Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol-gel membrane for glucose and ATP detections

The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins.     

Application of a quinohaemoprotein alcohol dehydrogenase in bio-electrocatalysis: Immobilization of the enzyme and mediated electron transfer between the enzyme and an electrode

Summary Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni was immobilized on polypyrrole-coated track-etch and microporous membranes. On the track-etch membrane, 3.4 to 4.8 × 10^–3 Units of enzyme/cm² was immobilized whilst on the microporous membrane 0.05 U/cm² was immobilized. The track-etch membrane was then used in electrochemical studies using ferricyanide as a redox mediator giving a maximum catalytic current of 0.022 mA/cm² membrane with 1-pentanol as the substrate. The kinetic parameters (K_m and V_max) of the immobilized enzyme are of the same order of magnitude as those of the free enzyme.     

Fabrication of polymeric electron-transfer mediator/enzyme hydrogel multilayer on an Au electrode in a layer-by-layer process

The layer-by-layer (LBL) construction of an enzyme electrode covered with a multilayer structure alternately composed of a polymeric electron transfer mediator and a polymer-modified enzyme was examined. Poly(2-methacryloyloxyethyl phosphorylcholine-co-p-vinylphenylboronic acid-co-vinylferrocene) (PMVF) was synthesized and used as a polymeric electron transfer mediator. Glucose oxidase (GOx) was selected as a model enzyme and poly(vinyl alcohol) (PVA) chains were bound to the GOx (GOx-PVA) under mild conditions. The PMVF and PVA formed a gel spontaneously through a selective reaction between phenylboronic acid units and hydroxyl groups in both polymers. Using the spin coating technique, a repeating PMVF/GOx-PVA multilayer was fabricated on the surface of an Au electrode. The thickness of each PMVF/GOx-PVA layer was around 5.8 nm, corresponding to the dimensions of GOx. The electrochemical performance of the electrode was evaluated in glucose concentration measurement. The oxidation current of glucose by GOx was measured at 0.38 V (vs. Ag/AgCl), verifying that ferrocene units in the PMVF of the hydrogel electrically wired the immobilized GOx. Moreover, the current increased with the number of PMVF/GOx-PVA layers. That is, both intermolecular electron transfer between each individual layer and the presence of a freely diffusing substrate in the hydrogel were achieved. We conclude that a LBL structure constructed from PMVF and a PVA-modified enzyme is effective for use in developing bioelectronic devices that employ enzyme molecules.     

Immobilization of glucoamylase on ceramic membrane surfaces modified with a new method of treatment utilizing SPCP-CVD

Glucoamylase, as a model enzyme, was immobilized on a ceramic membrane modified by surface corona discharge induced plasma chemical process-chemical vapor deposition (SPCP-CVD). Characterizations of the immobilized enzyme were then discussed. Three kinds of ceramic membranes with different amounts of amino groups on the surface were prepared utilizing the SPCP-CVD method. Each with 1-time, 3-times and 5-times surface modification treatments and used for supports in glucoamylase immobilization. The amount of immobilized glucoamylase increased with the increase in the number of surface modification treatments and saturated to a certain maximum value estimated by a two-dimensional random packing. The operational stability of the immobilized glucoamylase also increased with the increase in the number of the surface treatment. It was almost the same as the conventional method, while the activity of immobilized enzyme was higher. The results indicated the possibility of designing the performance of the immobilized enzyme by controlling the amount of amino groups. The above results showed that the completely new surface modification method using SPCP was effective in modifying ceramic membranes for enzyme immobilization.     

Stable mediated amperometric biosensors using a graphite electrode embedded with tetrathiafulvalene in silicone oil

A new method has been developed to incorporate the mediator, tetrathiafulvalene (TTF), to the electrode/solution interface of an amperometric biosensor. TTF was dissolved in methylphenyl polysiloxane (silicone oil) and embedded in a graphite disc electrode. The mediator was able to diffuse to the electrode surface at an electrocatalytically significant speed. The storage of TTF in the inert polysiloxane provided a long-lasting and stable mediator supply.

TTF-silicone oil electrodes with immobilized glucose oxidase, xanthine oxidase, or amino acid oxidase exhibited sensitive, fast and reproducible responses. The glucose oxidase electrode was very stable for at least 2 months when stored at 4°C. Together with flow injection analysis (FIA), the enzyme electrodes were reused for at least 500 repeated analyses during a 25 h operation without losing their initial activity.     

Imaging of electrochemical enzyme sensor on gold electrode using surface plasmon resonance

Three types of imaging, namely layer structure, electrochemical reaction, and enzyme sensor response, were achieved by applying surface plasmon resonance (SPR) measurement to an electrochemical biosensor. We constructed glucose oxidase based mediator type sensors on a gold electrode by spotting the mediator that contained horseradish peroxidase and spin coating the glucose oxidase film. The layer structure of the sensor was imaged by means of angle scanning SPR measurement. The single sensor spot (about 1 mm in diameter) consisted of about 100 x 100 pixels and its spatial structure was imaged. The multilayer structure of the enzyme sensor had a complex reflectance-incident angle curve and this required us to choose a suitable incident angle for mapping the redox state. We chose an incident angle that provided the most significant reflection intensity difference by using data obtained from two angle scanning SPR measurements at different electrode potentials. At this incident angle, we controlled the electrochemical states of the spotted mediator in cyclic voltammetry and imaged the degree to which the charged site density changed. Finally, we mapped the enzymatic activity around the mediator spot by the enzymatic reoxidation of pre-reduced mediator in the presence of glucose.     

Covalent linkage of CYP101 with the electrode enhances the electrocatalytic activity of the enzyme: Vectorial electron transport from the electrode

Direct electrochemical transfer of electrons to the enzyme provides an excellent method of driving the catalytic reactions of cytochrome P450 enzymes that form a superfamily of vital heme enzymes involved in biological monooxygenation reactions. Covalent attachment of N-(1-pyrenyl) maleimide (pyrene maleimide) to the bacterial cytochrome P450, CYP101 has been carried out and the conjugated enzyme was shown to be specifically immobilized onto the glassy carbon electrode through the pyrene group. The electrode immobilized pyrene-conjugated enzyme showed quasi-reversible electrochemistry with a midpoint potential at −330 ± 10 mV versus Ag/AgCl. The unconjugated enzyme that did not have specific linkage with the pyrene maleimide was non-specifically adsorbed on the electrode surface and the electrochemical response was much weaker than that observed in case of the conjugated enzyme, though the midpoint potential was almost unchanged. The pyrene maleimide bound CYP101 was found to have surface coverage of 1.35 ± 0.3 × 10⁻¹⁰ mol/cm² and the heterogeneous rate of electron transfer was found to be 0.21 ± 0.02 s⁻¹, which is larger than that for the unconjugated enzyme. The pyrene maleimide linked immobilized enzyme was oriented to the electrode so that efficient electron transfer takes place from the electrode to the immobilized enzyme. The oxygenase activity of the immobilized conjugated enzyme was assayed from the enhancement of catalytic current in presence of oxygen and the natural substrate camphor. Mass spectrometric studies also showed enhanced formation of hydroxycamphor by electrochemically driven catalysis in the pyrene maleimide linked immobilized CYP101.     

Immobilization of glucose oxidase in conducting graft copolymers and determination of glucose amount in orange juices with enzyme electrodes

Glucose oxidase was immobilized in conducting copolymers of three different types of poly(methyl methacrylate-co-thienyl methacrylate). Immobilization of enzyme was carried out by the entrapment in conducting polymers during electrochemical polymerization of pyrrole on the copolymer electrodes. Maximum reaction rate, Michaelis-Menten constants, temperature, pH and operational stabilities were determined for immobilized enzyme. The amount of glucose in orange juices of Turkey was investigated by using enzyme electrodes.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.     

Immobilized glucose dehydrogenase for cofactor regeneration in heme-catalyzed demethylation and hydroxylation reactions

Glucose dehydrogenase (E.C. 1.1.1.47) from B. megaterium M 1286 was immobilized together with mutarotase (E.C. 5.1.3.3) on several organic carriers and by different methods. The storage stability of the enzyme at pH-values > 6 is slightly improved by immobilization and the pH-optimum is shifted from 8.3 to 8.0. Kinetic constants of the immobilized enzyme are: K′_M(NAD⁺) = 5.36 × 10^?4 mol/l K′_M(glucose) = 3.76 · 10^?2 mol/l and V′_max = 5.54 · 10^?5 mol/(l min g carrier) for the most active preparation (2.16 mg enzyme/g carrier). In reactor experiments the immobilized glucose dehydrogenase was used with glucose to regenerate NADPH in NADPH-dependent iron-III-protoporphyrin-IX-imidazole catalyzed hydroxylation and demethylation of model substrates of cytochrome P-450. The advantages of the coupling of both reactions with cofactor recycling are shown and discussed.     

Detection of two distinct substrate-dependent catabolic responses in yeast cells using a mediated electrochemical method

Mediated electrochemical detection of catabolism in prokaryotic cells is well documented; however, the application of this technique to eukaryotic cells has received less attention. Two catabolic substrate-dependent mediated electrochemical signals were detected in the yeast Saccharomyces cerevisiae. The signal using a single hydrophilic mediator (ferricyanide) is small whereas the response using a double mediator system comprising a hydrophilic and a lipophilic mediator (ferricyanide and menadione) is up to three orders of magnitude larger. The behaviour of each response during cell ageing is different: the single mediator response increases whereas the double mediator response decreases. This difference indicates that the two signals originate at different points in the catabolic pathways. In S. cerevisiae the double mediator response is proposed to originate from the reduction of the lipophilic mediator by NADPH produced in the pentose phosphate pathway. The single mediator signal arises from reduction of the hydrophilic mediator by an extracellular redox species produced in response to the presence of glucose.     

Application of an Electrochemical NAD+ Recycling System Involving a String-Like Carbon Fiber to an Enzyme Reactor

A string-like carbon fiber was found to be very suitable as a working electrode material for direct electrochemical oxidation of β-nicotinamide adenine dinucleotide reduced form (NADH), and direct use of it for an enzyme reactor was possible. The electrochemical NAD⁺ recycling system was applied to glucose dehydrogenase (GDH) and to the recombinant formate dehydrogenase (RFDH) reactors. The maximum oxidation current value increased to 3.9 mA in the case of the GDH reactor. The remaining GDH activity after the reaction for 10 h amounted to 57% of the initial level. The remaining NAD⁺ activity amounted to 78% of the initial level. The current efficiency was calculated to be 80%. Furthermore, RFDH, which was more stable than GDH, was applied to the system. The maximum current value reached 5.9 mA. The remaining RFDH activity after reaction for 10 h amounted to 81% of the initial level. The remaining NAD⁺ activity was 78% of the initial level. The current efficiency was calculated to be 73%. Based on these results, both the enzyme and NAD⁺ were found to be acceptably stable in the electrochemical NAD⁺ recycling system.     

Electrocatalysis with a Glucose-Oxidase-immobilized Graphite Electrode

Glucose oxidase was immobilized on the surface of a graphite electrode by irreversible adsorption. An electrocatalytic steady-state current for the oxidation of D-glucose was observed using this electrode in the presence of p-benzoquinone as an electron transfer mediator. The electrocatalytic current at 0.5 V vs. SCE was analyzed as a function of the concentrations of D-glucose and p-benzoquinone, and the maximum current, I_s^max, and the Michaelis constants (K₁ and K₂ for D-glucose and p-benzoquinone, respectively) of the electrocatalysis were determined. The dependence of the current on the electrode potential, pH, and temperature was also investigated. The results indicate that the kinetics of the immobilized enzyme are essentially the same as those of the enzyme in the solubilized state. The effect of various electron transfer mediators on the electrocatalytic current was also examined and evaluated in terms of I_s^max, K₁, and K₂ values.     

Development of Novel Glucose oxidase Immobilization on Graphene/Gold nanoparticles/Poly Neutral red modified electrode

Glucose oxidase (GOx) was immobilized onto glassy carbon electrode (GCE) that modified by reduced graphene oxide-gold nanoparticles- poly neutral red (RGO/AuNPs/PNR) nanocomposite. The composite was analyzed by scanning electron microscope (SEM), energy dispersive x-ray (EDX) spectroscopy, atomic force microscopy (AFM), attenuated total reflectance (ATR), cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). SEM/EDX analysis showed the morphological of the nanocomposite. AFM results showed the morphology and structure of the RGO/AuNPs and RGO surfaces. The covalent bonding between glucose oxidase and composite was confirmed by ATR technique. The electrochemical experiments were done in 100 mM phosphate buffer at pH 7 and temperature of 25 °C with three electrodes including Ag/AgCl, platinum wire and the modified GCE as the reference electrode, the auxiliary electrode and working electrode respectively. The electrochemical results confirmed the activity and direct electron transfer of immobilized enzyme. The immobilized electroactive GOx concentration was estimated 3.06 × 10⁻¹¹ mol cm⁻². The results showed the immobilized enzyme had a good stability and maintained 90% of its performance after two weeks. The nanocomposite bioanode in an air-birthing biofuel cell and 100 mM glucose concentration showed 176 μWcm⁻² Power density. This strategy could be used for GOx-based biofuel cells.     

Glucose/O2 biofuel cell based on enzymes, redox mediators, and multiple-walled carbon nanotubes deposited by AC-electrophoresis then stabilized by electropolymerized polypyrrole

In this study, we developed an automated strategy to manufacture an enzyme BFC powered by glucose/O(2). The bioanode consists of GOx enzyme and PQQ redox mediator adsorbed over night on MWCNTs then deposited by means of AC-electrophoresis at 30 Hz and 160 V(p-p) and, finally stabilized by electropolymerized polypyrrole. The biocathode is constructed from LAc enzyme and ABTS redox mediator adsorbed over night on MWCNTs, then electrophoretically deposited under AC-electric field at 30 Hz and 160 V(p-p) and, finally stabilized by electrodeposited polypyrrole. The BFC was studied under air in phosphate buffer solution pH 7.4 containing 10 mM glucose and in human serum with 5 mM glucose addition at the physiological temperature of 37°C. Under these conditions, the maximum power density reaches 1.1 μW · mm(-2) at a cell voltage of 0.167 V in buffer solution and 0.69 μW · mm(-2) at cell voltage of 0.151 V in human serum. Such automated BFCs have a great potential to be optimized, miniaturized to micro and nanoscale devices suitable for in vivo studies.     

Microfluidic electroporation for delivery of small molecules and genes into cells using a common DC power supply

The end-product profile of the glucose fermentation by Propionibacterium freudenreichii ET-3 changed on an electrochemical treatment, in which the culture vessel was filled with a carbon felt anode. Acetate and propionate were produced as final end products in a molar ratio of 2:3 without any electrochemical treatments at the point of the consumption of lactate as an intermediate of the glucose fermentation. The ratio was changed to 1:1 at the point of the lactate consumption by the electrochemical incubation at an electrode potential of 0.4 V versus Ag|AgCl for 100 h. During further electrochemical incubation, propionate was oxidized to acetate as a final end-product in the microbe-containing anode chamber. 1,4-Dihydroxy-2-naphthoic acid produced by P. freudenreichii ET-3 itself would receive electrons from the metabolic pathway and serve as an electron transfer mediator from the microbial cells to the electrode.     

Electrospun gold nanofiber electrodes for biosensors

A new form of high surface area bioelectrode, based on nanofibers of electrospun gold with immobilized fructose dehydrogenase, was developed. The gold fibers were prepared by electroless deposition of gold nanoparticles on an electrospun poly(acrylonitrile)-HAuCl(4) fiber. The material was characterized using electron microscopy, XRD and BET, as well as cyclic voltammetry and biochemical assay of the immobilized enzyme. The electrochemical surface area of the gold microfibers was 0.32 ± 0.04 m(2)/g. Fructose dehydrogenase was covalently coupled to the gold surface through glutaraldehyde crosslinks to a cystamine monolayer. The enzyme exhibited mediated electron transfer directly to the gold electrode and catalytic currents characteristic of fructose oxidation in the presence of a ferrocene methanol mediator were observed. The limit of detection of fructose was 11.7 μM and the K(M) of the immobilized enzyme was 5mM. The microfiber electrode was stable over 20 cycles with a 3.05% standard deviation. The response time of the sensor was less than 2.2s and reached half maximum value within 3.6s. The sensor was proven to be accurate and precise in both serum and popular beverages sweetened with high fructose corn syrup. The addition of glucose isomerase enabled the sensor to perform with glucose, thus expanding the available analyte selection for the sensor.     

On-line monitoring of glucose in mammalian cell culture using a flow injection analysis (FIA) mediated biosensor

A flow injection analysis (FIA) biosensor system has been developed for on-line determination of glucose during mammalian cell cultivation. The culture sample was peristaltically withdrawn from the bioreactor and after cell separation by a steam sterilizable ceramic microfilter, the filtrate was continuously fed to the FIA mediated-biosensor system at 4 mLh(-1), whereas the cell-containing retentate was recirculated to the bioreactor. In the amperometric biosensor system, glucose oxidase was covalently immobilized onto a preactivated nylon membrane and attached to the sensing area of a platinum working electrode. The enzyme reaction was coupled with the mediator 1,1'-dimethylferricinium (DMFe(+))-cyclodextrin inclusion complex to recycle the reduced glucose oxidase to its original active state. 1,1'-Dimethylferrocene (DMFe) was then reoxidized to DMFe(+) at the surface of the platinum electrode poised at + 0.15 V vs silver/silver chloride. The FIA mediated-biosensor was linear up to 6 mM glucose, with a detection limit of 0.1 mM, and possessed excellent reproducibility (+/- 0.4 %, 95 % confidence interval) over 123 repeated analyses during a 62 h continuous operation. The immobilized glucose oxidase was stable for up to 7 days when applied to glucose measurement during 5-10 day fed-batch cultivation of 293S mammalian cells. The results obtained from the mediated-biosensor system compared well with the hexokinase and HPLC data. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 497-504, 1997.     

Production of high-fructose syrup by a heat-fixed Lactobacillus sp.

A Lactobacillus sp. isolated from soil and capable of growing on xylose-containing medium exhibited high glucose isomerase activity. The enzyme was thermostable, stable toward dialysis, and activated by heat treatment. It did not show the presence of xylose or ribose isomerase activities; the K_m for glucose and xylose substrates were 0.48M and 0.513M, respectively. The heat treatment of ultrasonic crude extract gave insoluble fixed active glucose isomerase enzyme. The properties of free and immobilized enzyme in heat-fixed whole cells differed in many respects. The optimum temperature for enzyme activity changed from 70 to 85°C, the optimum substrate concentration changed from 1.0M to 2.4M, and the optimum pH from 7.4 to 6.0. Co²⁺ and Mg²⁺ ions activated the enzyme when used singly, but in combination they inhibited the enzyme and Mn²⁺ had no effect on the enzyme. Free and immobilized enzymes, when used in the used in the conversions of corn and bagasse hydrolysates to fructose, gave 58, 25.6%, and 50, 27.6% conversions, respectively. Immobilized enzyme retained a significant activity for more than 30 hr and was able to operate at higher glucose concentrations showing less products inhibition effect as compared to free enzyme. In the batch process it was able to operate for about eight cycles.     

Comparison of Immobilized and Free Amyloglucosidase Process in Glucose SyrupsProduction from White Sorghum Starch

Optimum conditions for glucose syrups production from white sorghum were studied through sequential liquefaction and saccharification processes. In the liquefaction process, a maximum dextrose equivalent (DE) of 10.98 % was achieved using 30 % (w/v) of starch and Termamyl ɑ-amylase from Bacillus licheniformis. Saccharification was performed by free and immobilized amyloglucosidase from Rhizopus mold at 1 % (w/v). DE values of 88.32 % and 79.95 % were obtained from 30 % (w/v) of starch with, respectively, free and immobilized enzyme. The immobilized Amyloglucosidase in calcium alginate beads showed reusable capacity for up to 6 cycles with 46 % of the original activity retained. The kinetic behaviour of immobilized and free enzyme gives K_m value of 22.13 and 16.55 mg mL⁻¹ and V_max of 0.69 and 1.61 mg mL⁻¹ min⁻¹, respectively. The hydrolysis yield using immobilized amyloglucosidase were lower than that of the free one. However, it is relevant to reuse enzyme without losing activity in order to trim down the overall costs of enzymatic bioprocesses as starch transformation into required products in industrial manufacturing. Hydrolysis of sorghum starch using immobilized amyloglucosidase represents a promising alternative towards the development of the glucose syrups production process and its utilization in various industries.