Micromol Dm Research Articles

Spectrophotometric Determination of Trace Phosphate Ions by Amplitude-Modulated Flow Analysis Coupled with Malachite Green Method

An amplitude-modulated flow analysis coupled with lock-in detection is proposed for the determination of trace phosphate. The flow rate F(S) of sample solution is varied in response to a periodic signal V(c) of alternating waveform. A coloring reagent solution (ammonium molybdate + Malachite Green) is delivered at a constant flow rate F(R). Under the constant total flow rate F(T), both solutions are merged with a diluent (water). Phosphate ion in the sample reacts with molybdate and then with Malachite Green to form green ion pairs in acidic media. Downstream, the absorbance of the mixed solution is measured at 625 nm. The output voltage V(d) from the detector is sent to a lock-in amplifier, where the wave component of V(d) that has the same frequency as that of V(c) is distinguished from background signals. Phosphate ion can be determined from the amplitude of the component thus extracted. The calibration curve is linear (r(2) > 0.998) and the limit of detection (3.3sigma) is 0.17 micromol dm(-3). Compared with a conventional flow-based method with no modulation, the present method is less susceptible to the baseline drift due mainly to the adsorption of the ion-pair on the optical window, because the quantification is based not on the absorbance itself but on the amplitude of the absorbance. Good recoveries around 100% are obtained for the phosphate ions spiked into real water samples.

Analytical Parameters for Amplitude-Modulated Multiplexed Flow Analysis

Analytical conditions of amplitude-modulated multiplexed flow analysis, the basic concept of which was recently proposed by our group, are investigated for higher sample throughput rate. The performance of the improved system is evaluated by applying it to the determination of chloride ions. The flow rates of two sample solutions are independently varied in accordance with sinusoidal voltage signals, each having different frequency. The solutions are merged with a reagent solution and/or a diluent, while the total flow rate is held constant. Downstream, the analytical signal V(d) is monitored with a spectrophotometer. The V(d) shows a complicated profile resulting from amplitude modulated and multiplexed information on the two samples. The V(d) can, however, be deconvoluted to the contribution of each sample through fast Fourier transform (FFT). The amplitudes of the separated wave components are closely related to the concentrations of the analytes in the samples. By moving the window for FFT analysis with time, a temporal profile of the amplitudes can be obtained in real-time. Analytical conditions such as modulation period and system configuration have been optimized using aqueous solutions of Malachite Green (MG). Adequate amplitudes are obtained at the period of as low as 5 s. At this period, the calibration curve for the MG concentration of 0-30 micromol dm(-3) has enough linearity (r(2) = 0.999) and the limit of detection (3.3sigma) is 1.3 micromol dm(-3); the relative standard deviation of repeated measurements (C(MG) = 15 micromol dm(-3), n = 10) is 2.4%. The developed system has been applied to the determination of chloride ions by a mercury(II) thiocyanate method. The system can adequately follow the changes in analyte concentration. The recoveries of chloride ion spiked in real water samples (river and tap water) are satisfactory, around 100%.

Effect of (C6H5)3PbCl and (C6H5)3SnCl on Delayed Luminescence Intensity, Evolving Oxygen and Electron Transport Rate in Photosystem II of Chlorella vulgaris

The effect of the organometallic compounds containing lead, (C6H5)3PbCl , and tin, (C6H5)3SnCl, on Chlorella green algae photosystem II was studied. Suspension of the algae treated with (C6H5)3SnCl at concentrations of 1.0 and 4.0 micromol dm(-3) for 22 h revealed a decrease in most physiological parameters studied, particularly in decasecond component of delayed chlorophyll luminescence, photosynthetic electron transport rate and diluted oxygen concentration, which implies an inhibition of photosynthetic electron transport as well as oxygen evolving system. On the other hand, (C6H5)3PbCl caused stronger inhibition than (C6H5)3SnCl, particularly in the higher concentration.

Measurement of the Binding of Cholera Toxin to GM1 Gangliosides on Solid Supported Lipid Bilayer Vesicles and Inhibition by Europium (III) Chloride

In this paper the immobilization of small unilamellar DMPC/GM1 lipid vesicles containing a water-soluble bodipy dye is described. The binding of the complete alphabeta toxin expressed by Vibrio cholerae to the attached vesicles was measured using Surface Plasmon Resonance (SPR) and a value of the dissociation constant K d obtained. Further measurements showed that the interaction of both the alphabeta-toxin and the beta-subunit alone resulted in the permeation of the lipid membrane, with release of a fluorophore contained within the vesicle being measured by combined SPR and Surface Plasmon enhanced Fluorescence Spectroscopy (SPFS). The leakage of dye through the membrane, measured by following the change in fluorescence, was fitted to a simple diffusion model. Finally, SPFS measurements of the effect of europium(III) chloride (EuCl 3) showed that cholera toxin binding and subsequent membrane permeation could be blocked by 1 micromol dm (-3) europium chloride. In view of the low oral toxicity of europium chloride, we speculate on the potential pharmaceutical applications of this molecule in the treatment of cholera infection.

An improved sensitivity non-enzymatic glucose sensor based on a CuO nanowire modified Cu electrode

CuO nanowires have been prepared and applied for the fabrication of glucose sensors with highly enhanced sensitivity. Cu(OH)(2) nanowires were initially synthesised by a simple and fast procedure, CuO nanowires were then formed simply by removing the water through heat treatment. The structures and morphologies of Cu(OH)(2) and CuO nanowires were characterised by X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The direct electrocatalytic oxidation of glucose in alkaline medium at CuO nanowire modified electrodes has been investigated in detail. Compared to a bare Cu electrode, a substantial decrease in the overvoltage of the glucose oxidation was observed at the CuO nanowire electrodes with oxidation starting at ca. 0.10 V vs. Ag/AgCl (saturated KCl). At an applied potential of 0.33 V, CuO nanowire electrodes produce high and reproducible sensitivity to glucose with 0.49 microA/micromol dm(-3). Linear responses were obtained over a concentration range from 0.40 micromol dm(-3) to 2.0 mmol dm(-3) with a detection limit of 49 nmol dm(-3) (S/N = 3). The CuO nanowire modified electrode allows highly sensitive, low working potential, stable, and fast amperometric sensing of glucose, thus is promising for the future development of non-enzymatic glucose sensors.

Liquid Core Waveguide Spectrophotometry for the Sensitive Determination of Nitrite in River Water Samples

A flow injection analysis system was built with a liquid core waveguide spectrophotometric detector using an 80 cm Teflon AF-1600 capillary tube (2,2-bistrifluoromethyl-4,5-difluoro-1,3-dioxole/tetrafluoroethylene). The system was applied to determine nitrite ion in river water samples. The lower limit of detection for nitrite was 2.1 nmol dm(-3) (0.1 ng dm(-3) as NO(2)(-)) and the relative standard deviation of measurements was typically 0.56% (n = 5) at 0.21 micromol dm(-3).

Arsenic speciation in xylem sap of cucumber (Cucumis sativus L.)

Flow injection analysis (FIA) and high-performance liquid chromatography double-focusing sector field inductively coupled plasma mass spectrometry (HPLC-DF-ICP-MS) were used for total arsenic determination and arsenic speciation of xylem sap of cucumber plants (Cucumis sativus L.) grown in hydroponics containing 2 micromol dm(-3) arsenate or arsenite, respectively. Arsenite [As(III)], arsenate [As(V)] and dimethylarsinic acid (DMA) were identified in the sap of the plants. Arsenite was the predominant arsenic species in the xylem saps regardless of the type of arsenic treatment, and the following concentration order was determined: As(III) > As(V) > DMA. The amount of total As, calculated taking into consideration the mass of xylem sap collected, was almost equal for both treatments. Arsenite was taken up more easily by cucumber than arsenate. Partial oxidation of arsenite to arsenate (<10% in 48 h) was observed in the case of arsenite-containing nutrient solutions, which may explain the detection of arsenate in the saps of plants treated with arsenite.

Adsorption Behavior of Metal Ions onto a Bovine Serum Albumin (BSA) Membrane Monitored by Means of an Electrode-Separated Piezoelectric Quartz Crystal

The interaction between metal ions and bovine serum albumin (BSA) was studied by using a piezoelectric quartz crystal (PQC) arranged in the electrode-separated configuration. A silanized surface of the PQC was coated with a BSA membrane via a coupling reaction with glutaraldehyde. The frequency shifts obtained from PQC coated with a BSA membrane suggested that various kinds of metal ions could be adsorbed onto the BSA membrane from aqueous solutions containing a low concentration of metal ions (2 or 10 micromol dm(-3)), only when the BSA was denatured with an alkaline solution. Anionic species of Pt(IV) and Au(III) were adsorbed onto the denatured BSA membrane from an acetic acid solution at pH 2.2, and cationic species of Cd(II), Zn(II), Co(II), Ni(II), Cu(II), and Ag(I), and cations, such as Ca2+, Ba2+, and Mg2+, were adsorbed from ammonia buffer at pH 9.5, whereas Al(III), Cr(III), Fe(III), Hg(II), and Pb(II) were hardly adsorbed. The adsorption mechanisms of these metal ions are discussed, based on the electrostatic interaction between the metal ions and the denatured BSA membrane, and complex formation between the metal ions and amino acid residues of the denatured BSA. Further, the PQC coated with a denatured BSA membrane was applied to the determination of Pt and Cd, using large frequency shifts for Pt(IV) and Cd(II).

Sensitive determination of heroin based on electrogenerated chemiluminescence of tris(2,2′-bipyridyl)ruthenium(ii) immobilized in zeolite Y modified carbon paste electrode

A novel method for rapid, inexpensive, sensitive and selective determination of heroin was proposed by flow injection electrogenerated chemiluminescence (ECL). Zeolite Y sieves were used for the preparation of a ECL sensor by immobilizing tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) in their supercages, which was achieved through the ion exchange properties of the sieves. The electrochemical and ECL behaviors of Ru(bpy)3(2+) immobilized in zeolite Y modified carbon paste electrode was investigated. The immobilized Ru(bpy)3(2+) displayed a pair of surface-controlled redox peaks with an electron transfer rate constant of 1.2 +/- 0.1 s(-1) in 0.1 mol dm(-3) pH 6.3 phosphate buffer. The modified electrode showed an electrocatalytic response to the oxidation of heroin, producing a sensitized ECL signal. The ECL sensor showed a linear response to flow injection of heroin in the range of 2.0-80 micromol dm(-3) with a detection limit of 1.1 micromol dm(-3). This method for heroin determination possessed good sensitivity and reproducibility with a coefficient of variation of 1.99% (n = 15) at 50.0 micromol dm(-3). The ECL sensor showed good selectivity and long-term stability. Its surface could be renewed quickly and reproducibly by a simple polish step.

Drought-induced changes in flavonoids and other low molecular weight antioxidants in Cistus clusii grown under Mediterranean field conditions.

Mediterranean plants have evolved a complex antioxidant defense system to cope with summer drought. Flavonoids, and particularly flavanols and flavonols, are potent in vitro antioxidants, but their in vivo significance within the complex network of antioxidant defenses remains unclear, especially in plant responses to stress. To gain insight into the role of flavonoids in the antioxidant defense system of Cistus clusii Dunal, we evaluated drought-induced changes in flavonoids in leaves and compared the response of these compounds with that of other low molecular weight antioxidants (ascorbic acid, tocopherols and carotenoids). Among the antioxidant flavonoids analyzed, epigallocatechin gallate was present in the greatest concentrations (up to about 5 micromol dm(-2)). Other flavanols, such as epicatechin and epicatechin gallate, were found at concentrations below 0.25 and 0.03 micromol dm(-2), respectively. Neither of the antioxidant flavonols analyzed, quercetin and kaempferol, were detected in C. clusii leaves. Epigallocatechin gallate, ascorbic acid and alpha-tocopherol concentrations increased to a similar extent (up to 2.8-, 2.6- and 3.3-fold, respectively) in response to drought, but the kinetics of the drought-induced increases differed. Epigallocatechin gallate, epicatechin and epicatechin gallate concentrations increased progressively during drought, reaching maximum values after 30 days of stress. Ascorbic acid concentrations increased twofold after 15 days of drought, and maximum values were attained after 50 days of drought. In contrast, alpha-tocopherol concentrations remained constant during the first 30 days of drought, but increased sharply by 3.3-fold after 50 days of drought. The maximum efficiency of photosystem II photochemistry and the extent of lipid peroxidation remained constant throughout the drought period, whereas the redox state of ascorbic acid and alpha-tocopherol shifted toward their reduced forms in response to drought, indicating that the concerted action of low molecular weight antioxidants may help prevent oxidative damage in plants.

Heteroclitic recognition of combinatorial TX1TX2T peptide mixtures by mucin-2 protein specific monoclonal antibody.

The mucin-2 (MUC2) glycoprotein secreted by the epithelial cells of human colon may be abnormally under-glycosylated in the case of cancer. Monoclonal antibody (mAb) 994 raised against the immunogenic part of the protein core, recognizes malignant human colon tissues as well as pentapeptides with TX1TX2T motif present in MUC2. Using a combinatorial approach and ELISA experiments it was found that mAb 994 is able to recognize peptides of the sub-library TQTX2T very strongly, and to some extent also peptides from TETX2T, TLTX2T and TVTX2T sub-libraries. Binding studies with peptides corresponding to the TQTX2T and TETX2T sub-libraries showed that mAb 994 recognized only six peptides (IC50 = 9-208 micromol dm(-3)) from the 19 compounds of the TQTX2T sub-library and only three peptides (IC50 = 3500-16700 micromol dm(-3)) from the 'second-best' TETX2T sub-library. The most pronounced mAb binding occurred when Gln was in position X1 and it was much weaker in the case of Glu, Val or Leu. As for X2 amino acids, the presence of Pro, Ala can provide a strong, while Tyr, Trp, Phe and Ser a weaker, peptide-antibody interaction. Data from this study suggest that pentapeptide TQTPT, whose sequence is present in the native protein, is bound most strongly. However, almost identical binding properties were observed with peptide TQTAT, whose sequence is not present in the protein. Apart from this, some other 'heteroclitic' peptides were found with a different rank in the binding-hierarchy. Based on these peptides artificial compounds can be prepared as potential candidates for vaccine development. Results of this study also provide a rationale for understanding the molecular background of the heteroclitic nature of the MUC2 protein core specific mAb 994.

Nitrite reduction and detection at a carbon paste electrode containing hemoglobin and colloidal gold.

A novel renewable reagentless nitrite biosensor based on the direct electron transfer of hemoglobin (Hb) and a new sensing mechanism was proposed by combining the advantageous features of colloidal gold nanoparticle and carbon paste technology. The direct electrochemistry of immobilized Hb displayed a pair of redox peaks with a formal potential of -42 mV (vs. NHE) in 0.2 mol dm(-3) NaAc-HAc buffer (pH 5.5). The immobilized Hb displayed an excellent response to the reduction of NO2(-) with one interfacial charge transfer followed by a chemical reaction (EC) mechanism. Under optimal conditions, the interfacial EC process could be used for the sensitive determination of NO2(-) with a linear range from 0.1 to 9.7 micromol dm(-3) and a detection limit of 0.06 [micro sign]mol dm(-3) at 3sigma. The amperometric determination of high concentrations of NO2(-) based on the irreversible reduction of NO could be performed at pH 4.0 with a linear range from 0.1 to 1.2 mmol dm(-3). The surface of biosensor could be renewed quickly and reproducibly by a simple polish step. The biosensor has been used satisfactorily for nitrite determination in native water samples.

A novel fluorescence reagent, 10-hydroxybenzo[h]quinoline-7-sulfonate, for selective determination of beryllium(II) ion at pg cm(-3) levels.

A facile method has been developed for the highly sensitive and selective determination of ultratrace Be(II) ion using a new fluorimetric reagent, 10-hydroxybenzo[h]quinoline-7-sulfonate (HBQS), under extremely alkaline conditions, at pH 12.0. This reagent is quite suitable for the very small ion, Be(II), to form a 6-membered chelate ring, compatible with a high fluorescence yield. The stoichiometry of the chelate is 1:1 for Be-HBQS at pH 12.0. The calibration graph gave a wide linear dynamic range, 2-100 nmol dm(-3) of Be(II) ion with the detection limit (3s blank) of 0.52 nmol dm(-3), or 4.7 pg cm(-3). The excellent sensitivity and toughness toward the matrix influence were demonstrated using the artificial sample solutions for air-dust. Coupled with the simple masking procedure using EDTA, the method enables one to determine Be(II) ion at nanomolar levels in the presence of metals at the natural abundance levels in air-dust samples, typically Al, Ca, Cu, Fe, Mg, Pb, and Zn at 130, 150, 1.0, 70, 33, 3.0, and 8.0 micromol dm(-3), respectively, in the final solution. The proposed method was successfully applied to the determination of Be in urban air.

Inactivation of antioxidant enzymes by peroxynitrite

Exposure of hemolysates and whole erythrocytes to peroxynitrite (bolus of 50 micromol dm(-3)-2 mmol dm(-3)) was found to inactivate erythrocyte antioxidant enzymes: glutathione peroxidase > superoxide dismutase > catalase. Inactivation of antioxidant enzymes by peroxynitrite may be one reason for the secondary oxidative stress in peroxynitrite-treated cells. When hemoglobin was not converted into the cyanmet form, an apparent activation of glutathione peroxidase activity by peroxynitrite was observed in hemolysates; this effect was artifactual and due to the pseudoenzymatic glutathione peroxidase activity of hemoglobin.