Biocomposites of covalently linked glucose oxidase on carbon nanotubes for glucose biosensor

The formation of covalently linked composites of multi–walled carbon nanotubes (MWCNT) and glucose oxidase (GOD) with high-function density for use as a biosensing interface is described. The reaction intermediates and the final product were characterized by using FT–IR spectroscopy, and the MWCNT-coated GOD nanocomposites were examined by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Interestingly, it was found that the GOD–MWCNT composites are highly water soluble. Electrochemical characterization of the GOD–MWCNT composites that were modified on a glassy carbon electrode shows that the covalently linked GOD retains its bioactivity and can specifically catalyze the oxidation of glucose. The oxidation current shows a linear dependence on the glucose concentration in the solution in the range of 0.5–40 mM with a detection limit of 30 μM and a detection sensitivity of 11.3 μA/mMcm². The present method may provide a way to synthesize MWCNT related composites with other biomolecules and for the construction of enzymatic reaction-based biofuel cells and biosensors. Supported by grants from the National Natural Science Foundation of China (NSFC, No. 20125515; 90206037; 20375016) and the Natural Science Foundation of Jiangsu Province (Grant No. BK 2004210)     

Determination of the thiol redox state of organisms: new oxidative stress indicators

This study describes a new methodology by which the concentrations of non-protein (NP) thiols glutathione (GSH), cysteine (CSH), N-acetylcysteine (AcCSH), and protein (P) thiols (PSH), as well as the contribution of these components to symmetric and mixed disulfides (NPSSR, NPSSC, NPSSCAc, PSSR, PSSC, PSSCAc, PSSP) can reliably be measured. The methodology consists of a strict sequence of methods which are applied to every sample. Free thiols at any given state of the procedure are measured by Ellmans assay, the CSH fraction is measured by its unique response in the ninhydrin assay, AcCSH is selectively measured with ninhydrin after enzymatic deacylation, proteins are separated from non-protein thiols/disulfides by precipitation with trichloroacetic or perchloric acid, disulfides are reduced into free thiols with borohydride, mixed disulfides between a protein and a non-protein component are measured by extracting the non-protein thiol from the protein pellet after borohydride treatment, and protein thiols/disulfides are measured after resolubilization of the protein pellet.When this method was applied to animal and fungal tissue, new molecular indicators of the thiol redox state of living cells were identified. The findings of the present study clearly show that the new parameters are very sensitive indicators of redox state, while at the same time the traditional parameters GSH and GSSG often remain constant even upon dramatic changes in the overall redox state of biological tissue. Therefore, unbiased assessment of the redox state also requires explicit measurement of its most sensitive thiol indicators.Electronic Supplementary Material Supplementary material is available in the online version of this article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Synthesis of macrocyclic tetraamides derived from α-amino acids and their investigations using ESI-MS technique

Chiral α,ω-diesters react under high-pressure conditions (10 kbar) with α,ω-diamines to give chiral cyclic tetraamides of C₂-symmetry. The complexation properties of tetraamides towards alkali metal cations (Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺) were estimated on the basis of ESI-MS spectra.     

SULFOPHTHALOCYANINES FOR PHOTODYNAMIC INACTIVATION OF VIRUSES IN BLOOD PRODUCTS: EFFECT OF STRUCTURAL MODIFICATIONS

Abstract— Transmission of infectious diseases through blood transfusions is well known. Ultraviolet irradiation, solvents and detergents provide a means of sterilizing noncellular blood components. However these harsh methods are not applicable to cellular blood products. Recently, attempts have been made to sterilize biological fluids using photodynamic treatment and phthalocyanine (Pc) dyes have been advanced as photosensitizers for this purpose. We have evaluated a series of water-soluble Pc, chelated with different central metal ions, substituted to different degrees with sulfonato and r-butyl groups, for their effectiveness to reduce virus infectivity in red blood cell suspensions. Vaccinia virus cytopathogenicity was determined by endpoint serial dilutions in the CV-1 cell line. Anti-viral activity increased with the central metal ion in the following order: Ga(III) < Al(III) < Zn(II), and varied inversely with the degree of sulfonation. Furthermore, addition of a t -butyl group onto the trisulfonated dyes (PcS₃[ t -Bul) resulted in a 5–40-fold increase in anti-viral potency, suggesting that amphiphilicity enhances the photodynamic activity of the dye. Strong anti-viral photosensitizing properties cannot be the sole selection criterion. Of equal importance is the preservation of blood component integrity. Accordingly, the photohemolytic activity of the dyes was evaluated using the rate of hemolysis as a parameter and a toxicity index was defined. Among the most active dyes, the AlPcS₃( t -Bu) complex exhibited the most favorable anti-viral properties combined with a low toxicity index. Our results suggest that trisulfophthalocyanines, bearing an additional t -butyl group to enhance amphiphilicity, are particularly promising dyes for photodynamic blood sterilization.     

Skeletal Isomerization of <Emphasis Type="Italic">n</Emphasis>-butenes over Solid Acid Catalysts

Conversion reactions of n-butenes over zeolites and amorphous catalysts have been investigated to deduce the factor that determines the selectivity for the skeletal isomerization producing isobutene. The effects of pore structure and acid site concentration on the selectivity for the skeletal isomerization are discussed on various catalysts. The pore structures of FER and CLI zeolites induce the distant locations of butene molecules, accelerating monomolecular skeletal isomerization. On the other hand, acid site concentration determines the preferred reaction path of n-butenes on amorphous catalysts. Oligomerization followed by cracking that produces various hydrocarbons is suppressed on the catalyst with low acid site concentration, resulting in high selectivity for isobutene. The feasibility of monomolecular skeletal isomerization on zeolites and amorphous catalysts is confirmed by its reversibility with high selectivity.     

Enzymatic kinetic of cellulose hydrolysis

The ethanol effect on the Trichoderma reesei cellulases was studied to quantify and clarify this inhibition type. To determine inhibition parameters of crude cellulase and purified exoglucanase Cel7A, integrated Michaelis-Menten equations were used assuming the presence of two inhibitors: cellobiose as the reaction product and ethanol as a possible bioproduct of cellulose fermentation. It was found that hydrolysis of cellulose by crude enzyme follows a model that considers noncompetitive inhibition by ethanol, whereas Cel7A is very slightly competitively inhibited. Crude cellulase is much more inhibited (K _iul=K _icl=151.9 mM) than exoglucanase Cel7A (K _icl=1.6 × 10¹⁵ mM). Also, calculated inhibition constants showed that cellobiose inhibition is more potent than ethanol inhibition both for the crude enzyme as well as exoglucanase Cel7A.     

Oxygenolysis of flavonoid compounds: DFT description of the mechanism for quercetin.

Flavonoids are naturally occurring phenol derivatives present in substantial amounts in a large variety of plants, fruits and vegetables daily eaten by humans. Most of these compounds exhibit several interesting biological activities, such as antiradical and antioxidant actions. Indeed, by complexation with specific enzymes, flavonoids are notably liable to metabolize molecular dioxygen. On the basis of experimental results describing oxygenolysis of the flavonoid quercetin, activated by the enzyme quercetin 2,3-dioxygenase (2,3-QD),ur attention has focused on the role of metal center in the activation of the substrate quercetin. Thus, in the present study, by means of DFT calculations at the B3LYP/ 6-31(+)G* level on model molecular systems, we describe different mechanisms for dioxygen metabolization by quercetin. Stationary points are described, and energetic and structural analyses along the reaction paths are reported. Our calculations show that the copper cation must act as an oxidant towards the substrate and that the reaction proceeds through a 1,3-cycloaddition.     

Trace analysis of rapamycin in human blood by micellar electrokinetic chromatography

A capillary electrophoretic method with UV detection at 278 nm has been developed for analysis of the immunosuppressant rapamycin (sirolimus) in human blood at low microgram per liter levels. Separation has been achieved in an acidic carrier electrolyte containing sodium dodecylsulfate and 30% (v/v) acetonitrile. For sample clean-up and preconcentration, an off-line solid-phase extraction step using a silica-based reversed-phase material and an on-capillary focussing technique were employed. The latter allows the injection of increased sample volumes without excessive band broadening. Although this new method is less sensitive than existing liquid chromatographic procedures combined with mass spectrometry, it is fully suited to routine analysis of rapamycin in blood from patients treated with this drug. Last but not least the low costs make it an attractive alternative to established methods.