Asymmetrical Coupling of Enzymatic Systems on Collagen Membranes Application to Multienzyme Electrodes

Abstract

An original method of preparation of enzyme collagen membranes has been developed which enables the asymmetrical coupling of multienzymatic systems working in sequence. One or several enzymes are immobilized on one face of the acyl-azide activated membranes, another enzyme being bound on the other face. When the first enzymatic face is exposed to the adequate reaction mixture, the products diffuse through the collagen membrane and act as substrates for the enzyme present on the other side. This possibility has been applied to the design of new and polyvalent multienzyme electrodes.     

Anti-T2 Monoclonal Antibody Immobilization On Quartz Fibers: Stability and Recognition of T2 Mycotoxin

Abstract

Several methods for immobilizing anti-T2 mycotoxin monoclonal antibodies on quartz fibers, for use in optical sensor development, have been evaluated with respect to the surface density and stability of the immobilized proteins. the first method activates matrix hydroxyl groups using p-toluenesulfonyl chloride (TSC). the second method activates these groups using p-nitrophenyl chloroformate (NPCF). the third method requires an initial silanization using 3-aminopropyltriethoxysilane (APTES) followed by carrier activation with glutaraldehyde. the activated carrier in all three methods is then reacted with the amino groups of the protein. the first two non-silanizing coupling methods are simple, inexpensive and non-hazardous compared to the third, more complex method in which an initial Correspondance: to PVS     

Piezoelectric Crystal Biosensor System for Detection of Escherichia Coli

Abstract

A piezoelectric crystal biosensor system was applied to the detection of Escherichia coli. the system consists of an oscillator, a frequency counter, a flow cell and a modified piezoelectric crystal. Anti-E. coli antibody is immobilized on the surface of the crystal. It is used as an E. coli detection by measuring its resonant frequency shift due to a mass change caused by specific binding of the micro organisms to the surface. the frequency shift correlates with an E. coli concentration in the range of 10⁶?10⁸ cells·cm^?3. the resonant frequency shift is increased by further treatment to bind micro-particles modified with anti-E. coli antibody. This method allows us to improve the determination limit to 10⁵ cells · cm^?3.     

Plasma-Polymerized Thin Films for Enzyme Immobilization in Biosensors

Abstract

Polymer films deposited on thin film noble metal electrodes on silicon chips, utilizing plasma polymerization are shown to be suited for immobilization of enzymes. Glucose oxidase is covalently coupled via amino- or carboxyl-groups of polymer films made by plasma polymerization of 2-amino-benzotrifluoride, acrylic or methacrylic acid. The concentration of these active surface groups increases by after-treatment in an ammonia- or oxygen-plasma. A biosensor consisting of a thin film metal electrode, plasma polymer film and immobilized glucose oxidase shows an amperometric response to glucose with a fast response time.     

Hybridization of binary monolayers of single stranded oligonucleotides and short blocking molecules

We have studied the immobilization of single stranded (ss) DNA oligonucleotides of 16-27 base pairs on gold. The oligonucleotides were thiol-modified (SH-ssDNA) or disulfide-modified via a dimethoxytrityl-group (DMT-S-S-ssDNA). Immobilization was performed by adsorption of the probes on the gold surface for 10-15 min, a time within which saturation coverage was obtained for both thiol- and disulfide-modified probes. Hereafter the layer was post-treated with hydroxyalkyl substituted lipoamides also for a time of 10-15 min. The surface density of layers with shorter probes (16-18 mer) was twice (2.4 ± 0.2 × 10¹³ probes/cm²) that of the longer probes (25-27 mer) as studied with surface plasmon resonance. Hybridization of single stranded polymerase chain reaction (PCR) amplified products with a length above 300 base pairs gave a very low hybridization response. For amplicons with about 100 base pairs the response was high. The surface coverage was comparable to that of complementary ssDNA binding (3.0 × 10¹² strands/cm²). Surfaces made from SH-ssDNA showed a 30% higher hybridization response than surfaces made from DMT-S-S-ssDNA. The PCR amplified products used are of relevance in breast cancer diagnosis. The results clearly demonstrate that the single stranded PCR products might be used in label-free cancer diagnostics.     

A novel method for immobilization of Co tetraphenylporphyrins on P(4VP-co-St)/SiO2: Efficient catalysts for aerobic oxidation of ethylbenzenes

In this paper, poly(4vinylpyridine-co-styrene) (P(4VP-co-St)) was grafted on silica gel particles in the manner of “grafting from”, and the grafting particle P(4VP-co-St)/SiO₂ was gained. The grafting particle P(4VP-co-St)/SiO₂ is a novel kind of supports for immobilizing metalloporphyrin catalysts. Then, the immobilization of cobalt tetraphenylporphyrin (CoTPP) on the supports P(4VP-co-St)/SiO₂ was carried out via the axial coordination reaction between CoTPP and the pyridine groups of the grafted P(4VP-co-St), resulting in the heterogenised catalysts CoTPP-P(4VP-co-St)/SiO₂. The synthesized catalysts were characterized by FTIR and the axial coordination process between CoTPP and the grafted P(4VP-co-St) was confirmed by UV-vis. The effects of various factors on the immobilization reaction of CoTPP were studied in detail. Finally, the catalytic performance of CoTPP-P(4VP-co-St)/SiO₂ in the catalytic oxidation process of ethyl benzene was investigated. The experimental results show that the axial coordination reaction is a very easy and novel method for favorably immobilizing CoTPP onto the P(4VP-co-St)/SiO₂ surfaces. During the immobilization process of CoTPP on P(4VP-co-St)/SiO₂, the most bonding amount of CoTPP (0.19 g/g) is obtained under the lower temperature (5 °C) and the higher concentration of CoTPP(6.0 mg/ml) lasting 4 h. Moreover, the supported catalyst CoTPP-P(4VP-co-St)/SiO₂ can effectively activate the dioxygen, and obviously catalyze the transform of ethylbenzene into acetophenone. So it exhibits the fine catalytic activity.     

Heterogeneous chiral Mn(III) salen catalysts for the epoxidation of unfunctionalized olefins immobilized on mesoporous materials with different pore sizes

Two chiral Mn(III) salen complexes were immobilized onto a series of mesoporous MCM-41 and MCM-48 materials with different pore sizes and the as-synthesized catalysts were active and enantioselective for the asymmetric epoxidation of styrene and indene. The results of XRD, FTIR, DR UV-vis, and N₂ sorption showed that the chiral Mn(III) salen complexes were anchored in the channels of mesoporous materials. The influence of organic silicane dosage on the catalytic performance was studied and the optimum dosage of organic silicane for preparing heterogeneous catalysts was determined. Furthermore, the effect of the fine-tuning of pore size on the performance of heterogeneous catalysts was discussed. In general, larger pore size of the supports could lead to higher conversions and the compatible pore size with substrate may be responsible for the improved enantiomeric excess (ee) values.     

A search of an optimal carrier for immobilization of chitin deacetylase

Chitin deacetylase is an enzyme that can play an important role in enzymatic deacetylation of chitosan to obtain polymers with a lower degree of acetylation. As this enzyme has never been immobilized up to now, efforts were directed towards determining both the most suitable carrier and the best method of covalent attachment to the selected carrier. In the preliminary experiments several different carriers were tested that were based on acrylic, silica-gel, agarose, dextran or cellulose materials. The best results were obtained for cellulose-based Granocel matrix. DEAE- and NH₂-Granocel activated with divinyl sulfone or glutaraldehyde were chosen for optimization of the immobilization procedure and the carrier’s superstructure. It was found that covalent binding of chitin deacetylase on DEAE-Granocel-2000 via divinyl sulfone offers preparations with the highest activity and stability. The characteristics of the selected preparation and comparison with the native enzyme show that optimal conditions are close to those for the free enzyme: the optimal pH is 4.0 for both enzymes and the optimal temperatures are 55 °C and 50 °C for native and immobilized forms, respectively. The kinetics of chitosan deacetylation for both enzymes follow the Michaelis–Menten relationship, but significant differences in the values of the equation parameters were observed.     

Direct electrochemistry of cytochrome c on a phosphonic acid terminated self-assembled monolayers

The direct electrochemistry of cytochrome c (cyt c) on a gold electrode modified with 3-mercaptopropylphosphonic acid [HS-(CH₂)₃-PO₃H₂, MPPA] self-assembled monolayers (SAMs) was for the first time investigated. Electrochemical measurements and surface-enhanced infrared absorption spectroscopic reveal that the adsorption kinetics of cyt c on the MPPA-SAMs is very fast (saturation adsorption is completed within 5 s) and the immobilized cyt c molecules retain their native secondary protein structure. The nature of interaction between cyt c and -PO₃H₂ groups is mainly the electrostatic interaction. The direct electrochemistry of the immobilized cyt c on the -PO₃H₂ terminated SAMs with short chain is nearly reversible. Its formal potential (E⁰′ = 18 ± 3 mV vs. SCE) is very close to that of cyt c in an aqueous solution (E⁰′ = 18-22 mV vs. SCE). In addition, the electron transfer rate of cyt c immobilized on -PO₃H₂ terminated SAMs is relatively slow as compared to -SO₃H and -COOH terminated SAMs, indicating excess negative charge density on the SAMs surface will decrease the electron transfer rate of cyt c.     

Concomitant polymorphism of an antiferromagnetically coupled dicopper(II,II) complex with single strand helical assembly: Synthesis,structure, DSC,magnetic and heterogeneous catalytic studies

Two concomitant polymorphic coordination complexes (dark blue – I and black – II) with the formula (Cu₂C₄₄H₆₀N₄O₄) have been synthesized and characterized crystallographically. Magnetic measurements show the presence of a strong antiferromagnetic interaction and the 2J value corresponds extremely well to the theoretically calculated one, indicating the fact that it follows nicely the magneto-structural relationship. Immobilization of the copper(II) complex 1 on a 2D-hexagonal mesoporous silica showed good catalytic efficiency in the liquid phase partial oxidation of olefins in the presence of TBHP as an oxidant.