Urea potentiometric enzymatic biosensor based on charged biopolymers and electrodeposited polyaniline

A potentiometric biosensor based on urease was developed for the quantitative determination of urea concentration in aqueous solutions for biomedical applications. The urease was either physisorbed onto an electrodeposited polyaniline film (PANI), or immobilized on a layer-by-layer film (LbL) assembled over the PANI film, that was obtained by the alternate deposition of charged polysaccharides (carboxymethylpullulan (CMP) and chitosan (CHI)). In the latter case, the urease (Urs) enzyme was either physically adsorbed or covalently grafted to the LbL film using carbodiimide coupling reaction. Potentiometric responses of the enzymatic biosensors were measured as a function of the urea concentration in aqueous solutions (from 10(-6) to 10(-1) mol L(-1) urea). Very high sensitivity and short response time were observed for the present biosensor. Moreover, a stability study showed a higher stability over time for the potentiometric response of the sensor with the enzyme-grafted LbL film, testifying for the protective nature of the polysaccharide coating and the interest of covalent grafting.     

Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers

A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe₃O₄/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe₃O₄/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization.     

Chitosan-modified poly(acrylonitrile-co-acrylic acid) nanofibrous membranes for the immobilization of concanavalin A

Lectin affinity membranes have been receiving much attention for the separation and detection of various glycoconjugates. In this work, we present a simple and efficient method for the preparation of lectin affinity nanofibrous membranes. Chitosan-modified poly(acrylonitrile-co-acrylic acid) (PANCAA) nanofibrous membranes were first prepared by a coupling reaction between the primary amino groups of chitosan and the carboxyl groups of PANCAA electrospun membranes. Surface characterizations by attenuated total reflectance Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscopy (XPS) and field-emission scanning electron microscopy (FESEM) confirm the chemical and morphological changes of the studied nanofibrous membranes. Fluorescence-labeled concanavalin A (FL-Con A) was then immobilized on these membranes via noncovalent binding. Analyses by fluorescence spectrophotometer (FS) and confocal laser scanning microscopy (CLSM) reveal that the immobilization of Con A onto the modified nanofibrous membranes has been successfully achieved on the basis of the electrostatic interaction and the specific recognition between Con A and chitosan. The results show that the amount of adsorbed FL-Con A increases dramatically with the increasing coupling degree of chitosan (CDC) on the nanofibrous membrane. Moreover, Con A immobilized on the chitosan-modified nanofibrous membranes (CMNMs) can remain relatively stable at pH 5.3. Therefore, it is believed that this work may provide a new kind of material for affinity application.     

Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4–chitosan nanoparticles

A novel and efficient immobilization of β-d-galactosidase from Aspergillus oryzae has been developed by using magnetic Fe₃O₄–chitosan (Fe₃O₄–CS) nanoparticles as support. The magnetic Fe₃O₄–CS nanoparticles were prepared by electrostatic adsorption of chitosan onto the surface of Fe₃O₄ nanoparticles made through co-precipitation of Fe²⁺ and Fe³⁺. The resultant material was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, vibrating sample magnetometry and thermogravimetric analysis. β-d-Galactosidase was covalently immobilized onto the nanocomposites using glutaraldehyde as activating agent. The immobilization process was optimized by examining immobilized time, cross-linking time, enzyme concentration, glutaraldehyde concentration, the initial pH values of glutaraldehyde and the enzyme solution. As a result, the immobilized enzyme presented a higher storage, pH and thermal stability than the soluble enzyme. Galactooligosaccharide was formed with lactose as substrate by using the immobilized enzyme as biocatalyst, and a maximum yield of 15.5% (w/v) was achieved when about 50% lactose was hydrolyzed. Hence, the magnetic Fe₃O₄–chitosan nanoparticles are proved to be an effective support for the immobilization of β-d-galactosidase.     

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.     

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.     

Layer-by-layer assembly to modify poly(l-lactic acid) surface toward improving its cytocompatibility to human endothelial cells

A novel technique to introduce free amino groups onto polyester scaffolds via aminolyzing the ester groups with diamine has been developed recently. Positively charged chitosan was then deposited onto the aminolyzed poly(l-lactic acid) (PLLA) membrane surface in a layer-by-layer assembly manner using poly(styrene sulfonate, sodium salt) (PSS) as a negatively charged polyelectrolyte. The layer-by-layer deposition process of PSS and chitosan was monitored by UV-vis absorbance spectroscopy, energy transfer by fluorescence spectroscopy, and advancing contact angle measurements. The existed chitosan obviously improved the cytocompatibility of PLLA to human endothelial cells. The cell attachment, activity, and proliferation on the PLLA membranes assembled with three or five bilayers of PSS/chitosan with chitosan as the outermost layer were better than those with one bilayer of PSS/chitosan or the control PLLA. The cells also showed morphology of an elongated shape with abundant cytoplasm, and a confluent cell layer was reached after being cultured for 4 days. Measurement of von Willebrand factor secreted by these endothelial cells (ECs) verified the endothelial function. Hence, better ECs compatible PLLA were produced.     

Adsorption of bovin serum albumin (BSA) onto the magnetic chitosan nanoparticles prepared by a microemulsion system

The adsorption characteristics of BSA onto the magnetic chitosan nanoparticles have been investigated in this paper. The magnetic chitosan nanoparticles were prepared by adding the basic precipitant of NaOH solution into a W/O microemulsion system. The morphology of magnetic chitosan nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of magnetic chitosan nanoparticles was from 10nm to 20 nm, and the nanoparticles suspending in the aqueous solution could easily aggregate by a magnet, which suggested that the nanoparticles had good magnetic characteristics. The BSA adsorption experiment indicated that when pH of BSA solution was equal to 4, the maximum adsorption loading reached 110 mg/g. Through measuring the zeta potential of BSA solution and the magnetic nanoparticles, it was found that under this situation the surface of BSA took the negative charge, but the magnetic nanoparticles took the positive charge. Due to the small diameter, the adsorption equilibrium of BSA onto the nanoparticles reached very quickly within 10 min. The adsorption equilibrium of BSA onto the magnetic chitosan nanoparticles fitted well with the Freundlich model. The experimental results showed that the magnetic chitosan nanoparticles have potential to be used for the quick pretreatment in the protein analysis process.     

Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis

In this work, a novel and facile route was developed for the immobilization of enzyme on nanosized magnetic particles, and its application to fast protein digestion via a direct MALDI-TOF mass spectrometry analysis was demonstrated. At first, amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The obtained trypsin-immobilized magnetic nanoparticles were conveniently applied for protein digestion. The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins. The process of digestion is very facile due to the easy manipulation of magnetic nanoparticles. Complete protein digestion was achieved in a short time (5 min), without any complicated reduction and alkylation procedures. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. Additionally, it is worth noting that, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research.     

Application of cellulase-polyamidoamine dendrimer-modified silica for microwave-assisted chitosan enzymolysis

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with a precise molecular structure, high geometric symmetry, and a large number of terminal groups. In this study, PAMAM was grafted onto the surface of silica by microwave irradiation and characterized by Fourier transform infrared spectroscopy and elemental analysis. A novel immobilized cellulase was developed based on enzyme immobilization onto the prepared PAMAM-grafted silica and applied in microwave-assisted chitosan enzymolysis. The results show that the efficiency of cellulase immobilization increased with increasing generations of PAMAM. A high enzymatic hydrolysis efficiency was obtained for a 7 mg ml^?1 chitosan solution at pH 6.2 and 50 °C with 40 W microwave-assisted enzymolysis (20 min) compared with a conventional enzymolysis protocol (3 h). The experimental results indicate that this rapid and efficient enzymolysis method combines the advantages of both PAMAM and microwave-assisted technology, which can be adapted to high-throughput enzyme assay in biochemical and clinical research.     

Activity of glucose oxidase functionalized onto magnetic nanoparticles

BACKGROUND: Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties. METHODS: Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy. RESULTS: The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94-100% while thiophene acetylation was 66-72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles CONCLUSION: Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications.     

Improved synthesis of CMP-sialates using enzymes from frog liver and equine submandibular gland.

An efficient method for the preparation of CMP-N-acetylneuraminic acid using crude or partially purified CTP:N-acylneuraminate cytidylyltransferase from equine submandibular gland or frog liver is described. The yield of the sugar nucleotide after purification by ion-exchange chromatography and gel filtration was 95%. The compound was studied by 360 MHz 1H NMR spectroscopy in addition to the usual chemical and physical analyses. The preparation of radioactive or unlabelled CMP 4-O-methyl-N-acetylneuraminic acid, which is not known to occur in nature, was achieved in 17% yield with the aid of the equine enzyme.     

Magnetic catechol-chitosan with bioinspired adhesive surface: preparation and immobilization of ω-transaminase

The magnetic chitosan nanocomposites have been studied intensively and been used practically in various biomedical and biological applications including enzyme immobilization. However, the loading capacity and the remained activity of immobilized enzyme based on existing approaches are not satisfied. Simpler and more effective immobilization strategies are needed. Here we report a simple catechol modified protocol for preparing a novel catechol-chitosan (CCS)-iron oxide nanoparticles (IONPs) composites carrying adhesive moieties with strong surface affinity. The ω-transaminase (ω-TA) was immobilized onto this magnetic composite via nucleophilic reactions between catechol and ω-TA. Under optimal conditions, 87.5% of the available ω-TA was immobilized on the composite, yielding an enzyme loading capacity as high as 681.7 mg/g. Furthermore, the valuation of enzyme activity showed that ω-TA immobilized on CCS-IONPs displayed enhanced pH and thermal stability compared to free enzyme. Importantly, the immobilized ω-TA retained more than 50% of its initial activity after 15 repeated reaction cycles using magnetic separation and 61.5% of its initial activity after storage at 4°C in phosphate buffered saline (PBS) for 15 days. The results suggested that such adhesive magnetic composites may provide an improved platform technology for bio-macromolecules immobilized.     

Enhanced resistance of polyelectrolyte multilayer microcapsules to pepsin erosion and release properties of encapsulated indomethacin

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly using polysaccharide sodium alginate (ALG) and chitosan (CHI). After incubation in an enzyme pepsin solution, the multilayer film was partially destroyed as detected by the decrease in fluorescent intensity because of the enzymatic degradation of CHI. The enzymatic desorption was also observed from the microcapsule wall made of the ALG/CHI multilayer film directly deposited on indomethacin (IDM) microcrystals through LbL self-assembly. After pepsin erosion, the IDM release from the microcapsules monitored by UV absorbance was obviously accelerated because of desorption. To enhance the stability of the ALG/CHI multilayer film to the enzymatic erosion, some physical and chemical methods were established to increase film thickness or to cross-link the polysaccharides within the film. Increasing the layer number and raising the deposition temperature effectively slowed down the enzymatic desorption and release rate. Especially, increasing deposition temperature was more effective because of producing a more perfect structure in the ALG/CHI multilayer film. Cross-linking the neighboring layers of ALG and CHI with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide in the ALG/CHI multilayer film significantly reduced the enzymatic desorption and release rate. Therefore, increasing deposition temperature and cross-linking neighboring layers are effective methods to protect the multilayer film fabricated using LbL assembly from the enzymatic erosion and to prolong the release of the encapsulated drug.     

Chitosan gelation induced by the in situ formation of gold nanoparticles and its processing into macroporous scaffolds

This work describes a simple synthetic route to induce chitosan (CHI) gelation by the in situ formation of gold nanoparticles (AuNPs). AuNPs were obtained by thermal treatment (e.g., 40 and 80 °C) of CHI aqueous solutions containing HAuCl(4) and in the absence of further reducing agents. The CHI hydrogels resulting after AuNP formation were submitted to unidirectional freezing and subsequent freeze-drying via ISISA (ice-segregation-induced self-assembly) process for the preparation of CHI scaffolds. The study of AuNP-CHI scaffolds by SEM and confocal fluorescence microscopy revealed a morphological structure characteristic of the hydrogel nature of the samples subjected to the ISISA process. Interestingly, not only the morphology but also the dissolution and swelling degree of the resulting CHI scaffolds were strongly influenced by the strength of the hydrogels obtained by the in situ formation of AuNP. We have also studied the catalytic activity AuNP-CHI scaffolds in the reduction of p-nitrophenol. The negligible dissolution and low swelling degree obtained in certain AuNP-CHI scaffolds allowed them to be used for more than four cycles with full preservation of the reaction kinetics.     

Synthesis and magnetic properties of biocompatible hybrid hollow spheres

Magnetic hybrid hollow spheres of about 200 nm were prepared by a core-template-free route, that is, adding Fe3O4 nanoparticles stabilized by poly(vinyl alcohol) (PVA) to an aqueous solution of polymer-monomer pairs composed of a cationic polymer, chitosan (CS), and an anionic monomer, acrylic acid (AA), followed by polymerization of acrylic acid and selective cross-linking of chitosan at the end of polymerization. The obtained hybrid spheres were characterized by dynamic light scattering (DLS) in aqueous solution and observed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) in the solid state. Fourier transform infrared spectroscopy (FTIR) and X-ray and electron diffractions revealed that the Fe3O4 nanoparticles were incorporated into the shells of chitosan-poly(acrylic acid) (CS-AA) hollow spheres. Magnetization studies and M?ssbauer spectroscopy suggested that the chains (or islands) of iron oxide nanoparticles were most likely formed in the walls of the hollow spheres. The phantom test of magnetic resonance imaging showed that the synthesized hybrid hollow spheres had a significant magnetic resonance signal enhancement in T2-weighted image.     

Purification and Characterization of Exo-beta-d-Glucosaminidase from a Cellulolytic Fungus, Trichoderma reesei PC-3-7

Chitosan-degrading activities induced by glucosamine (GlcN) or N-acetylglucosamine (GlcNAc) were found in a culture filtrate of Trichoderma reesei PC-3-7. One of the chitosan-degrading enzymes was purified to homogeneity by precipitation with ammonium sulfate followed by anion-exchange and hydrophobic-interaction chromatographies. The enzyme was monomeric, and its molecular mass was 93 kDa. The optimum pH and temperature of the enzyme were 4.0 and 50 degrees C, respectively. The activity was stable in the pH range 6.0 to 9.0 and at a temperature below 50 degrees C. Reaction product analysis from the viscosimetric assay and thin-layer chromatography and H nuclear magnetic resonance spectroscopy clearly indicated that the enzyme was an exo-type chitosanase, exo-beta-d-glucosaminidase, that releases GlcN from the nonreducing end of the chitosan chain. H nuclear magnetic resonance spectroscopy also showed that the exo-beta-d-glucosaminidase produced a beta-form of GlcN, demonstrating that the enzyme is a retaining glycanase. Time-dependent liberation of the reducing sugar from partially acetylated chitosan with exo-beta-d-glucosaminidase and the partially purified exo-beta-d-N-acetylglucosaminidase from T. reesei PC-3-7 suggested that the exo-beta-d-glucosaminidase cleaves the glycosidic link of either GlcN-beta(1-->4)-GlcN or GlcN-beta(1-->4)-GlcNAc.     

Optimized immobilization of endoglucanase Cel9A onto glutaraldehyde activated chitosan nanoparticles by response surface methodology: The study of kinetic behaviors

Immobilization of enzyme onto nanoparticles such as chitosan can have biotechnological importance. In this study, chitosan nanoparticles (ChNPs) were prepared by Ionic gelation method and Endoglucanase Cel9A from Alicyclobacillus acidocaldariius (AaCel9A) immobilized on the nanoparticles. The FTIR results showed that the enzymes were immobilized on the ChNPs. The dynamic light scattering and scanning electron microscope (SEM) results illustrated that the AaCel9A-ChNPs approximately had 40 nm diameters. For optimizing enzyme immobilization, response surface methodology was employed using different variables (pH, enzyme immobilization time, and enzyme to ChNPs ratio [E/Cs]). The results showed that the high immobilization efficiency was achieved in pH 7, E/Cs of 0.4 in 2.63 hr. The enzyme activity results showed that, immobilization increased optimum pH for activity (from 6.5 to 7.5) and the enzyme K_m (from 3.703 to 12.195 [mg/ml]), which make it suitable to use in some industries such as detergents.     

N-terminal region of chitinase I of Bacillus circulans KA-304 contained new chitin-biding domain

Chitinase I (CHI1) of Bacillus circulans KA-304 forms protoplasts from Schizophyllum commune mycelia when the enzyme is combined with α-1,3-glucanase of B. circulans KA-304. CHI1 consists of an N-terminal unknown region and a C-terminal catalytic region classified into the glycoside hydrolase family-19 type. An N-terminal region-truncated mutant of CHI 1 (CatCHI1), which was expressed in Escherichia coli Rosetta-gami B (DE3), lost colloidal chitin- and powder chitin-binding activities. The colloidal chitin- and the powder chitin-hydrolyzing activities of CatCHI1 were lower than those of CHI1, and CatCHI1 was not effective in forming the protoplast. A fusion protein of the N-terminal region of CHI1 and green fluorescent protein (Nterm-GFP) was expressed in E. coli, and the fusion protein was adsorbed to colloidal chitin, powder chitin, and chitosan. Fluorescence microscopy analysis showed that Nterm-GFP bound to the S. commune cell-wall.     

Adsorption of humic acid from aqueous solution onto irradiation-crosslinked carboxymethylchitosan

This paper presents the adsorption of humic acid from aqueous solution onto crosslinked chitosan derivative (carboxymethylchitosan), formed by additionless irradiation technique. The surface charge and swelling properties of crosslinked samples were investigated. The adsorption of humic acid onto crosslinked carboxymethylchitosan was carried out by the batch method at room temperature, and it was found to be strongly pH-dependent. Maximum amount of humic acid was adsorbed under acidic conditions at the optimum pH value of 3.5. Adsorption kinetic studies indicated the adsorption process was transport-limited at the same pH. The adsorption isotherm analysis data under various initial humic acid concentrations confirms that experimental data fitted well into the Langmuir equation. X-ray photoelectron spectroscopy (XPS) revealed that the amino groups of carboxymethylated chitosan were protonated, suggesting the formation of organic complex between the protonated amino groups and humic acid. From these preliminary evaluations, it was concluded that crosslinked carboxymethylated chitosan derivatives have a great potential in water treatment for the removal of humic acid and other polarized or electrically charged species.