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The resonance light scattering (RLS) spectral characteristics of the interaction between rose Bengal and mexiletine hydrochloride in the presence of cetylpyridinium bromide were investigated. A dual-wavelength resonance light scattering (DWO-RLS) method for the determination of mexiletine hydrochloride in drugs was established. In a weakly acidic solution, rose Bengal interacts with mexiletine hydrochloride and cetylpyridinium bromide to form a red ternary ion association complex, which led to a significantly enhanced resonance light scattering signal and produced two strong characteristic scattering peaks at 372 nm and 596 nm. In these two wavelengths the mass concentration of mexiletine hydrochloride was in the range of 0.004 to 0.65 mg·L-1 and had a good linear relationship with the resonance light scattering enhancement intensity (ΔIRLS), with detection limits of 0.003 2 mg·L-1 (372 nm) and 0.003 8 mg·L-1 (596 nm), respectively. When measured by the dual-wavelength resonance light scattering (DWO-RLS) technique, the detection limit was lower, only 0.001 8 mg·L-1. When the DWO-RLS method was applied to the determination of mexiletine hydrochloride in commercially available mexiletine hydrochloride tablets, and the recovery was 98.5%-103%, and the relative standard deviation was 2.0%-2.7%.
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OBJECTIVE@#To develop a fast, sensitive and cost-effective method based on resonance light scattering (RLS) for characterization of protein solubility to facilitate detection of changes in solubility of mutant proteins.@*METHODS@#We examined the response curve of RLS intensities to the protein concentrations in synchronous scanning mode. The curve intersection points were searched to predict the maximal concentrations of the protein in dispersion state, which defined the solubility of the protein in this given state. Bovine serum albumin (BSA, 0-50 g/L) was used as the model to investigate the influences of pH values (6.5, 7.0, and 7.4) and salt concentrations (0.05, 0.10, 0.15, and 0.20 mol/L) on the determined solubility. The solubility of glutathione S-transferase isoenzymes alpha (GSTA, 0-27.0 g/L) and Mμ (GSTM, 0-20.0 g/L) were estimated for comparison. The RLS-based method was used to determine the solubility of uricase (MGU, 0-0.4 g/L) to provide assistance in improving the solubility of its mutants.@*RESULTS@#We identified two intersection points in the RLS response curves of the tested proteins, among which the lower one represented an approximation of the maximal concentration (or the solubility of the protein) in single molecular dispersion, and the higher one the saturated concentration of the protein in multiple molecular aggregation. In HEPES buffer, the two intersection points of BSA (isoelectric point 4.6) both increased with the increase of pH (6.5-7.4), and their values were ~1.2 g/L and ~33 g/L at pH 7.4, respectively; the latter concentration approached the solubility of commercial BSA in the same buffer at the same pH. The addition of NaCl reduced the values of the two intersection points, and increasing salt ion concentration decreased the values of the lower intersection points. Further characterizations of GSTA and GSTM showed that the low concentration intersection points of the two proteins were ~0.7 g/L and ~0.8 g/L, and their high concentration intersection points were ~10 g/L and ~11 g/L, respectively, both lower than those of BSA, indicating the feasibility of the direct characterization of protein solubility by RLS. The two concentration intersection points of MGU were 0.24 g/L and 0.30 g/L, respectively, and the low concentration intersection point of its selected mutant was increased by 2 times.@*CONCLUSIONS@#RLS allows direct characterization of the solubility of macromolecular proteins. This method, which is simple and sensitive and needs only a small amount of proteins, has a unique advantage for rapid comparison of solubility of low-abundance protein mutants.
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Concentración de Iones de Hidrógeno , Luz , Dispersión de Radiación , Solubilidad , Análisis EspectralRESUMEN
A peptide microarray-based fluorescence and resonance light scattering ( RLS ) two readout assay was developed for screening thrombin inhibitors in blood samples. In this assay, the biotinylated peptide microarray was used as the platform. The peptide C-terminal fragments carried biotin sites departed from the slide when the biotinylated peptides were digested by thrombin hydrolysis reaction. The hydrolysis progress was labeled by fluorescence and 30 nm peptide-stabilized gold nanoparticles through the biotin-avidin reaction. In the presence of thrombin inhibitors, the hydrolysis reactions were blocked, and the inhibition capability of inhibitors could be detected by the fluorescent and RLS signal changes. The order of the half maximal inhibitory concentration ( IC50 ) of thrombin inhibitors in pure thrombin solution and spiked human serum were argatrobanHAT-Ⅲ>trypsin inhibitor>E-64>AEBSF. The reversible or irreversible characters of argatroban and HAT-Ⅲ had been estimated in human plasma. Compared with the experimental data of fluorescent and RLS assay in blood sample, the RLS assay labeled by 30 nm gold nanoparticles are more suitable for the inhibitor detection in complicated blood sample.
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In this study, based on its enhancement effect on resonance light scattering (RLS) of fluorosurfactant (FSN)-capped gold nanoparticles (GNPs), we reported a simple approach for the rapid sensing of captopril. Under optimum conditions, the lowest detectable concentration of captopril through this approach (S/N=3) was 0.01μg/mL. The calibration curve was linear over the range of 0.08-4.0μg/mL for the detection of captopril. The recoveries of captopril were found to fall in the range between 99% and 100%. We have validated the applicability of our method through the analyses of captopril in pharmaceutical formulations. Good agreements were obtained for the determination of captopril between the present approach and official method.
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A resonance light scattering(RLS) method for the determination of critical micelle concentration(CMC) of sodium dodecyl benzene sulfonate(SDBS) was proposed. Under room temperature, the RLS intensity of the SDBS system increased with increasing SDBS concentration. And when the concentration of SDBS approached CMC, the RLS intensity had increased sharply. The RLS peaks were appeared at 330 nm and 396 nm, respectively. The plot of the RLS intensity at 396 nm versus SDBS concentration was S-Curve. The concentration of SDBS at the intersection point of two tangents to S-curve was considered as SBDS CMC. This result was consistent with the results of the pyrene probe fluorescence spectrometry and electrical conductivity method. The influences of the concentration of Ca2+ on the aggregation behave of SDBS and SDBS-emulsion OP(OP) systems were studied by the RLS method. The results indicated that the mixed
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Objective To establish a rapid and sensitive method for detection of urinary protein.Methods In B-R buffer solution with pH 4.2,the signals of resonance light scattering by Poncesu S (PS) combined with protein in ?ex=?em=306nm were detected.Results There was a linear relation between the scattering signals of resonance light,and the protein concentration ranged from 0 to-1500 mg/l. The regression equation was ?I=2.24c-0.41,r=0.999 and the detection limit was 1.48 mg/l. The average recovery was 102.8% and the between-and within-subject coefficients of variation were 2.09% and 5.40% respectively.No significant difference was found compared with the method of PS.Conclusion The established method in this study is a simple,rapid and high sensitive method for determination of urinary protein.
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Objective To understand the spectroscopic property of AO-H2SO4-KBrO3-naphthol system and the enhanced mechanism of resonance light scattering(RLS),and to develop a method for the determination of trace naphthols in urine.Methods In the dilute H2SO4 medium,naphthols could react with KBrO3 and AO to form ion-association complexes,which produced a new RLS spectrum and resulted in the great enhancement of RLS.The characteristics of RLS spectrum,three-dimensions fluorescence spectrum,absorption spectrum and fluorescence spectrum and the optimum conditions of reaction were studied.Results The enhanced intensity of RLS was 468 nm.The linear range was at 1.41?10-7-2.80?10-5 mol/L for ?-naphthol,1.28?10-7-3.00?10-5 mol/L for ?-naphthol.The relative coefficient and the limits were 0.999 6 and 0.422?10-7 mol/L,0.999 3 and 0.385?10-7 mol/L for ?-naphthol and ?-naphthol,respectively.The urine samples analysis of the relative standard deviation was 4.8%-7.3% and the average recovery rate was 90.7%(n=6).Conclusion This method is sensitive,simple,rapid and applicable to the determination of trace naphthols in human urine.