Amperometric glucose sensor based on coimmobilization of glucose oxidase and Poly(p-phenylenediamine) at a platinum microdisk electrode

A miniaturized glucose biosensor in which glucose oxidase (GOD) and poly(p-phenylenediamine) (poly-PPD) were coimmobilized at the surface of a platinum microdisk electrode was developed and used successfully for amperometric determination of glucose. The performance of sensors prepared at different monomer concentrations and polymerization potentials with different media was investigated in detail. It was found that similarly to poly(o-phenylenediamine) (poly-OPD), (poly-PPD) noticeably eliminated the electrochemical interference of ascorbic acid, uric acid, and l-cysteine. The amperometric response of glucose with the biosensor under optimal conditions exhibited a linear relationship in the range of 5.0 x 10(-5) to 3.0 x 10(-3) M with correlation coefficient 0.9995. According to the Michaelis-Menten equation, the apparent Michaelis constant for glucose and the maximum steady-state current density of the poly-PPD/GOD-modified microelectrode were 3.94 mM and 607.5 microA cm(-2), respectively. The current density of the sensor responding to glucose in the linear range can reach 160 microA cm(-2) mM(-1), which is far greater than that obtained using poly-OPD and poly(phenol) film. In addition, the stability of the sensor was examined over a 2-month period.     

Electron transferase activity of diaphorase (NADH: acceptor oxidoreductase) from Bacillus stearothermophilus

Electrochemical kinetic measurements were carried out for electron-transfer between NADH and the oxidized forms of mediators (ferrocenylmethanol (FMA), ferrocenyl-1-ethanol (FEA), N,N,N',N'-tetramethylphenylenediamine (TMPD), Co(Phen)2+(3) and Fe(CN)4-(6)) catalyzed by diaphorase (NADH: acceptor oxidoreductase, EC 1.6.99.-) purified from Bacillus stearothermophilus. Cyclic voltammograms for the mediators with excess NADH in the presence of diaphorase gave steady-state currents. The quantitative analysis of the dependence of the current on the mediator concentration yielded a Michaelis constant (Km) and molecular activity (ko), which are difficult to determine by the conventional spectrophotometric method. Small Km and large ko values were observed for the oxidized forms of FMA, FEA and TMPD compared to those for Co(Phen)3+(3) and Fe(CN)3-(6). It is suggested that the reaction pocket of the present diaphorase is hydrophobic. The present electrochemical procedure for the determination of the kinetic parameters is applicable widely to similar enzyme reactions.     

Electrochemical study of ferrocenemethanol-modified layered double hydroxides composite matrix: application to glucose amperometric biosensor

A novel amperometric glucose sensor based on co-immobilization of ferrocenemethanol (MeOHFc) and glucose oxidase (GOD) in the layered double hydroxides (LDHs) was described. MeOHFc immobilized in LDHs played effectively the role of an electron shuttle and allowed the detection of glucose at 0.25 V (versus SCE), with dramatically reduced interference from easily oxidizable constituents. The sensor (LDHs/MeOHFc/GOD) exhibited a relatively fast response (response time was about 5s), low detection limit (3 microM), and high sensitivity (ca. 60 mA M(-1)cm(-2)) with a linear range of 6.7 x 10(-6) to 3.86 x 10(-4)M of glucose. Apparent Michaelis-Menten constant was calculated to be 2.25 mM.     

Enzymatically induced formation of neodymium hexacyanoferrate nanoparticles on the glucose oxidase/chitosan modified glass carbon electrode for the detection of glucose

The formation of neodymium hexacyanoferrate (NdHCF) nanoparticles (NPs) on the surface of glucose oxidase/chitosan (GOx/CHIT) modified glass carbon electrode induced by enzymatic reaction was described and characterized. CHIT can be used not only as enzyme immobilizer, but also to provide active sites for NPs growth. Results showed that the optimized conditions of the GOx/CHIT film induced NdHCF NPs for the biosensing of glucose were 1.0mM Nd(3+) and 20.0mM Fe(CN)(6)(3-). The biocatalyzed generation of NdHCF NPs enabled the development of an electrochemical biosensor for glucose. The calculated apparent Michaelis-Menten constant was 7.5mM. The linear range for glucose detection was 0.01-10.0mM with the correlation coefficient of 0.9946, and the detection limit was 5muM (S/N=3). Furthermore, this system avoids the interferences of other species during the biosensing process and can be used for the determination of glucose in human plasma samples.     

The carbon paste electrode encrusted with a microreactor as glucose biosensor.

The amperometric biosensors based on carbon paste electrodes (CPEs) encrusted with single microreactor (MR) have been constructed for the determination of glucose. The MRs were prepared from CPC-silica carrier (CPC) and were loaded with glucose oxidase (GO), mediator (M) and acceptor (A). As the mediator cation radical of 5,10-dimethyl-5, 10-dihydrophenazine (DMDHP), N-methylphenazonium methyl sulfate (PMS) and o-benzoquinone (BQ) and as the acceptor Fe[EDTA]- or Fe(CN)6(3-) was used. The biosensors acted at electrode potential 0.15-0.27 V versus Ag-AgCl electrode. The calibration graphs of the biosensors were linear in the range from 1.5 to 50 mM of glucose. The sensitivity of the biosensors did not change at pH 6-8. The dissolved oxygen little (7%) influenced the biosensors response and 1 mM of ascorbic acid produced the response that was of equal value to 0.5 mM of glucose. The biosensors showed high stability; no change of the response of the biosensors prepared by using the novel microreactor was observed at least for 6 months by keeping the loaded CPC at room temperature in silica container. An optimization of the biosensors response against the GO, the mediator and the polymer amount was performed. The digital modeling of the biosensors action is following.     

Voltammetric detection of inorganic phosphate using ion-channel sensing with self-assembled monolayers of a hydrogen bond-forming receptor

A voltammetric ion-channel sensing for phosphate based on gold electrodes modified with the self-assembled monolayers of a bis-thiourea receptor was developed to detect phosphate. The working principle of this voltammetric sensor conceptually mimics that of ligand gated ion-channel proteins, as to chemically stimulated changes in membrane permeability. The response to analytes is based on the change in electron transfer rate constant of the redox reaction of [Fe(CN)(6)](4-/3-) marker, before and after binding of phosphate to the receptor on the electrode surface; where the electrostatic repulsion between a phosphate-receptor complex and the marker induced the decrease in the rate constant. In a solution of pH 7.0, a high selectivity was observed for phosphate and the sensor was virtually insensitive at all to many of other anions, such as SO(4)(2-), AcO(-), NO(3)(-), and Cl(-). The sensor response was obtained with phosphate concentrations above 5.0 x 10(-4) M using cyclic voltammetry and differential pulse voltammetry.     

Amperometric glucose biosensor with extended concentration range utilizing complexation effect of borate.

Borate buffer strongly decreases amperometric response of a glucose oxidase linked pO2 or H2O2 sensing electrode, extending substantially its linear calibration range. With increasing pH and concentration of the buffer the upper limit for glucose can be varied between 1 and 30 mmol l-1 glucose. The effect of borate ion is explained by the rapid complexation of glucose decreasing the equilibrium concentration of free beta-anomer, the specific substrate of glucose oxidase. The high loading of cross-linked enzyme inside the sensor membrane is necessary for the measurement to ensure an almost constant response factor (delta i per 1 mmol l-1) between pH 5 and 10. Analysis in stirred solution and in a flow-through system has been employed for the measurement of elevated glucose levels in heparinized human blood or plasma samples.     

Glucose metabolism of lactic acid bacteria changed by quinone-mediated extracellular electron transfer

It can be expected that extracellular electron transfer to regenerate NAD+ changes the glucose metabolism of the homofermentative lactic acid bacteria. In this work, the glucose metabolism of Lactobacillusplantarum and Lactococcus lactis was examined in resting cells with 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as the electron transfer mediator and ferricyanide (Fe(CN)6(3-)) as the extracellular electron acceptor. NADH in the cells was oxidized by ACNQ with the aid of diaphorase, and the reduced ACNQ was reoxidized with Fe(CN)6(3-). The extracellular electron transfer system promoted the generation of pyruvate, acetate, and acetoin from glucose, and restricted lactate production. Diaphorase activity increased when cultivation was aerobic, and this increased the concentrations of pyruvate, acetate, and acetoin relative to the concentration of lactate to increase in the presence of ACNQ and Fe(CN)6(3-)     

A porous poly(acrylonitrile-co-acrylic acid) film-based glucose biosensor constructed by electrochemical entrapment

A novel glucose biosensor was constructed by electrochemical entrapment of glucose oxidase (GOD) into porous poly(acrylonitrile-co-acrylic acid), which was synthesized via radical polymerization of acrylonitrile and acrylic acid. The obtained biosensor showed a better stability and higher sensitivity than the biosensor prepared by simple physical adsorption. Effects of some experimental variables such as immobilization time, enzyme concentration, pH, applied potential, and temperature on the amperometric response of the sensor were investigated. The biosensor exhibited a rapid response to glucose (< 30s) with a linear range of 5 x 10(-6) to 3 x 10(-3)M and a sensitivity of 6.82 mAM(-1)cm(-2). The apparent Michaelis-Menten constant (K(M)(app)) was 7.3mM.     

An acoustic glucose sensor

In vivo glucose monitoring is required for tighter glycaemic control. This report describes a new approach to construct a miniature implantable device based on a magnetic acoustic resonance sensor (MARS). A ≈ 600-800 nm thick glucose-responsive poly(acrylamide-co-3-acrylamidophenylboronic acid) (poly(acrylamide-co-3-APB)) film was polymerised on the quartz disc (12 mm in diameter and 0.25 mm thick) of the MARS. The swelling/shrinking of the polymer film induced by the glucose binding to the phenylboronate caused changes in the resonance amplitude of the quartz disc in the MARS. A linear relationship between the response of the MARS and the glucose concentration in the range ≈ 0-15 mM was observed, with the optimum response of the MARS sensor being obtained when the polymer films contained ≈ 20 mol% 3-APB. The MARS glucose sensor also functioned under flow conditions (9 μl/min) with a response almost identical to the sensor under static or non-flow conditions. The results suggest that the MARS could offer a promising strategy for developing a small subcutaneously implanted continuous glucose monitor.     

Electron transfer kinetics and mechanistic study of the thionicotinamide coordinated to the pentacyanoferrate(III)/(II) complexes: a model system for the in vitro activation of thioamides anti-tuberculosis drugs

The mechanism of activation thioamide-pyridine anti-tuberculosis prodrugs is poorly described in the literature. It has recently been shown that ethionamide, an important component of second-line therapy for the treatment of multi-drug-resistant tuberculosis, is activated through an enzymatic electron transfer (ET) reaction. In an attempt to shed light on the activation of thioamide drugs, we have mimicked a redox process involving the thionicotinamide (thio) ligand, investigating its reactivity through coordination to the redox reversible [Fe(III/II)(CN)(5)(H(2)O)](2-/3-) metal center. The reaction of the Fe(III) complex with thionicotinamide leads to the ligand conversion to the 3-cyanopyridine species coordinated to a Fe(II) metal center. The rate constant, k(et)=10 s(-1), was determined for this intra-molecular ET reaction. A kinetic study for the cross-reaction of thionicotinamide and [Fe(CN)(6)](3-) was also carried out. The oxidation of thionicotinamide by [Fe(CN)(6)](3-) leads to formation of mainly 3-cyanopyridine and [Fe(CN)(6)](4-) with a k(et)=(5.38+/-0.03) M(-1)s(-1) at 25 degrees C, pH 12.0. The rate of this reaction is strongly dependent on pH due to an acid-base equilibrium related to the deprotonation of the R-SH functional group of the imidothiol form of thionicotinamide. The kinetic results reinforced the assignment of an intra-molecular mechanism for the ET reaction of [Fe(III)(CN)(5)(H(2)O)](2-) and the thioamide ligand. These results can be valuable for the design of new thiocarbonyl-containing drugs against resistant strains of Mycobacterium tuberculosis by a self-activating mechanism.     

Iron oxide nanoparticles-chitosan composite based glucose biosensor

Iron oxide (Fe(3)O(4)) nanoparticles prepared using co-precipitation method have been dispersed in chitosan (CH) solution to fabricate nanocomposite film on indium-tin oxide (ITO) glass plate. Glucose oxidase (GOx) has been immobilized onto this CH-Fe(3)O(4) nanocomposite film via physical adsorption. The size of the Fe(3)O(4) nanoparticles estimated using X-ray diffraction (XRD) pattern and transmission electron microscopy (TEM) has been found to be approximately 22 nm. The CH-Fe(3)O(4) nanocomposite film and GOx/CH-Fe(3)O(4)/ITO bioelectrode have been characterized using UV-visible and Fourier transform infrared (FTIR) spectroscopic and scanning electron microscopy (SEM) techniques, respectively. This GOx/CH-Fe(3)O(4)/ITO nanocomposite bioelectrode has response time of 5s, linearity as 10-400 mgdL(-1) of glucose, sensitivity as 9.3 microA/(mgdLcm(2)) and shelf life of about 8 weeks under refrigerated conditions. The value of Michaelis-Menten (K(m)) constant obtained as 0.141 mM indicates high affinity of immobilized GOx towards the substrate (glucose).     

Improved specificity of reagentless amperometric PQQ-sGDH glucose biosensors by using indirectly heated electrodes

Indirectly heated electrodes operating in a non-isothermal mode have been used as transducers for reagentless glucose biosensors. Pyrroloquinoline quinone-dependent soluble glucose dehydrogenase (PQQ-sGDH) was entrapped on the electrode surface within a redox hydrogel layer. Localized polymer film precipitation was invoked by electrochemically modulating the pH-value in the diffusion zone in front of the electrode. The resulting decrease in solubility of an anodic electrodeposition paint (EDP) functionalized with Osmium complexes leads to precipitation of the redox hydrogel concomitantly entrapping the enzyme. The resulting sensor architecture enables a fast electron transfer between enzyme and electrode surface. The glucose sensor was operated at pre-defined temperatures using a multiple current-pulse mode allowing reproducible indirect heating of the sensor. The sensor characteristics such as the apparent Michaelis constants K(M)(app) and maximum currents I(max)(app) were determined at different temperatures for the main substrate glucose as well as a potential interfering co-substrate maltose. The limit of detection increased with higher temperatures for both substrates (0.020 mM for glucose, and 0.023 mM for maltose at 48 degrees C). The substrate specificity of PQQ-sGDH is highly temperature dependent. Therefore, a mathematical model based on a multiple linear regression approach could be applied to discriminate between the current response for glucose and maltose. This allowed accurate determination of glucose in a concentration range of 0-0.1mM in the presence of unknown maltose concentrations ranging from 0 to 0.04 mM.     

Biosensor based on polyaniline-Prussian Blue/multi-walled carbon nanotubes hybrid composites

In this work, a novel route for fabrication polyaniline (PANI)-Prussian Blue (PB) hybrid composites is proposed by the spontaneous redox reaction in the FeCl(3)-K(3)[Fe(CN)(6)] and the aniline solution. With the introduction of multi-walled carbon nanotubes (MWNTs), the PANI-PB/MWNTs system shows synergy between the PANI-PB and MWNTs which amplified the H(2)O(2) sensitivity greatly. A linear range from 8 x1 0(-8) to 1 x 10(-5)M and a high sensitivity 508.1 8 microA microM cm(2) for H(2)O(2) detection are obtained. The composites also show good stability in neutral solution. A glucose biosensor was further constructed by immobilizing glucose oxidase (GOD) with Nafion and glutaraldehyde on the electrode surface. The performance factors influencing the resulted biosensor were studied in detail. The biosensor exhibits excellent response performance to glucose with the linear range from 1 to 11 mM and a detection limit of 0.01 mM. Furthermore, the biosensor shows rapid response, high sensitivity, good reproducibility, long-term stability and freedom of interference from other co-existing electroactive species.     

Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase

In this work, a novel chemiluminescence (CL) flow biosensor for glucose was proposed. Glucose oxidase (GOD), horseradish peroxidase (HRP) and gold nanoparticles were immobilized with sol-gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and H(2)O(2), and then the generated H(2)O(2) oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The enhanced effect was closely related to the sizes of gold colloids, and the smaller the size of gold colloids had the higher CL respond. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0 x 10(-5)molL(-1) to 1.0 x 10(-3)molL(-1), and the detection limit was 5 x 10(-6)molL(-1) (3sigma). The apparent Michaelis-Menten constant of GOD in gold nanoparticles/sol-gel matrix was evaluated to be 0.3mmolL(-1), which was smaller than that of GOD immobilized in sol-gel matrix without gold nanoparticles. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

Role of the glucose cycle in control of net glucose flux in exercising humans

The hepatic glucose cycle involves the production of plasma glucose from glucose 6-phosphate and the simultaneous conversion of glucose back to glucose 6-phosphate. We have evaluated the role of the glucose cycle in the regulation of plasma glucose concentration during exercise at 70% of maximal O2 uptake and during recovery in five normal volunteers. Total glucose flux was measured by use of [2-2H]glucose (Ra2), net glucose flux through the glucose cycle was determined with [6,6-2H2]glucose (Ra6), and the rate of glucose cycling was determined by Ra2 - Ra6. Gas chromatography-mass spectrometry was used for analysis of isotopic enrichment. At rest, 33% of total glucose flux was recycled. In exercise, total flux increased 300%, but so did glucose cycling, which means that there was no change in the percentage of flux recycled. In recovery, both total flux and the rate of recycling returned rapidly to the resting value. We therefore conclude that whereas total glucose production can respond extremely quickly to large changes in energy requirements caused by exercise, thereby enabling maintenance of a constant blood glucose concentration, glucose cycling does not have an important role in amplifying the control of net hepatic glucose flux through the glucose cycle.     

Bacterial sensors based on Acidithiobacillus ferrooxidans Part I. Fe2+ and S2O32- determination

An amperometric bacterial sensor with current response to Fe(2+) and S(2)O(3)(2-) ions has been designed by immobilizing an acidophilic biomass of Acidithiobacillus ferrooxidans on a multi disk flat-front oxygen probe. The bacterial layer was located between the oxygen probe and a membrane of cellulose. A filtration technique was used to yield the bacterial membranes having reproducible activity. The decrease of O(2) flow across the bacterial layer is proportional to the concentration of the dosed species. The dynamic range appeared to be linear for the Fe(2+) ions up to 2.5 mmol L(-1) with a detection limit of 9 x 10(-7) mol L(-1) and a sensitivity of 0.25 A L mol(-1). The response of the biosensor is 84 s for a determination of 2 x 10(-4) mol L(-1) Fe(2+). Optimizing the Fe(2+) determination by A. ferrooxidans sensor was carried out owing to Design of Experiments (DOE) methodology and empirical modelling. The optimal response was thus obtained for a pH of 3.4, at 35 degrees C under 290 rpm solution stirring. S(2)O(3)(2-) concentration was determined at pH 4.7, so avoiding its decomposition. The concentration range was linear up to 0.6 mmol L(-1). Sensitivity was 0.20 A L mol(-1) with a response time of 207 s for a 2 x 10(-4) mol L(-1) S(2)O(3)(2-) concentration.     

Minimizing tissue-material interaction in microsensor for subcutaneous glucose monitoring

A new implantable electrocatalytic glucose sensor for subcutaneous glucose monitoring has been fabricated by immobilizing glucose oxidase on a chemically modified carbon fiber. The sensor was inserted subcutaneously on a male spraguely rat without any incision after dipping the microsensor in the rat's serum for 3 days. The so called "stained" microsensor, operated in the amperometric mode with an applied potential of +0.23 V versus Ag|AgCl, was able to directly measure the glucose concentration upon infusion of glucose. The results obtained were encouraging, with the response time was less than 2s and the apparent Michaelis-Menten value at 5.1+/-0.5mM. The "stained" microsensor shows good stability and reproducibility with constant response spanned over 25 days. Most common interferences in glucose analysis were minimized by the outerlayer Nafion. Hematology examinations showed minimal material-tissue interaction. Use of such mechanical devices will allow a more refined understanding towards glucose control in diabetic patients as the implanted microsensor was not effected by biocompatibility failures.     

A bioelectrochemical polypyrrole-containing Fe(CN)6(3-) interface for the design of a NAD-dependent reagentless biosensor

Ferricyanide ions were immobilized on a platinum electrode surface by means of an electrochemically grown polypyrrole film. The entrapped Fe(CN)6(3-)/Fe(CN)6(4-) redox system displayed a high heterogeneous electron transfer rate. The resulting modified electrode was efficient for the ferricyanide-mediated NADH oxidation catalyzed by a diaphorase. The bioelectrochemical interface was applied to the design of a reagentless amperometric D-lactate biosensor. A weakly polarized two polypyrrole-containing Fe(CN)6(3-) modified electrode system was involved without any reference. An enzymatic solution containing D-lactate dehydrogenase, diaphorase and NAD-dextran was further confined on the sensing electrode using a semi-permeable membrane. The sensitivity and the response time of the reagentless biosensor were similar to those of the analogous sensor working with soluble mediator and cofactor, i.e. 25 microA mM(-1) cm(-2) and 120 s, respectively. The other analytical performances were less satisfactorily: the detection limit was 5 x 10 mmol L(-1) and the linearity range was comprised between 0.1 and 0.5 mmol L(-1).     

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.