positive correlation was found between the amount of olive extract’s feeding of the rats and the level of their plasma biophenols.
In this work, a rapid and sensitive method, based on ultrasound-assisted extraction of samples with a small volume of toluene, has been developed for the quantification of C60 and C70 fullerenes in soil and sediments. Good extraction efficiencies were obtained at all of the fortification levels studied (200, 100, 20 and 2 ng/g), which ranged from 72 to 104%. An important effect of the extraction temperature was observed on C60 recoveries, and an acceptable decrease in the recovery rates was observed in aged samples. In the case of wet samples, the extraction can be done by percolating a small volume of acetonitrile before extraction. The extraction yields of the developed method for the extraction of C60 and C70 from soil were compared with those obtained by pressurized liquid extraction. Finally, analyses of fullerenes in agricultural and industrial soils and sediments were carried out, but no fullerene residues were found.
We investigated the efficacy and utility of a post-column derivatization method for the detection of amines with o-phthalaldehyde (OPA) in ultra high-performance liquid chromatography (UHPLC). Since it was anticipated that a derivatization reaction system placed downstream of the column would give considerable band broadening to narrow peaks generated by a UHPLC column, we examined the contributions of the dimensions of the reaction tube, the fluorescence (FL) detector flow cell, and the column to the extra-column peak broadening, and optimized the post-column derivatization system in UHPLC. We successfully separated amines within 6 min with gradient elution by using the post-column derivatization system connected to a UHPLC system. The analysis time was reduced by a factor of 7 compared with that by conventional HPLC. The detection limits were 18.0 – 105.7 fmol, depending on the compounds; the reproducibilities were 0.11 – 0.82% RSD for the retention times and 0.78 – 1.66% RSD for the peak areas (n = 10), respectively. The linear dynamic ranges were up to 12, 20, and 40 pmol/μL, depending on the compounds, respectively.
An acid dissociation constant of tetrabromophenolphthalein ethyl ester (TBPE) was determined through the measurement of electrophoretic mobility by capillary zone electrophoresis (CZE). Although TBPE is degradable in acidic pH region and it gradually degraded at pH conditions around and below its pKa values in the time scale of the CZE measurement, equilibrium species of interest were detected as a peak-shaped signal with tailing of the degraded species. Changes in electrophoretic mobility of the equilibrium species of TBPE were analyzed at its detectable pH range in the presence of phenol red as a mobility standard. An acid dissociation constant of 3.47 ± 0.06 (pKa, I = 0.01, 25°C, standard error) was determined for TBPE.
Dissociation kinetic analysis of a complex of Ce3+ with a polyaminocarboxylic ligand, 8-amino-2-[(2-amino-5-methylphenoxyl)methyl]-6-methoxyquinoline-N,N,N′,N′,-tetraacetic acid (Quin2), was studied by microchip capillary electrophoretic reactor. Dissociation rate constants, kd, of Ce3+-Quin2 complex in alkaline conditions at pH 8.3 – 9.8 were determined. The linear relationship of kd with the concentration of hydroxide ion indicates the existence of a hydroxide ion-assisted path in the dissociation reaction of Ce3+-Quin2 complex in alkaline conditions. The solvolytic dissociation rate constant, and the hydroxide ion-assisted dissociation rate constant of Ce3+-Quin2 complex were determined to be 1.55 × 10−3 and 3.24 × 102 s−1 in the analysis of the dependence of kd with the concentration of hydroxide ion, respectively.
In the present work, all steps of the sulfate reduction procedures, such as reduction, distillation and sulfate determination were joined in only one FIA system. The formed sulfide was on-line determined by employ the Fisher reaction. The proposed method presented a LOD of 1.00 μg L−1 and a LOQ of 3.33 μg L−1, well lower than the most commonly used turbidimetric determination, with no significant interferences. Additionally, we reached an analytical frequency of 6 measurements per hour, including all steps, beginning with the introduction of the sample up to the signal reading. Therefore, it revealed very fast, low reagent/sample consumption, agreeing with green-chemistry statements. This method has also a wide linear range (3.33 up to 1000 μg L−1), being useful for both low and high sulfate concentrations.
In this work, aiming to develop a simple, inexpensive method for the determination of low bromate levels in water samples, a liquid waveguide capillary cell (LWCC) was coupled to a FIA system. The long optical path (100 cm) of the LWCC was used to improve the sensitivity and the limit of detection without resorting to any off-line or in-line preconcentration processes. The spectrophotometric determination was based on the oxidation of chlorpromazine by bromate in an acidic medium, resulting in the formation of a colored radical product. Sulfamic acid was added to the reagent for minimizing the interference of nitrite, and a chelating ion exchange resin was used to remove major cationic interferences. The developed system allowed the determination of bromate within the range between 1 – 20 μg L−1 with a detection limit of 0.2 μg L−1.
A novel electrochemical assay for lead (Pb2+) detection was developed in this study, involving the use of screen-printed electrodes (SPEs). The sensor was used with selected supporting electrolytes in conjunction with square wave anodic stripping voltammetry (ASV) to study the redox characteristics of lead. Good response currents were obtained using a supporting electrolyte comprising acetate buffer pH 5.0 with 0.5 mol L−1 NH4Cl. Without a deposition process, the anodic peaks for higher lead concentrations (ranging from 0.7 to 10 mg L−1) were sharp and symmetrical on the bare electrode surface. Optimal ASV conditions were obtained with a well-defined stripping peak for 200 μL deposition solution volumes, a deposition potential of –1.2 V for Ag/AgCl, and a deposition time of either 300 or 400 s. Under optimal conditions, a calibration curve ranging from 6.25 to 500 μg L−1 was obtained for lead determination. Furthermore, the performance of a Hg-free sensor for Pb2+ determination was compared with that of a Hg-coated electrode, and the detection limits were approximately 0.9 – 1.5 μg L−1.
The preparation and characterization of Sb-doped Bi2UO6 solid solutions, in a limited composition range, is reported for the first time. The solid solutions were prepared by solid-state reactions of Bi2O3, Sb2O3 and U3O8 in the required stoichiometry. The reaction products were characterized by X-ray diffraction (XRD) and X-ray absorption spectroscopy (XAS) measurements at the Bi and U L3 edges. The XRD patterns indicate the precipitation of additional phases in the samples when Sb doping exceeds 4 at%. The chemical shifts of the Bi absorption edges in the samples, determined from the XANES spectra, show a systematic variation only up to 4 at% of Sb doping and support the results of XRD measurements. These observations are further supported by the local structure parameters obtained by analysis of the EXAFS spectra. The local structure of U is found to remain unchanged upon Sb doping indicating that Sb+3 ions replace Bi+3 during the doping of Bi2UO6 by Sb.
In glow discharge optical emission spectrometry, two-dimensional emission images for iron atomic lines were measured by using an imaging spectrograph equipped with a CCD detector, when a radio-frequency (r.f.) power source was employed for excitation. Emission images at the Fe I 371.99-nm and the Fe I 375.82-nm lines, having different excitation energies, were analyzed by the two-line method to obtain the spatial distribution of the excitation temperature in the plasma. Their emission intensities had a concentric-circle-like distribution along the radial direction of the plasma to become weaker towards the surrounding portion, which was very similar to a direct-current (d.c.) glow discharge plasma. On the other hand, the spatial distribution in the excitation temperature became relatively uniform over the central portion of the plasma, also being analogous between the r.f. and the d.c. glow discharge plasmas. These results imply that there is a major excitation process that occurs in a glow discharge plasma regardless of the power modes.