Preparation of filled temperature-sensitive poly(N-isopropylacrylamide) gel beads

Temperature-sensitive filled poly(N-isopropylacrylamide) (PNIPAAm) gel beads with diameters in the range of millimeters were prepared using the alginate technique. The polymerization and cross-linking reaction of NIPAAm in the presence of inorganic filling particles was performed in spherical networks of Ca-alginate forming interpenetrating networks (IPN). Thermo-sensitive gel beads could be obtained by washing these IPN with EDTA solution. The PNIPAAm gel beads were analyzed by optical methods to observe there swollen diameter in dependence on the temperature. The diameters of the swollen gel beads were in the range of 0.1 - 2 mm. The influence of the monomer to cross-linker ratio (MCR) and the filling materials (ferrofluid, BaTiO₃, TiO₂, and Ni,) were studied. The phase transition temperature (T_pt) was only weakly influenced by the MCR and the filling material remaining at around 34°C.     

Alginate beads containing pH-sensitive liposomes and glucose oxidase: glucose-sensitive release

Egg PC (EPC) liposomes bearing a copolymer of N-isopropylacrylamide, methacrylic acid, and octadecylacrylate (P(NIPAM-co-MAA-co-ODA)) were prepared as pH-sensitive liposomes. They were embedded in glucose oxidase (GOD)-immobilized alginate beads. The ratio of EPC/GOD/alginate in the beads was 7.8:1.0:140.4, and the beads were added to glucose solutions so that the concentration of GOD was 0.0068 mg/ml. The enzymatic activity of the immobilized GOD was one fifth to half of that of native enzyme. As the glucose concentration increased from 0 to 400 mg/dl, the degree of calcein release increased from 17% to 75%. The acidification induced by the enzymatic reaction would be responsible for the glucose-triggered release.     

Self-Assembled Supramolecular Hybrid Hydrogel Beads Loaded with Silver Nanoparticles for Antimicrobial Applications

This Full Paper reports the formation of silver (Ag) NPs within spatially resolved two-component hydrogel beads, which combine a low-molecular-weight gelator (LMWG) DBS-CONHNH₂ and a polymer gelator (PG) calcium alginate. The AgNPs are formed through in situ reduction of Ag^I, with the resulting nanoparticle-loaded gels being characterised in detail. The antibacterial activity of the nanocomposite gel beads was tested against two drug-resistant bacterial strains, often associated with hospital-acquired infections: vancomycin-resistant Enterococcus faecium (VRE) and Pseudomonas aeruginosa (PA14), and the AgNP-loaded gels showed good antimicrobial properties against both types of bacteria. It is suggested that the gel bead format of these AgNP-loaded hybrid hydrogels makes them promising versatile materials for potential applications in orthopaedics or wound healing.     

Hybrid Self-Assembled Gel Beads for Tuneable pH-Controlled Rosuvastatin Delivery

This article describes the fabrication of new pH-responsive hybrid gel beads combining the polymer gelator calcium alginate with two different low-molecular-weight gelators (LMWGs) based on 1,3 : 2,4-dibenzylidene-d -sorbitol: pH-responsive DBS-COOH and thermally responsive DBS-CONHNH₂, thus clearly demonstrating that different classes of LMWG can be fabricated into gel beads by using this approach. We also demonstrate that self-assembled multicomponent gel beads can be formed by using different combinations of these gelators. The different gel bead formulations exhibit different responsiveness – the DBS-COOH network can disassemble within those beads in which it is present upon raising the pH. To exemplify preliminary data for a potential application for these hybrid gel beads, we explored aspects of the delivery of the lipid-lowering active pharmaceutical ingredient (API) rosuvastatin. The release profile of this statin from the hybrid gel beads is pH-dependent, with greater release at pH 7.4 than at pH 4.0 – primary control of this process results from the pK_a of the API. The extent of pH-mediated API release is also significantly further modified according to gel bead composition. The DBS-COOH/alginate beads show rapid, highly effective drug release at pH 7.4, whereas the three-component DBS-COOH/DBS-CONHNH₂/alginate system shows controlled slow release of the API under the same conditions. These initial results indicate that such gel beads constitute a promising, versatile and easily tuned platform suitable for further development for controlled drug-delivery applications.     

Synthesis of polyacrylamide gel beads with electrostatic functional groups for the molecular imprinting of bovine serum albumin

Synthetic materials capable of recognizing proteins are important in separation, biosensors and biomaterials. In this study, bovine serum albumin-imprinted soft-wet polyacrylamide gel beads were prepared via inverse-phase suspension polymerization, using acrylamide and N,N′-methylene diacrylamide as polymeric matrix components and methacrylic acid as functional monomer. The adsorption study showed, through the imprinting process, that the imprinted gel beads had much higher adsorption capacity than the nonimprinted gel beads, and that the matching of the surface zeta-potential between the templates and the imprinted gel beads can enhance the imprinting effect. Adsorption kinetics indicated that the adsorption process could be described as an apparent first-order kinetic process for the gel beads. From the adsorption isotherm curve, we found that the adsorption of the imprinted gel beads was in agreement with the Langmuir adsorption model. Moreover, selectivity testing of the imprinted gel beads showed that imprinted gel beads exhibited good recognition for BSA as compared to the control protein. We speculate that the formation of complementary shapes and multiple-point electrostatic interactions between the imprinting cavities and the template proteins are the two factors that lead to the imprinting effect.      

Tailoring Zinc Oxide Nanowires for High Performance Amperometric Glucose Sensor

ZnO nanowire was tailored both physically and chemically to immobilize the enzyme glucose oxidase (GOD) for construction of a glucose sensor with high performance, which was ascribed to its high specific surface area and high isoelectric point value for efficient immobilization of high concentration of acidic enzymes and the mediating effect by the redox reaction of ZnO nanowires. The apparent Michaelis constants J_max, and K_M were adjusted in a large scope by tailoring the thickness of the GOD/ZnO nanowire layer and the enzyme load in the nanowired network. Thus, a variety of linear region, sensitivities and reaction rates of the sensor could be easily achieved. Moreover, the glucose sensor showed long term stability with the incorporation of the inorganic zinc oxide nanowire.     

Temperature-sensitive poly(vinyl methyl ether) hydrogel beads

Temperature-sensitive hydrogel beads were prepared by radiation crosslinking of poly(vinyl methyl ether) PVME spheres wrapped in Ca-alginate. The obtained gel beads have diameters in the sub-millimeter or millimeter range (depending on the PVME concentration). They were characterized by sol-gel analysis, swelling measurements, and differential scanning calorimetry. The gel content g increases with increasing radiation dose D. The swelling degree Q_v decreases with increasing PVME concentration c_p and increasing D. In comparison to PVME bulkgels the phase-transition temperature of the synthesized PVME gel beads is a little decreased.     

l-DOPA production by immobilized tyrosinase

The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U _C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U _IMO). The optimal conditions were t=24 h, G=2% (v/v), and U _C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U _IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h).     

Amperometric Detection of Glucose with Glucose Oxidase Immobilized in Layered Double Hydroxides

A novel cheap and simple amperometric biosensor, based on the immobilization of glucose oxidase (GOD) into anionic clay; layered double hydroxides (LDHs) [Zn₃‐Al‐Cl] is presented. GOD can be entrapped in the LDHs gel via electrostatic interaction. Amperometric detection of glucose with an unmediated sensor at 0.6 V (vs. SCE) results in a rapid response (5 s), a wide linear range of 0.001–12 mM, as well as good operational stability. The low detection limit was 0.1 μM at 3σ. The apparent Michaelis‐Menten constant (K is 4.4 mM. The general interferences that coexisted in blood serum do not affect glucose determination, except for uric acid. In addition, optimization of the biosensor construction and the effects of the applied potential on the amperometric response of the sensor were investigated and discussed herein.     

Production ofL-glutamate oxidase andin situ monitoring of oxygen uptake in solid state fermentation ofstreptomyces sp. Nl

Effects of water content and carbon and nitrogen sources on the production ofL-glutamate oxidase (GOD) by solid state fermentation (SSF) ofStreptomyces sp. N₁ were investigated in a 250-mL shake flask. The results show that in the solid medium containing wheat bran 98% (w/w), KCl 0.2% (w/w), and MgCl₂ 0.2% (w/w), addition of 2.0-mL water per gram solid medium and 0.4% (w/w) (NH₄)₂SO₄ was the best for GOD production. In this work, we also developed a simple technique forin situ measuring oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) in SSF in a shake flask based on the principle of Warburg manometer. The method was successfully applied to determine OUR and CER values in SSF ofStreptomyces sp. N₁. The results indicate that the largest OUR value was detected about one or two days ahead of the highest GOD activity reached depending on the fermentation conditions, and the OUR may be used as anin situ indicator of GOD production in the SSF process.     

Apatite-Silica Gel Composite Materials Prepared by a New Alternate Soaking Process

Novel apatite/silica composite materials were synthesized by modifying the surface of silica gel beads or plates with apatite. An alternate soaking process, which involves alternate soaking in a CaCl₂/tris-HCl aqueous solution (pH 7.4) and a Na₂HPO₄ aqueous solution, was utilized to prepare a composite of silica gel and apatite. The characteristics of apatite formed on the surface of silica gel have been studied using X-ray diffraction, Fourier-transform infrared spectrophotometry (FT-IR) and scanning electron microscopy (SEM). Data clearly showed surface modification of silica with the crystalline apatite. Pore volume and specific total surface area, which were measured using nitrogen gas adsorption apparatus, were decreased after apatite deposition on the surface of silica gel beads, whereas the external surface area was increased drastically. The alternate soaking process made it possible to prepare apatite/silica gel composites in a remarkably short period of time, i.e. several hours.     

Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol–gel silica thin films on glassy carbon

This work points out that electrogeneration of silica gel (SG) films on glassy carbon electrodes (GCEs) can be applied to immobilize biomolecules – hemoglobin (Hb) or glucose oxidase (GOD) or both of them in mixture – without preventing their activity. These proteins were physically entrapped in the sol–gel material in the course of the electro-assisted deposition process applied to form the thin films onto the electrode surface. SG films were prepared from a precursor solution by applying a suitable cathodic potential likely to induce a local pH increase at the electrode/solution interface, accelerating thereby polycondensation of the silica precursors with concomitant film formation. Successful immobilization of proteins was checked by various physico-chemical techniques. Both Hb and GOD were found to undergo direct electron transfer, as demonstrated by cyclic voltammetry. GCE–SG–Hb gave rise to well-defined peaks at potentials E_c = −0.29 V and E_a = −0.17 V in acetate buffer, corresponding to the Fe^III/Fe^II redox system of heme group of the protein, while GCE–SG–GOD was characterized by the typical signals of FAD group at E_c = −0.41 V and E_a = −0.33 V in phosphate buffer. These two redox processes were also evidenced on a single voltammogram when both Hb and GOD were present together in the same SG film. Hb entrapped in the silica thin film displayed an electrocatalytic behavior towards O₂ and H₂O₂ in solution, respectively in the mM and μM concentration ranges. Immobilized GOD kept its biocatalytic properties towards glucose. Combined use of these two proteins in mixture has proven to be promising for detection of glucose in solution via the electrochemical monitoring of oxygen consumption (decrease of the oxygen electrocatalytic signal).     

Unadulterated Glucose Biosensor Based on Direct Electron Transfer of Glucose Oxidase Encapsulated Chitosan Modified Glassy Carbon Electrode

Glucose oxidase (GOD) was encapsulated in chitosan matrix and immobilized on a glassy carbon electrode, achieving direct electron transfer (DET) reaction between GOD and electrode without any nano‐material. On basis of such DET, a novel glucose biosensor was fabricated for direct bioelectrochemical sensing without any electron‐mediator. GOD incorporated in chitosan films gave a pair of stable, well‐defined, and quasireversible cyclic voltammetric peaks at about ?0.284 (E_pa) and ?0.338 V (E_pc) vs. Ag/AgCl electrode in phosphate buffers. And the peak is located at the potentials characteristic of FAD redox couples of the proteins. The electrochemical parameters, such as midpoint potential (E_1/2) and apparent heterogeneous electron‐transfer rate constants (k_s) were estimated to ?0.311 V and 1.79 s^?1 by voltammetry, respectively. Experimental results indicate that the encapsulated GOD retains its catalytic activity for the oxidation of glucose. Such a GOD encapsulated chitosan based biosensor revealed a relatively rapid response time of less than 2 min, and a sufficient linear detection range for glucose concentration, from 0.60 to 2.80 mmol L^?1 with a detection limit of 0.10 mmol L^?1 and electrode sensitivity of 0.233 μA mmol^?1. The relative standard deviation (RSD) is under 3.2% (n=7) for the determination of practical serum samples. The biologic compounds probably existed in the sample, such as ascorbic acid, uric acid, dopamine, and epinephrine, do not affect the determination of glucose. The proposed method is satisfactory to the determination of human serum samples compared with the routine hexokinase method. Both the unique electrical property and biocompatibility of chitosan enable the construction of a good bio‐sensing platform for achieved DET of GOD and developed the third‐generation glucose biosensors.     

Preparation of the glucose sensor based on three-dimensional ordered macroporous gold film and room temperature ionic liquid

A novel type of glucose sensor was fabricated based on a glucose oxidase (GOD)-N,N-dimethtylformamide (DMF)-[BMIm][BF₄] composites modified three-dimensional ordered macroporous (3DOM) gold film electrode. The immobilized GOD exhibits a pair of well-defined reversible peaks in 50 mM pH 7.0 phosphate buffer solutions (PBS), which could be attributed to the redox of flavin adenine dinucleotide (FAD) in GOD. The research results show that ionic liquid ([BMIm][BF₄]), DMF and 3DOM gold film are crucial for GOD to exhibit a pair of stable and reversible peaks. It is believed that the large active area of 3DOM gold film can increase the amount of immobilized GOD. Simultaneously, the application of IL enhances the stability of GOD and facilitates the electron transfer between GOD and the electrode. The synergetic effect of DMF can help the GOD to maintain its bioactivity better. GOD immobilized on the electrode exhibits the favorable electrocatalytic property to glucose, and the prepared sensor has a linear range from 10 to 125 nM with a detection limit of 3.3 nM at a signal-to-noise ratio of 3σ. The apparent K _m (Michaelis- Menten constant) for the enzymatic reaction is 0.018 mM.     

Stratiform Protein Microtube Reactors Containing Glucose Oxidase Layer

This paper describes the synthesis and catalytic activities of stratiform protein microtube reactors containing a glucose oxidase (GOD) enzyme layer. The microtubes were fabricated by layer‐by‐layer assembly using a microporous polycarbonate membrane with human serum albumin (HSA), poly(l ‐arginine) (PLA), and GOD. The GOD component was introduced into the tube wall as the innermost layer, the intermediate layer, or all internal protein layers. SEM observations revealed the formation of uniform hollow cylinders with ca. 1.17 μm outer diameter and ca. 135 nm wall thickness. In aqueous medium, each microtube catalyzed β‐d ‐glucose oxidation with high efficiency. We first ascertained the enzyme parameters (K_m and k_cat) of these microtube reactors. Different catalytic activities that have dependent on the GOD layer position in the cylindrical wall have been elucidated.     

A Biosensor Composed of Glucose Oxidase‐Containing Liposomes and MnO2‐Based Layered Nanocomposite

Glucose oxidase (GOD) was encapsulated in liposomes, and then the GOD‐containing liposomes were immobilized to a MnO₂‐based multilayered nanocomposite film grown electrochemically. Oxidation of glucose took place on the encapsulated GOD in the manganese oxide film, and the generated H₂O₂ molecules were oxidized catalytically at high valent Mn sites (4+) in the film. Anodic currents due to reoxidation of the reduced Mn ions (3+) were in proportion to the concentration of glucose from 19.6 to 107.1 mM. Such a simple construction of biosensor is applicable to a variety of combinations of liposomal enzymes and substrates.     

Cloning and Heterologous Expression of Glucose Oxidase Gene from <Emphasis Type="Italic">Aspergillus niger</Emphasis> Z-25 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting revealed that rGOD exhibited a K _m value of 16.95 mM and a K _cat value of 484.26 s⁻¹.     

Preparation of GOD/Sol‐Gel Silica Film on Prussian Blue Modified Electrode for Glucose Biosensor Application

A novel amperometric glucose biosensor was fabricated by in situ incorporating glucose oxidase (GOD) within the sol‐gel silica film on a Prussian blue (PB) modified electrode. The method is simple and controllable, which combined the merits of in situ immobilizing biomolecules in sol‐gel silica film by electrochemical method and the synergic catalysis effects of PB and GOD molecules. Scanning electron microscopy (SEM) showed that the GOD/sol‐gel silica film was homogeneous with a large number of three‐dimensional nanopores, which not only enhanced mass transport, but also maintained the active configuration of the enzyme molecule and prevented the leakage of enzyme, therefore improved the stability and sensitivity of the biosensor. The fabricated biosensor showed fast response time (10 s), high sensitivity (26.6 mA cm^?2 M^?1), long‐term stability, good suppression of interference, and linear range of 0.01 mM–5.8 mM with a low detection limit of 0.94 μM for the detection of glucose. In addition, the biosensor was successfully applied to determine glucose in human serum samples.     

Calorimetric study of the isotropic to nematic phase transition in an aligned liquid crystal nano‐colloidal gel

A high‐resolution calorimetric study of the specific heat (C_p ) has been carried out for the isotropic to nematic phase transition in an aligned liquid crystal (octylcyanobiphenyl ‐ 8CB) and aerosil nano‐colloid gel. A stable alignment was achieved by repeated thermal cycling of the samples in the presence of a strong uniform magnetic field, which introduces anisotropy to the quenched random disorder of the silica gel. In general, the specific heat features of the I?N transition in aligned (anisotropic) gel samples are consistent with those seen in random (isotropic) gel samples, namely the observance of two C_p peaks and non‐monotonic transition temperature shifts with increasing silica concentration. However, larger transition temperature shifts with silica density, modification of the phase conversion process in the two‐phase coexistence region, and a larger effective transition enthalpy are observed for the aligned samples. The lower‐temperature aligned C_p peak is larger and broader while exhibiting less dispersion than the equivalent peak for the random gel. This may be a consequence of the alignment altering the evolution from random‐dilution‐dominated to random‐field‐dominated effects. The exact origin of the larger transition temperature shifts is uncertain but the larger enthalpy suggests that the nematic state is different in the aligned system than in random gels. The general non‐monotonic behaviour of the transition temperature is interpreted using dimensional analysis as a combination of an effective elastic stiffening of the liquid crystal combined with a liquid crystal and aerosil surface interaction energy.     

Superparamagnetic behavior of iron oxides supported on porous silica gels

Spinel iron oxide (Fe₃O₄-γ-Fe₂O₃) particles were supported on microbeads of silica gel by the calcination of the silica gel base adsorbing citric acid and Fe³⁺ ions. The X-ray diffraction patterns and the⁵⁷Fe Mössbauer spectra measured for the spinel iron oxide indicated that the particle size of the oxide was regulated by the mean pore diameter (4–82 nm) of the silica gel support employed. In the case of α-Fe₂O₃ particles prepared by using the same silica gel beads, it was revealed by the Mössbauer spectra and the electron micrographs that there were relatively large particles of the oxide on the surface of the beads, in addition to the particles in the silica gel micropores.