Presence Of Glutaraldehyde Research Articles

Surface functionalization via in situ interaction of plasma-generated free radicals with stable precursor-molecules on cellulose

The surface functionalization process was accomplished in a consecutive 3 step process including: (1) Argon- and oxygen-plasma enhanced generation of free radical sites on cellophane surfaces; (2) “In situ” gas phase derivatization in the absence of plasma using hydrazine, ethylene diamine, or propylene diamine; (3) Second “in situ”, gas phase derivatization in the absence of plasma using oxallyl chloride or “ex situ” derivatization in the presence of glutaraldehyde. The presence of free radical sites on the plasma exposed cellophane surfaces was demonstrated using “in situ” sulfur dioxide and nitric oxide labeling techniques. It was shown that the free radical sites readily react under “in situ” conditions with the stable chain-precursor components and generate the desired spacer-chain molecules. ESCA, ATR-FTIR analysis and dying techniques were used to monitor the cellophane surface changes. A factorial design was used for selecting the optimal plasma parameters. Functionalized cellophane substrates were used for immobilization of α-chymotrypsin in the presence of spacer-chain molecules. The activity of the immobilized α-chymotrypsin was found to be lower in comparison to the activity of the free enzyme and the presence of virgin cellophane in the free enzyme solution also reduced significantly the activity of the enzyme. It is suggested that the swollen state of the cellophane plays a significant role in the decrease of the immobilized enzyme activity.

Biosensor for acrylamide based on an ion-selective electrode using whole cells of Pseudomonas aeruginosa containing amidase activity

The aim of this work was to develop a biosensor for toxic amides using whole cells of Pseudomonas. aeruginosa containing amidase activity, which catalyses the hydrolysis of amides such as acrylamide producing ammonia and the corresponding organic acid. Whole cells immobilized in several types of membrane in the presence of glutaraldehyde and an ammonium ion-selective electrode, were used for biosensor development. This biosensor exhibited a linear response in the range of 0.1–4.0×10−3 M of acrylamide, a detection limit of 4.48×10−5 M acrylamide, a response time of 55 s, a sensitivity of 58.99 mV mM−1 of acrylamide and a maximum t1/2 of 54 days. The selectivity of this biosensor towards other amides was investigated, which revealed that it cross-reacted with acetamide and formamide, but no activity was detected with phenylacetamide, p-nitrophenylacetamide and acetanilide. It was successfully used for quantification of acrylamide in real industrial effluents and recovery experiments were carried out which revealed an average substrate recovery of 93.3%. The biosensor is cheap since whole cells of P. aeruginosa can be used as source of amidase activity.

Preparation and characterization of molecular photoresists: Crosslinkable positive and water developable negative tones

AbstractThe development of a water‐developable negative photoresist from β‐CD using an acid‐catalyzed chemical amplification method is investigated here. Tertiary butoxyl protected β‐cyclodextrin (t‐BOC‐CD) is also synthesized and used to prepare a positive photoresist. Glutaraldehyde is added as a crosslinking agent for both positive and negative photoresists. Deprotection of t‐BOC‐CD is accelerated by a photo‐induced acid. In the presence of glutaraldehyde and acid, both the deprotected t‐BOC‐CD and β‐cyclodextrin are crosslinked. The introduction of a t‐butoxyl group into the β‐CD molecule and the addition of glutaraldehyde into the β‐CD molecules are both found to decrease the crystallinity of the molecules, improving the resist film properties. The etching resistance of both positive and negative photoresist films is improved by the crosslinking method. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Immobilization of acetylcholinesterase on new modified acrylonitrile copolymer membranes

The three new dual-layer matrices (polyacrylonitrile (PAN) membranes coated with physically bound chitosan (CHI)—PANCHI-A and chemically bound chitosan—PANCHI-B and PANCHI-C) for immobilization of acetylcholinesterase (AChE) were obtained. The chemical-modified PAN membrane (PAN-NaOH + ethylenediamine (EDA)) was used as a base for the prepared dual-layer membranes. For chemical chitosan bound membrane, chitosan was tethered onto the membrane surface to form a dual-layer biomimetic membrane in the presence of glutaraldehyde (GA). The basic characteristics (amount of amino groups, hydrophilicity and transport characteristics) of the chitosan-modified membranes were investigated. The SEM analyses were shown essential morphology change in the different chitosan membranes. The relative activities and V max of the covalently immobilized enzyme on PANCHI-B and PANCHI-C membranes were higher than that on PANCHI-A membrane and chemical-modified membrane with NaOH + EDA. K m values for the different modified membranes are lower for the chitosan-treated membranes. The pH and temperature optimum of immobilized enzyme were determined. The bound enzymes on PANCHI-B and PANCHI-C have higher thermal and storage stability in comparison with AChE on PANCHI-A membrane and free enzyme.

Formation of a cross-linking complex of dinitrogenase reductase-activating glycohydrolase (DRAG) with membrane proteins from Rhodospirillum rubrum chromatophores

Association of dinitrogenase reductase-activating glycohydrolase (DRAG) with membrane proteins of chromatophores has been investigated. The formation of a multicomponent complex between DRAG and membrane proteins was demonstrated in the presence of glutaraldehyde and EDC/NHS (N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride/hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid sodium salt). Complex formation was observed both in native chromatophore membrane and in chromatophores treated with 0.5 M NaCl in the presence of homogeneous DRAG and glutaraldehyde in cross-reaction. The molecular weight of the complex was around 200 kD, which is consistent with the association of DRAG with three or more chromatophore membrane proteins. A specific complex with molecular weight of about 75 kD was formed only in the presence of EDC/NHS in the cross-linking reaction. It was demonstrated that ammonium transport protein and P11 protein are possible candidates for association with DRAG in chromatophore membranes.

Development of a Faradic Impedimetric Immunosensor for the Detection of Salmonella typhimurium in Milk

The development of a faradic impedimetric immunosensor for the detection of S. typhimurium in milk is described for first time. Polyclonal anti-Salmonella was cross-linked, in the presence of glutaraldehyde, on gold electrodes modified with a single 11-amino-1-undecanethiol (MUAM) self-assembled monolayer (SAM) or a mixed SAM of MUAM and 6-mercapto-1-hexanol at a constant 1 + 3 proportion, respectively. The mixed SAM was also deposited in the presence of triethylamine, which was used to prevent the formation of interplane hydrogen bonds among amine-terminated thiols. The effect of the different surface modifications on both the sensitivity and the selectivity of the immunosensors was investigated. The alteration of the interfacial features of the electrodes due to different modification or recognition steps, was measured by faradic electrochemical impedance spectroscopy in the presence of a hexacyanoferrate(II)/(III) redox couple. A substantial amplification of the measuring signal was achieved by performing the immunoreaction directly in culture samples. This resulted in immunosensors with great analytical features, as follows: (i) high sensitivity; the response of the immunosensors increases with respect to the detection time as a consequence of the simultaneous proliferation of the viable bacteria cells in the tested samples; (ii) validity; the response of the immunosensors is practically insensitive to the presence of dead cells; (iii) working simplicity; elimination of various centrifugation and washing steps, which are used for the isolation of bacteria cells from the culture. The proposed immunosensors were successfully used for the detection of S. typhimurium in experimentally inoculated milk samples. The effect of different postblocking agents on the performance of the immunosensors in real samples was also examined.

Oligomerization of the potato virus X 25-kD movement protein

A 25-kD movement protein (25K protein) encoded by the first gene of the potexvirus Potato virus X triple gene block of transport genes is essential for the viral movement in infected plants. The 25K protein belongs to superfamily 1 of NTPase/helicases and exhibits in vitro RNA helicase, Mg2+-dependent NTPase, and RNA-binding activities. In the present work, the ability of 25K protein for homologous interactions was studied using the yeast two-hybrid system, protein chemical cross-linking in the presence of glutaraldehyde, far-Western blotting, and ultracentrifugation in sucrose density gradients. The 25K protein was shown to form homodimers and homooligomers. Sites of homologous protein-protein interactions were found in both the N- and C-terminal portions of the protein.

The N-terminal Extensions of Deinococcus radiodurans Dps-1 Mediate DNA Major Groove Interactions as well as Assembly of the Dodecamer

Dps (DNA protection during starvation) proteins play an important role in the protection of prokaryotic macromolecules from damage by reactive oxygen species. Previous studies have suggested that the lysine-rich N-terminal tail of Dps proteins participates in DNA binding. In comparison with other Dps proteins, Dps-1 from Deinococcus radiodurans has an extended N terminus comprising 55 amino acids preceding the first helix of the 4-helix bundle monomer. In the crystal structure of Dps-1, the first approximately 30 N-terminal residues are invisible, and the remaining 25 residues form a loop that harbors a novel metal-binding site. We show here that deletion of the flexible N-terminal tail obliterates DNA/Dps-1 interaction. Surprisingly, deletion of the entire N terminus also abolishes dodecameric assembly of the protein. Retention of the N-terminal metal site is necessary for formation of the dodecamer, and metal binding at this site facilitates oligomerization of the protein. Electrophoretic mobility shift assays using DNA modified with specific major/minor groove reagents further show that Dps-1 interacts through the DNA major groove. DNA cyclization assays suggest that dodecameric Dps-1 does not wrap DNA about itself. A significant decrease in DNA binding affinity accompanies a reduction in duplex length from 22 to 18 bp, but only for dodecameric Dps-1. Our data further suggest that high affinity DNA binding depends on occupancy of the N-terminal metal site. Taken together, the mode of DNA interaction by dodecameric Dps-1 suggests interaction of two metal-anchored N-terminal tails in successive DNA major grooves, leading to DNA compaction by formation of stacked protein-DNA layers.

Novel interpenetrating polymer network microspheres of chitosan and methylcellulose for controlled release of theophylline

Interpenetrating polymer network (IPN) microspheres of chitosan (CS) and methylcellulose (MC) were prepared by emulsion-crosslinking in the presence of glutaraldehyde (GA) as a crosslinker. Theophylline (THP), an antiasthmatic drug was encapsulated into IPN microspheres under varying ratios of MC and CS, % drug loading and amount of GA added. IPNs have shown better mechanical properties than pure CS. Cross-link density of the matrices was significantly affected by the amount of GA and MC. Microspheres were characterized by Fourier transform infrared (FTIR) spectroscopy to assess the formation of IPN structure and to confirm the absence of chemical interactions between drug, polymer and crosslinking agent. Particle size was measured by laser light scattering technique. Microspheres with the average particle sizes ranging from 119 to 318 μm were produced. Differential scanning calorimetry (DSC) and X-ray diffraction (X-RD) studies were performed to understand the crystalline nature of drug after encapsulation into IPN microspheres. Theophylline encapsulation of up to 82% was achieved as measured by UV spectrometer. Equilibrium swelling was performed in distilled water. In vitro release studies were performed in both 0.1 N HCl and pH 7.4 buffer solutions. These data indicated a dependence of drug release on the extent of crosslinking and amount of MC added during the preparation of microspheres. The release was extended up to 12 h and release rates were fitted to an empirical equation to compute the diffusional parameters, which indicated a slight deviation from the Fickian trend for the release of theophylline.

Development of a conductometric nitrate biosensor based on Methyl viologen/Nafion ® composite film

A conductometric biosensor for nitrate analytical determination was developed using nitrate reductase (NR) from Aspergillus niger immobilized on a thin-film electrode by cross-linking with bovine serum albumin in the presence of glutaraldehyde, Nafion ® cation-exchange polymer and methyl viologen mediator. The process parameters for the fabrication of the enzyme electrode and various experimental variables were investigated with regard to their influence on sensitivity, limit of detection, dynamic range, operational and storage stability. The biosensor can reach 95% of steady-state conductance value in about 13 s. Linear calibration in the range of 0.02 and 0.25 mM with detection limits of 0.005 mM nitrate was obtained with a signal-to-noise ratio of 3. When stored in 5 mM phosphate buffer (pH 7.5) at 4 °C, the sensor showed good stability over two weeks. The electrode is suitable for use in real samples.

Electrochemical reduction and determination of Cibacron Blue F3GA at poly- l-lysine modified glassy carbon electrode

The oxidation of a reactive dye, Cibacron Blue F3GA, CB, (C.I. 61211), widely used in the textile industries to color natural fibers, was studied by electrochemical techniques. The oxidation on glassy carbon electrode occurs in two steps at 2.0 < pH < 10 involving one electron transfer each to the amine group leading to the imide derivative. Stable films of poly- l-lysine (PLL) in the presence of glutaraldehyde (GA) 97.5%:2.5% on glassy carbon electrode can be used to detect low levels of dye using its oxidation peak at +0.75 V by voltammetry. Linear calibration graphs were obtained for the CB reactive dye, from 1.0 × 10 −6 to 1.0 × 10 −5 mol L −1 in B–R buffer, pH 2.0, using a pre-concentration off-line during 10 min. The detection limit (3 σ/slope) was calculated to be 4.5 × 10 −8 mol L −1. Films of PLL can readily be applied for the determination of CB dye bearing aminoanthraquinone as chromophore and chlorotriazinyl as reactive group at concentrations at least 100 times lesser than using a glassy carbon electrode without modification. The method described was applied for the determination of CB dye in tap water and raw water collected from the municipal treatment plant with a recovery of 89.2% ± 5.4 and 88.0% ± 6.5, respectively.

Preparation, characterization, swelling and in vitro drug release behaviour of poly[ N-acryloylglycine-chitosan] interpolymeric pH and thermally-responsive hydrogels

Novel biodegradable pH- and thermal-responsive interpenetrating polymer network (IPN) hydrogels were prepared for controlled drug delivery studies. The IPN hydrogels were obtained in mild aqueous acid media by irradiation of solutions of N-acryloylglycine (NAGly) mixed with chitosan, in the presence of glutaraldehyde as a crosslinking agent and using 2,2-dimethoxy-2-phenyl acetophenone as photoinitiator. These hydrogels were subjected to equilibrium swelling studies at different temperatures (25 °C, 37 °C and 45 °C) in buffer solutions of pH 2.1 and 7.4 (similar to that of gastric and intestinal fluids respectively). 5-Fluorouracil (5-FU) was entrapped in the hydrogels, and drug release studies carried out at 37 °C in buffer solutions at pH 2.1 and 7.4.

Enzyme immobilization procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides: Comparative study

A comparison between several acetylcholinesterase (AChE) immobilization procedures on the 7,7,8,8-tetracyanoquinodimethane (TCNQ)-modified graphite working electrodes is presented. The immobilization methods employed crosslinking with glutaraldehyde in presence of BSA protein and photopolymerization with poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ). The main variations were related to the enzyme charge in each electrode and the enzyme conditioning and storage conditions after immobilization. Initially, the enzyme–substrate reaction was carried out and the following parameters were chrono-amperometrically and -coulometrically monitored: current intensities, time to stabilize the current response, and the mass transfer represented by the Coulomb charge. The screen-printed biosensors that presented best characteristics were then used to perform the inhibition assays and to verify the sensitivity against the following NMC insecticides: aldicarb, carbaryl, carbofuran, and methomyl. In general, diffusion of electrons into the sensitive layer, mass transfer, and time to stabilize the current were adequate in all cases. The Cottrell law was followed before the 1 min of enzyme–substrate reaction. Adequate reproducibility within electrochemical measurements was also observed, with relative standard deviations varying from 6.5 to 18.6%. AChE immobilization with glutaraldehyde allow to obtain robust and reproducible biosensors, but they need a much higher enzyme content (80 mUA per electrode) to achieve current values comparable to that constructed by immobilizing the AChE through photopolymerization with PVA-SbQ (0.7 to 1 mUA per electrode). The limits of detection were determined with a minimum 10% inhibition, and varied from 10 −9 to 8×10 −9 M (0.2 to 1.5 ppb) by employing the enzyme immobilization through photopolymerization with PVA-SbQ. In practice, this kind of immobilization procedure is much simpler and produces good results: fast response, adequate reproducibility, large pesticides working ranges, and excellent sensitivities to N-methylcarbamates (NMCs) which in general do not present enzyme inhibition power as elevated as for the organophosphate pesticides.

Synthesis and characterization of konjac glucomannan/poly(vinyl alcohol) interpenetrating polymer networks

AbstractNovel interpenetrating polymer networks (IPNs) coded as KP were synthesized successfully from poly(vinyl alcohol) (PVA) and konjac glucomannan (KGM) in the presence of glutaraldehyde as a crosslink agent. The transparent IPN films that were 40 μm thick were prepared by means of conventional solvent‐casting technique and dried at room temperature for 2 days. The structure and miscibility of the KP films were studied by Fourier transformed infrared spectra, scanning electron microscopy, differential scanning calorimetry, wide‐angle X‐ray diffraction, and ultraviolet visible spectroscopy (UV–Vis). The results indicated that strong intermolecular interaction caused by crosslink bonding between PVA and KGM occurred in the IPN films, resulting in wonderful miscibility when the reaction time is 4 h. The tensile strength, elongation at break, and moisture uptake was much higher than that of the pure PVA film, KGM film, and uncrosslinked blend films. In other words, the structure of IPN endowed the films with excellent performance, so the new material has promising applications to food package film and agricultural film because of its biodegradability. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 2775–2780, 2004

Plasmid-DNA loaded chitosan microspheres for in vitro IL-2 expression

Interleukin-2 (IL-2) expression plasmid (pCXWN-hIL-2) loaded chitosan microspheres were evaluated for using in gene-based immunotherapy. Chitosan microspheres containing pCXWN-hIL-2 were prepared by using a precipitation technique. In addition, the effects of different factors such as the concentration (0.35–0.70%) and the molecular weight of chitosan (low and medium molecular weights), the plasmid amount (5–10 μg/ml) and the presence of glutaraldehyde during the encapsulation process, on microsphere characteristics were investigated. The size of microspheres changed between 1.45 and 2.00 μm. All the formulation factors affected the size of microspheres. The structure of plasmid remained unchanged during the encapsulation process and the release studies. Plasmid encapsulation efficiency of chitosan microspheres was high (82–92%). The zeta potential values of microspheres was approximately +5.2 to +12.4 mV. In vitro release properties of microspheres changed with formulation variables. In vitro release of DNA changed with the concentration and molecular weight of chitosan and initial plasmid amount. Addition of glutaraldehyde is not necessary for a formulation. MAT-LyLu, the rat prostate adenocarcinoma cell line, was used for the determination of the in vitro transfectional activity of IL-2 encoding plasmid DNA loaded chitosan microspheres and the level of IL-2 expressed into the cells was assayed using a ELISA kit. High level of IL-2 expression was obtained with plasmid-loaded chitosan microspheres. Microspheres showed similar IL-2 production as lipofectin. The molecular weight of chitosan used and the amount of plasmid influenced the in vitro IL-2 production in the cells. Encapsulation of IL-2 encoding gene into chitosan microspheres might be a useful strategy to increase the expression and to control the delivery of cytokine gene to cells.

Soymilk oligosaccharide hydrolysis by Aspergillus oryzae α-galactosidase immobilized in calcium alginate

α-Galactosidase was immobilized in calcium alginate in the presence of glutaraldehyde. The optimum pH of the soluble enzyme was 4.8, whereas that of the immobilized enzyme was 4.5. The optimum temperature of the soluble enzyme was 50 °C and that of the immobilized enzyme was increased to 57 °C. The immobilized enzyme retained its activity for a longer period. The immobilized enzyme exhibited higher K m and lower V max compared to the soluble enzyme. Immobilized α-galactosidase was used in batch, repeated batch and in continuous mode. After 12 h, soluble and immobilized enzyme resulted in 93 and 81% reduction in raffinose family oligosaccharide content in soymilk. Immobilized calcium alginate beads were used for repeated hydrolysis at 50 °C and showed good operational stability. The performance of immobilized α-galactosidase was also tested in a fluidized bed reactor at different flow rates and 90% reduction of raffinose family oligosaccharides in soymilk was obtained at 40 ml h −1 flow rate.

Synthesis, Characterization and Evaluation of IPN Hydrogels for Antibiotic Release

We prepared new ternary interpenetrating polymeric networks (IPN) systems containing chitosan, poly(N-vinylpyrrolidone) and poly(acrylamide) polymers. IPNs were synthesized by radical polymerization of acrylamide monomers in presence of glutaraldehyde (G) and N,N′-methylenebisacrylamide as crosslinkers and the other polymers. These IPNs were named as C-P-A. Glutaraldehyde were used in different concentration to control the network porous of IPNs. Spectroscopic and thermal analyses of these cylindrical shaped IPNs were made with fourier transform infrared spectroscopy analysis, thermogravimetric analysis, and thermomechanical analysis. Swelling studies of IPNs were carried out at pH 1.1 and pH 7.4 at 37°C. The swelling and diffusion parameters of IPNs in these solutions were calculated. Amoxicillin as a bioactive species was entrapped to the IPNs during synthesis. In vitro release kinetics of IPNs were investigated. The experimental data of swelling and release studies suggest clearly that the swelling and release process obeys second-order kinetics. The release of the entrapped bioactive species from IPNs depends on the degree of crosslinking of the polymer and pH of the medium at body temperature. We observed that amoxicillin release at pH 1.1 was higher than at pH 7.4. As a result, IPNs-based chitosan with different cross-linker concentration could be promising candidates for formulation in oral gastrointestinal delivery systems.

Preparation and adsorption properties of modified chitosan

AbstractChitosan has been modified by polyacrylate sodium or acrylate sodium in the presence of glutaraldehyde, which afforded four kinds of modified chitosan (CTS‐PA) with different ratios of reacting substance. They are used as an environmentally benign polymer adsorbent. The adsorption properties of these CTS‐PA to Pb2+ have been measured. The best adsorption to Pb2+ was CTS‐PA4, the removal efficiency was higher in higher concentration solution than in a lower one, the value of the removal efficiency is approximate 99.991%. Copyright © 2004 John Wiley &amp; Sons, Ltd.

Cholesterol amperometric biosensor based on cytochrome P450scc

A screen-printed enzyme electrode based on flavocytochrome P450scc (RfP450scc) for amperometric determination of cholesterol has been developed. A one-step method for RfP450scc immobilization in the presence of glutaraldehyde or by entrapment of enzyme within a hydrogel of agarose is discussed. The sensitivity of the biosensor based on immobilization procedures of flavocytochrome P450scc by glutaric aldehyde is 13.8 nA μM −1 and the detection limit is 300 μM with a coefficient of linearity 0.98 for cholesterol in the presence of sodium cholate as detergent. The detection limits and the sensitivity of the agarose-based electrode are 155 μM and 6.9 nA μM −1 with a linearity coefficient of 0.99. For both types of electrodes, the amperometric response to cholesterol in the presence of detergent was rather quick (1.5–2 min).

Single‐Molecular Imaging of Anticoagulation Factor I from Snake Venom by Atomic Force Microscopy

AbstractAnticoagulation factor I (ACF I) from the venom of Agkistrodon acutus is a binding protein to activated coagulation factor X (FXa) and possesses marked anticoagulant activity. Single ACF I molecule has been successfully imaged in air by tapping mode atomic force microscopy (AFM) with high‐resolution using glutaraldehyde as a coupling agent. The physical adsorption and covalent binding of ACF I onto the mica show very different surface topographies. The former exhibits the characteristic strand‐like structure with much less reproducibility, the latter displays a elliptic granular structure with better reproducibility, which suggests that the stability of ACF I molecules on the mica is enhanced by covalent bonding in the presence of glutaraldehyde. A small‐scale AFM amplitude‐mode image clearly shows that the covalently bonded ACF I molecule by glutaraldehyde has olive shape structure with an average size of 7.4 nm × 3.6 nm × 3.1 nm, which is very similar to the size determined from the crystal structure of ACF I.