Improvement of enzyme carbon paste-based biosensor using carbon nanotubes for determination of water-soluble analogue of vitamin E

The catalytic oxidation of a synthetic water-soluble analogue of vitamin E (α-tocopherol, Trolox) by tyrosinase enzyme in the presence of molecular oxygen was studied using electrochemical techniques. This specific enzymatic reaction was exploited for the preparation of a biosensor based on the amperometric reduction of the electroactive product (α-tocoquinone) formed. An electroactive surface of the transducers used was covered with a thin conductive layer of Nafion containing tyrosinase. Significant progress in sensitivity towards polyphenolic compounds such as Trolox was achieved at CPE with carbon nanotubes immobilised on its surface (CPE/CNTs) as electric transducers. The biosensor so developed can be used for the direct determination of total phenolic content (TPC). This important nutrition value can be expressed as the mass equivalent of Trolox, i.e. Trolox equivalent antioxidant capacity (TEAC), which could be used as an alternative to the evaluations currently used based on spectrophotometric methods such as total radical-trapping antioxidant parameter (TRAP), ferric reducing-antioxidant power (FRAP) or 1,1-diphenyl-2-picrylhydrazyl spectrometric assay (DPPH). The effects of the enzyme amount in the Nafion layer (3.0 µg), the influence of the nanoparticles present, the optimal pH value suitable for enzymatic activity (7.0), and the kinetics of enzymatic and electrochemical reactions were studied using cyclic voltammetry (CV). The determination of optimal conditions for amperometry in batch configuration (working potential, speed of stirring, volume of sample, calibration curve, etc.) was not a target of this electrochemical study.     

Synthesis of selectively deuterated and tritiated lupane derivatives with cytotoxic activity

The aim of this work was to synthesize deuterated and tritiated analogues of highly oxidized lupane derivatives known from our group. We selected compounds that previously showed very high cytotoxic activity on multiple cancer cell lines in order to further investigate the mechanism of their action. From starting material (compounds 1–4), we obtained benzyl platanate (5) and its reaction with deuteromethyltriphenylphosphonium iodide gave deuterated compound 6. Following benzyl deprotection gave free acid 7 and oxidation with SeO₂ gave 30-oxo-[29-²H₂]lup-20(29)-en-28-oic acid (8), which is one of the most active compounds synthesized in our group to date (IC₅₀ 6 μmol/L on CEM cell line). The alkylation of benzyl 2-hydroxy-3-oxolupa-1,20(29)-dien-28-oate (9) with methyliodide or deuteromethyliodide followed by a series of deprotection and hydrogenation steps gave compounds 10–14, where 2β-[31-²H₃]methoxy-3-oxolupan-20(29)-en-28-oic acid (13) is especially interesting, it showed lower activity on CEM cell line (IC₅₀ 10 μmol/L) however, it was very active against Ph1—positive human leukemia BV-173 (IC₅₀ 0.91 μmol/L) and against human myelogenous leukemia K562 (IC₅₀ 0.52 μmol/L). Selectively labelled [3α-²H] and [3α-³H] methyl 3β-acetoxy-21,22-dioxolup-18-en-28-oates 24, 25 were prepared in three steps by reduction of corresponding 3-oxo derivatives and they showed moderate activity on CEM cell line (IC₅₀ 10 μmol/L). In total, 11 labelled compounds (6–8, 11, 14, 18, 19, 21, 22, 24 and 25) have not been reported before.     

Equilibrium and kinetics of protein binding on ion-exchange cellulose membranes with grafted polymer layer

The performance of weak and strong anion- and cation-exchange membrane adsorbents with a grafted gel layer (Sartobind Q, D, S, and C) was investigated using six proteins: bovine serum albumin, human serum albumin, α-lactalbumin, β-lactoglobulin, lysozyme, and myoglobin. Static binding experiments were used to assess the effect of pH and buffer concentration and to determine the adsorption isotherms for selected membrane/protein combinations. The equilibrium data were duly described either by the Langmuir or Freundlich isotherms. Dynamic binding experiments were carried out for the same membrane/protein combinations in a broad range of linear flow velocity. Both the dynamic binding capacity at 10 % breakthrough and the final binding capacity at complete breakthrough were independent of the flow velocity despite strong dispersion of the adsorption zone. A good match between the equilibrium data from static and dynamic experiments was obtained for the anion exchangers. The correlation between the dynamic binding capacity and protein molecule size was observed for the strong cation exchanger. This was due to the different accessibility of the gel layer for the protein molecules.     

Synthesis,properties and crystal structures of nitrobenzoatocopper(II) complexes with pyrazinecarboxamide

The synthesis, spectral characterization and crystal structures of two nitrobenzoatocopper(II) complexes, namely [Cu(2-O₂Nbz)₂(pca)₂(H₂O)₂] (1) and [Cu(3,5-(O₂N)₂bz)₂(pca)₂(H₂O)₂] (2) (where 2-O₂Nbz = 2-nitrobenzoate, 3,5-(O₂N)₂bz = 3,5-dinitrobenzoate, pca = pyrazinecarboxamide), are reported. Complexes 1 and 2 consist of centrosymmetric molecules with the Cu(II) atom monodentately coordinated by a pair of anionic 2-nitrobenzoato (1) or 3,5-dinitrobenzoato (2) ligands and a pair of pyrazinecarboxamide ligands, forming a nearly tetragonal basal plane, and by a pair of water ligands that complete the tetragonal–bipyramidal coordination polyhedron. The molecules of both complexes are linked by N–H⋯O and O–H⋯O hydrogen bonds and lie in planes, which have different orientations depending on the space group. Similar experiments with 3-nitrobenzoic acid resulted in the isolation of the hydrolysis product [Cu(pyzCOO)₂] _n (3) (pyzCOO = pyrazinecarboxylate). The known crystal structure of complex 3 has been re-determined at low temperature with significantly higher precision. The crystal packing and C–H⋯O/C–H⋯N hydrogen bonds are discussed.     

Zinc catalyst recycling in the preparation of (all-rac)-α-tocopherol from trimethylhydroquinone and isophytol

Recycling of ZnCl₂ as a catalyst in the cyclocondensation of trimethylhydroquinone and isophytol to all-rac-α-tocopherol was studied. ZnCl₂ was recycled at over 98 % efficiency in required purity by repeated extraction of the reaction streams with water, followed by switching of solvent to butyl acetate and then back to water. Recycled ZnCl₂ had no negative effect on the yield of all-rac-α-tocopherol (maintaining over 90 %) at nearly total conversion of isophytol and only a 3 % molar excess of trimethylhydroquinone.     

HPLC determination of antilipoxygenase activity of a water infusion of Ligustrum vulgare L. leaves and some of its constituents

The aim of the study was a HPLC evaluation of the lipoxygenase activity inhibiting activity of a water infusion of Ligustrum vulgare L. leaves and selected isolates from it. The antiradical activity of the water infusion was determined using DPPH, ABTS and FRAP tests. Oleuropein and echinacoside concentrations in the water infusion were determined by HPLC. Water infusion, echinacoside and oleuropein were used for an antilipoxygenase activity assay using lipoxygenase isolated from rat lung cytosol fraction. Activity of 8-LOX, 12-LOX and 15-LOX were monitored through formation of 8-HETE, 12-HETE and 15-HETE, respectively. The water infusion exhibited the highest activity against all lipoxygenases, followed by oleuropein. Echinacoside was ineffective against LOXs in lower concentrations, while higher concentration showed similar inhibition on 8-LOX and 12-LOX. 15-LOX was affected more and the presence of echinacoside remarkably decreased its activity.     

Z‐DNA Recognition in B‐Z‐B Sequences by a Cationic Zinc Porphyrin

For the first time it has been shown by spectroscopic studies such as circular dichroism and UV/Vis that cationic zinc porphyrin serves as a selective spectroscopic sensor that is able to recognize short left‐handed Z‐DNA tracts embedded in the B‐Z‐B sequences.     

Simultaneous potentiometric determination of cationic and ethoxylated nonionic surfactants in liquid cleaners and disinfectants

A sensitive potentiometric surfactant sensor based on a highly lipophilic 1,3-didecyl-2-methyl-imidazolium cation and a tetraphenylborate (TPB) antagonist ion was used as the end-point detector in ion-pair potentiometric surfactant titrations using sodium TPB as a titrant. Several analytical and technical grade cationic and ethoxylated nonionic surfactants (EONS) and mixtures of both were potentiometrically titrated.The sensor showed satisfactory analytical performances within a pH range of 3-10 and exhibited satisfactory selectivity for all CS and EONS investigated. Ionic strength did not influence the titration except at 0.1 M NaCl, in which a slight distortion of the second inflexion corresponded with the nonionic surfactant.Two-component combinations of four CS and three EONS were potentiometrically titrated using the sensor previously mentioned as the end-point detector. The quantities of the surfactants varied between 2 and 6 μmol for CS and 2.50 and 7.50 μmol for EONS. The known addition methodology was used for determination of the surfactant with considerably lower concentration in the mixture.Three commercial products containing cationic surfactants as disinfectants and nonionic surfactants were potentiometrically titrated, and the results for both type of surfactants were compared with those obtained with standard conventional methods.     

Determination of 15 isomers of chlorobenzoic acid in soil samples using accelerated sample extraction followed by liquid chromatography

A study was conducted to elaborate a fast, simple and efficient method for determination of 15 isomers chlorobenzoic acids (CBAs) in soil using HPLC-UV. Artificially contaminated soil samples were extracted using accelerated solvent extraction (ASE) with 1% acetic acid in a mixture of hexane and acetone (1:1, V/V) under a pressure of 10.34 MPa and temperature of 150 °C. The recovery of the ASE method was above 82%. The extracts were concentrated; dimethyl sulfoxide was used to prevent CBA volatilization and the final analysis was performed with a C18 XBridge HPLC column employing a mobile phase consisting of acetonitrile and 0.1% trifluoracetic acid in water. A HPLC procedure with gradient elution and UV detection was developed and validated. The method exhibited a linear range for 2-CBA; 2,6-CBA; 3-CBA; 4-CBA; 2,3-CBA; 2,3,6-CBA; 2,5-CBA; and 2,4-CBA from 5 to 120 μg/mL with a limit of quantification (LOQ) of 5 μg/mL, RSD from 2.42 to 9.42% and accuracy from 82 ± 2 to 103 ± 3%. The linear range of determination of 2,4,6-CBA, 3,4-CBA, 2,3,5,6-CBA, 3,5-CBA, 2,3,5-CBA, 2,3,4,5,6-CBA and 2,3,4,5-CBA was 10-120 μg/mL with LOQ 10 μg/mL, RSD from 0.74 to 5.84% and accuracy from 94 ± 1 to 114 ± 1%. The optimized analytical procedure was finally applied on two historically PCB contaminated soils and 9 CBAs were quantified in the samples.