Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Electrochemical sensing platform based on covalent immobilization of thionine onto gold electrode surface via diazotization-coupling reaction

A novel electrochemical sensing platform by modification of electroactive thionine (Th) onto gold electrode surface was constructed, which was realized by diazotization of 4-aminothiophenol (ATP) self-assembled monolayer, followed by coupling of Th with the diazonium group to form a covalent diazo bond. A pair of well-defined redox peaks of Th was observed in the cyclic voltammetric measurement. The resulting diazo-ATP monolayer displayed superior electrical conductivity, which contributed to the sensitive detection of hydrogen peroxide (H₂O₂). The immobilized Th also showed a remarkable stability, which may benefit from the π-π stacking force and the covalent diazo bond between diazo-ATP and Th molecules. Under the optimized experimental conditions, the current fabricated non-enzyme and reagentless sensor could show a rapid response to H₂O₂ within 3 s and a linear calibration plot ranged from 1.0 × 10⁻⁶ to 6.38 × 10⁻³ M with a detection limit of 6.7 × 10⁻⁷ M. The current fabrication strategy of electroactive interface is expected to be used as a versatile route for the immobilization of more electroactive molecules and offer more opportunities for the applications in electrochemical sensor, biosensor, electrocatalysis, etc.     

Glucose biosensing based on the highly efficient immobilization of glucose oxidase on a Prussian blue modified nanostructured Au surface

We report on a novel glucose biosensor based on the immobilization of glucose oxidase (GOx) on a Prussian blue modified nanoporous gold surface. The amperometric glucose biosensor fabricated in this study exhibits a fast response and the very low detection limit of 2.5 μM glucose. The sensitivity of the biosensor was found to be very high, 177 μA/mM; the apparent Michaelis–Menten constant is calculated to be 2.1 mM. In addition, the biosensor has good reproducibility and remains stable over 60 days. The anti-interference ability of the biosensor was also assessed, showing little interference from possible interferents such as ascorbic acid (AA), acetaminophen (AP) and uric acid (UA).     

Glucose biosensors based on the immobilization of copper oxide and glucose oxidase within a carbon paste matrix

The performance of amperometric glucose biosensors based on the dispersion of glucose oxidase (GOx) and copper oxide within a classical carbon (graphite) paste composite is reported in this work. Copper oxide promotes an excellent electrocatalytic activity towards the oxidation and reduction of hydrogen peroxide, allowing a large decrease in the oxidation and reduction overpotentials, as well as an important enhancement of the corresponding currents. Therefore, it is possible to perform the glucose biosensing at low potentials where there is no interference even in large excess of ascorbic acid, uric acid or acetaminophen. The influence of the copper oxide and glucose oxidase content in the paste on the analytical performance of the bioelectrode is discussed. The resulting biosensor shows a fast response, a linear relationship between current and glucose concentration up to 1.35 × 10⁻² M (2.43 g L⁻¹) and a detection limit of 2.0 × 10⁻⁵ M. The effect of the presence of the enzyme in the composite material on the dispersion of the copper oxide particles is also discussed.     

Development of an amperometric glucose biosensor based on the immobilization of glucose oxidase in an ormosil-PVA matrix onto a Prussian Blue modified electrode

An amperometric glucose biosensor was developed based on the immobilization of glucose oxidase in the organically modified silicate (ormosil)-polyvinyl acetate (PVA) matrix onto a Prussian Blue (PB)-modified glassy carbon electrode. A higher stability PB-modified electrode was prepared by the electrochemical deposition of FeCl₃, K₃[Fe(CN)₆] and ethylenediamine tetraacetic acid (EDTA) under cyclic voltammetric (CV) conditions. The effects of the potential range of CV conditions, electrolyte cations, applied potential, pH, temperature and co-existing substances were investigated. The detection limit of the glucose biosensor was 8.1 μmol·L⁻¹ (S/N = 3) with a linear range from 20 μmol·L⁻¹ to 2 mmol·L⁻¹ (R = 0.9965). The biosensor presented a fast response and good selectivity. Additionally, excellent reproducibility and stability of the biosensor were observed. Supported by the National High Technical Development Project (863 project) Foundation (Grant No. 2006AA09Z160) and the National Natural Science Foundation of China (Grant No. 20775064)     

Preparation of interpenetrating polymer network composed of poly(ethylene glycol) and poly(acrylamide) hydrogels as a support of enzyme immobilization

Poly(ethylene glycol)(PEG)‐based interpenetrating polymeric network (IPN) hydrogels were prepared for the application of enzyme immobilization. Poly(acrylamide)(PAAm) was chosen as the other network of IPN hydrogel and different concentration of PAAm networks were incorporated inside the PEG hydrogel to improve the mechanical strength and provide functional groups that covalently bind the enzyme. Formation of IPN hydrogels was confirmed by observing the weight per cent gain of hydrogel after incorporation of PAAm network and by attenuated total reflectance/Fourier transform infrared (ATR/FTIR) analysis. Synthesis of IPN hydrogels with higher PAAm content produced more crosslinked hydrogels with lower water content (WC), smaller M_c and mesh size, which resulted in enhanced mechanical properties compared to the PEG hydrogel. The IPN hydrogels exhibited tensile strength between 0.2 and 1.2 MPa while retaining high levels of hydration (70–81% water). For enzyme immobilization, glucose oxidase (GOX) was immobilized to PEG and IPN hydrogel beads. Enzyme activity studies revealed that although all the hydrogels initially had similar enzymatic activity, enzyme‐immobilizing PEG hydrogels lost most of the enzymatic activity within 2 days due to enzyme leaching while IPN hydrogels maintained a maximum 80% of the initial enzymatic activity over a week due to the covalent linkage between the enzyme and amine groups of PAAm. Copyright © 2008 John Wiley & Sons, Ltd.     

New insights from X-ray photoelectron spectroscopy into the chemistry of covalent enzyme immobilization, with glutamate dehydrogenase (GDH) on silicon dioxide as an example

A three-step process for immobilization of glutamate dehydrogenase (GDH) on the surface of silicon dioxide has been studied by X-ray photoelectron spectroscopy (XPS). The enzyme layer was deposited on the silicon dioxide surface after first exposing the surface to 3-aminopropyltriethoxysilane (3-APTS) and reacting the silylated surface with glutaraldehyde (GA). Fine XPS analysis, performed after each step of the chemical procedure, revealed unknown details of the step-by-step construction of the enzyme layer under different experimental conditions.     

Co-immobilization of polymeric luminol,iron(II) tris(5-aminophenanthroline) and glucose oxidase at an electrode surface,and its application as a glucose optrode

The anodic polymerization of 3-aminophthalhydrazide (luminol) and iron(II) tris 5-aminophenanthroline (Fe(phen-NH2)3(2+)) has been reported in this paper. A bilayer electrode was developed based on these polymers and the ITO conductive glass (denoted ITO[Fe(phen-NH2)3(2+)]luminol electrode). This electrode emitted light (lambdaem: 430 nm) as it was brought into contact with H2O2. At pH 10, the resulting electrochemiluminescence (ECL) showed a linear relationship with the concentration of H2O2 in the range of 10 microM(-1) mM. This bilayer electrode also showed an application potential for the detection of glucose after being further modified with glucose oxidase (denoted ITO[Fe(phen-NH2)3(2+)]luminol]GOx electrode). Although the resulting ECL decayed more rapidly in concentrated glucose solutions (e.g., I M) because of the consumption of luminol during use, the decay became less severe in diluted glucose solutions (e.g., 10 mM). According to the flow injection analysis, a linear relationship existed between the ECL and the concentration of glucose from 10(-5)-10(-3) M at pH 9. The detection limit could reach a level of 5 x 10(-5) M at this pH.     

A highly sensitive non-enzymatic glucose sensor based on a simple two-step electrodeposition of cupric oxide (CuO) nanoparticles onto multi-walled carbon nanotube arrays

A novel, stable and highly sensitive non-enzymatic glucose (Glc) sensor was developed using vertically well-aligned multi-walled carbon nanotubes array (MWCNTs) incorporated with cupric oxide (CuO) nanoparticles. The MWCNTs array was prepared by catalytic chemical vapor deposition on a tantalum (Ta) substrate, while a simple and rapid two-step electrodeposition technique was used to prepare the CuO-MWCNTs nanocomposite. First, Cu nanoparticles were deposited onto MWCNTs at constant potential and then they were oxidized into CuO by potential cycling. The electrocatalytic activity of CuO-MWCNTs array was investigated for Glc under alkaline conditions using cyclic voltammetry and chronoamperometry. The sensor exhibited a linear response up to 3 mM of Glc and sensitivity of 2190 μA mM⁻¹ cm⁻², which is two to three orders of magnitude higher than that of most non-enzymatic Glc sensors reported in the literature. The sensor response time is less than 2 s and detection limit is 800 nM (at signal/noise = 3). When tested with human blood serum samples, the sensor exhibited high electrocatalytic activity, stability, fast response and good selectivity against common interfering species, suggesting its potential to be developed as a non-enzymatic Glc sensor.     

Enhanced mechanical properties of carbon fiber composites by grafting different structural poly(amido amine) onto fiber surface

The mechanical properties of carbon fiber composites depend on the interfacial strength between fiber and epoxy matrix. Different poly (amido amine) (PAMAM) dendrimers were grafted onto carbon fiber to improve the interfacial strength of the resulting composites. Functional groups on the carbon fiber surface were examined by X-ray photoelectron spectroscopy. The surface morphology of the resulting materials was characterized by scanning electron microscopy and atomic force microscope. The characterization results revealed that PAMAM dendrimers were chemically grafted onto the surface of carbon fiber. More importantly, the mechanical properties of the resulting composites were enhanced owing to the presence of sufficient functional groups on the carbon fiber surface. In addition, after PAMAM containing chair conformations were grafted, the interlaminar shear strength had the highest increase of 53.13%, higher than that of the fiber grafted with PAMAM containing terminated linear amine. This work provides an alternative approach to enhance the mechanical properties of fiber composites by controlling the interface between fiber and epoxy matrix.     

Cross-linked poly(4-vinylpyridine/styrene) copolymers as a support for immobilization of ytterbium triflate

Eight cross-linked poly(4-vinylpyridine/styrene) (P/S) resins (as beads) were prepared by radical suspension polymerization. Ytterbium triflate was immobilized in the range of 0.10-0.24 mmol/g by mixing with the P/S resins. The ytterbium triflate-immobilized P/S resins exhibited good activity in two Lewis acid-catalyzed reactions. Low pyridine containing resins were recycled with no loss of activity, while a slight loss of activity was observed with the higher pyridine containing resins.     

Inner filter effect (IFE) as a simple and selective sensing platform for detection of tetracycline using milk-based nitrogen-doped carbon nanodots as fluorescence probe

A simple, selective, and sensitive turn-off fluorescent assay for detecting of tetracycline in pharmaceutical dosage form based on inner filter effect (IFE) sensing platform has been described. In this IFE sensing strategy, N-doped carbon dots (CDs) were prepared by one-pot solvothermal synthesis using milk as a precursor and were directly used as a fluorophore in IFE. The prepared CDs were characterized by common spectroscopic and microscopic techniques. The CDs exhibited excitation-wavelength dependent emission with 10% as the fluorescence quantum yield. The fluorescence of CDs was decreased in correlation to the addition of absorber (tetracycline), as the excitation spectrum of the fluorophore (CDs) matches the absorption spectrum of the absorber. The present IFE-based sensing platform showed a good linear relationship from 2.0 µM to 200 µM (R² = 0.9960) and provided a detection limit of 0.6 µM (signal-to-noise ratio of 3). Additionally, the cytotoxic effects of CDs were determined using normal healthy male Balb/C mice model treated with various doses of CDs and at the end of the study, no mortality or even no sign of toxicity was observed at oral doses of 100 and 200 mg/kg CDs in all treated animals. The proposed nanoprobe assay is a free from interferences, low-cost, biocompatible, and accurate for the detection of tetracycline in pharmaceutical formulation.     

Modification of sodium aluminum silicate hydrate by thioglycolic acid as a new composite capable of removing and preconcentrating Pb(II), Cu(II), and Zn(II) ions from food and water samples

In this paper, sodium aluminum silicate hydrate was synthesized using rice husk as a silicon source and scrap aluminum cans as an aluminum source. Afterward, with the aid of microwave heating, a composite of sodium aluminum silicate hydrate and (3-aminopropyl)trimethoxysilane was facilely synthesized then modified by thioglycolic acid to produce a new composite. Besides, characterization of the synthesized composite was carried out using XRD, FT-IR, TEM, CHN elemental analyzer, nitrogen gas sorption analyzer, and SEM. The XRD pattern of the produced composite shows that there is a halo at 2θ = 25.0°, which means that there is a crystalline structure that is combined with an amorphous background. The SEM and TEM studies indicate that the synthesized composite has a structure similar to cotton. The synthesized composite was utilized for the efficient removal and preconcentration of Pb(II), Cu(II), and Zn(II) ions from food and water samples prior to determination by flame atomic absorption spectrometry. The produced composite has a maximum adsorption capacity of 185.53, 168.92, and 125.94 mg/g for Pb(II), Cu(II), and Zn(II) ions, respectively. The recovery findings demonstrate that the process is accurate, adaptable, and resulted in quantitative separation (greater than95 percent). Furthermore, the % RSD was less than 3.5 percent, indicating good reproducibility. The Langmuir isotherm and pseudo-second-order model fit the experimental results well. The thermodynamic studies established that the adsorption process is spontaneous, chemical, and exothermic. The produced composite was successfully regenerated and used multiple times to remove the metal ions under investigation from aqueous solutions.     

Electrochemistry at cobalt(II)tetrasulfophthalocyanine-multi-walled carbon nanotubes modified glassy carbon electrode: a sensing platform for efficient suppression of ascorbic acid in the presence of epinephrine

Electrochemistry of water-soluble cobalt(II) tetrasulfophthalocyanine (CoTSPc) electrodeposited on glassy carbon nanotube pre-modified with acid-functionalized multi-walled carbon nanotubes (MWCNT) is described. Both charge transfer resistances toward [Fe(CN)₆]^3−/4− redox probe and electrocatalytic responses toward epinephrine (EP) detection follow the trend: bare GCE < GCE-MWCNT < GCE-CoTSPc < GCE-MWCNT-CoTSPc. EP analysis was then carried out in details using GCE-MWCNT-CoTSPc. The catalytic rate constant value k _ch = 2.2 × 10⁷ (mol cm⁻³)⁻¹ s⁻¹ was obtained from rotating disk electrode experiment. Interestingly, GCE-MWCNT-CoTSPc efficiently suppressed the detection of ascorbic acid (the natural interference of neurotransmitters in physiological conditions) showing good sensitivity (0.132 ± 0.003 A l mol⁻¹), limit of detection (4.517 × 10⁻⁷ mol l⁻¹), and quantification (15.056 × 10⁻⁷ mol l⁻¹). In addition, GCE-MWCNT-CoTSPc was conveniently used to determine EP in epinephrine hydrochloric acid injection with recovery of 101.1 ± 2.2%.     

Electrocatalytic properties of [Ru(bpy)(tpy)Cl]PF6 at carbon ceramic electrode modified with nafion sol-gel composite: application to amperometric detection of l-cysteine

A two-step sol-gel technique was used here to prepare a carbon ceramic electrode modified with nafion and [Ru(bpy)(tpy)Cl]PF₆. This involves two steps: first, forming a bulk-modified carbon ceramic electrode with nafion, and then immersing the electrode into a Ru-complex solution (electroless deposition) for a short period of time (5-25 s). Cyclic voltammograms of the resulting surface-modified carbon ceramic electrode show stable and a well-defined redox couple due to Ru(II)/Ru(III) system with surface-confined characteristic. l-Cysteine (CySH) has been chosen as a model to elucidate the electrocatalytic ability of Ru-complex nafion sol-gel composite electrode. Not only the modified electrode shows excellent catalytic activity toward l-cysteine electrooxidation in pH range 3-9, but the antifouling effect of nafion film also increases the reproducibility of results in comparison with CCE modified with homogeneous mixing of graphite powder and Ru-complex (one step sol-gel method). Under the optimized conditions in amperometry method, the concentration calibration range, detection limit and sensitivity were 0.1-100 μM, 20 nM and 50 nA/μM, respectively. The advantages of this modified electrode are good reproducibility, excellent catalytic activity, simplicity of preparation and especially its antifouling properties towards l-cysteine and its oxidation products. Additionally, it is promising as a detector in flow system or chromatography systems.     

Nanoclay and carbon nanotubes as potential synergists of an organophosphorus flame-retardant in poly(methyl methacrylate)

This study explores whether nanoparticles incorporated in polymers always act as synergists of conventional flame-retardant additives. For this purpose, two different filler nanoparticles, namely organically modified layered-silicate clay minerals or nanoclays and multi-walled carbon nanotubes, were incorporated in poly(methyl methacrylate) filled with an organophosphorus flame-retardant that acts through intumescence. Effective dispersion techniques specific to each nanoparticle were utilized and prepared samples were thoroughly characterized for their nanocomposite morphologies. Nanoclays were shown to outperform carbon nanotubes in respect of improving the fire properties of intumescent formulations assessed by cone calorimeter analysis. An intriguing explanation for the observed behaviour was the restriction of intumescence by strong carbon nanotube networks formed on the flaming surfaces during combustion contrary to enhanced intumescent chars by nanoclays. Carbon nanotubes surpassed nanoclays considering the thermal stability of intumescent formulations in thermogravimetry whereas mechanical properties were significantly superior with nanoclays to those with carbon nanotubes.     

Structure and properties of porous composite membranes of regenerated silk fibroin and poly(vinyl alcohol) and biosensing of glucose via Meldola blue dispersed in polyester ionomer as an electron transfer mediator

Porous composite membranes of regenerated silk fibroin and poly(vinyl alcohol) were prepared by adding polyethyleneglycol to the composite solution to reduce the mass-transfer resistance to the diffusion of substrate material transport; their surfaces were visualized with scanning electron microscopy. An amperometric glucose biosensor employing Meldola blue dispersed in polyester ionomer as electron transfer mediator was prepared to test the feasibility and workability of the composite membrane as immobilization matrix for glucose oxidase. The cationic exchange property of the polyester ionomer was employed to provide high local concentrations of Meldola blue (MB⁺) in the polymer film via ion exchange. Performance and characteristics of the glucose biosensor were evaluated with respect to response time, detection limit, applied potential, thickness of polyester ionomer membrane, pH and temperature. The glucose biosensor possesses a variety of advantages including easy maintenance of enzyme, simplicity of construction, fast response time and high stability. Received: 13 May 1996 / Revised: 30 July 1996 / Accepted: 2 August 1996     

Characterisation of poly(neutral red) modified carbon film electrodes; application as a redox mediator for biosensors

The polymer redox mediator, poly(neutral red) (PNR), has been synthesised and characterised electrochemically to investigate the best electropolymerisation and mediation conditions for application in enzyme biosensors and to clarify the mechanism of action. Neutral red was electropolymerised by potential cycling on carbon film electrode substrates by allowing the monomer to be oxidised during the full 20 cycles of polymerisation or reducing the positive limit of the potential window after the first 2 cycles to impede monomer oxidation with a view to obtaining longer polymer chains and a lesser degree of branching. Comparison was made with glassy carbon substrates. The PNR films on carbon film electrodes were characterised using cyclic voltammetry and electrochemical impedance spectroscopy, as well as in glucose biosensors prepared with PNR. Glucose oxidase enzyme was immobilised by encapsulation in silica sol-gel and compared with that obtained by cross-linking with glutaraldehyde. The biosensors were evaluated by chronoamperometry in 0.1 M phosphate buffer saline solution, pH 7.0, and showed evidence of electron transfer between the enzyme cofactor flavin adenine dinucleotide and PNR dissolved in the enzyme layer competing with PNR-mediated electrochemical degradation of H₂O₂ formed during the enzymatic process. This paper is dedicated to Professor Dr. Algirdas Vaskelis on the occasion of his 70th birthday.     

Micro-perforated elastomeric poly(dimethylsiloxane) mask fabricated using high-aspect-ratio micro-pillar arrays for spatially defined surface modification: an unconventional method for establishing a microarray platform

Herein, we introduce the fabrication of a micro-perforated elastomeric poly(dimethylsiloxane) (PDMS) mask and employ it for spatially defined surface modification. To fabricate the micro-perforated PDMS mask, high-aspect-ratio micro-pillar arrays having millimeter scale height were first fabricated via direct photopolymerization using a thiol–ene-based UV-curable adhesive. Square pillars (500 × 500 μm) and 200 μm circular pillars with 5 and 12.5 in the aspect ratios, respectively, were successfully fabricated with high pattern fidelity, reaching 2.5mm in height. Next, using the micro-pillar-array platform as a master mold, PDMS prepolymer was cast and polymerized to form an elastomeric PDMS mask having micro-perforation arrays. Alternating hydrophilic and hydrophobic surfaces were successfully obtained by oxidizing PDMS-covered Si wafer with corona discharge. Spatially defined chemical functionalities obtained by selective oxidation and subsequent silanization were clearly distinguished via colorimetric detection methods employing ninhydrin and toluidine reagents. The micro-perforated elastomeric PDMS mask enables selective modification of a surface without utilizing photoreactive chemicals and a photomask. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.