Application of screen-printed microband biosensors to end-point measurements of glucose and cell numbers in HepG2 cell culture

Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37 °C, at an operating potential of +0.4 V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4 nA/mM and a linear range of 0.45 to 9.0 mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55 kJ mol⁻¹ was calculated. In culture medium (pH 7.3), a sensitivity of 13 nA/mM was obtained and the response was linear up to 5 mM with a detection limit of 0.5 mM. The working concentration was up to 20 mM glucose with a precision of 11.3% for replicate biosensors (n = 4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400 s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R² = 0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20 mM, with those determined spectrophotometrically (R² = 0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9 nmol/(10⁶ cells min) based on a 24-h period in culture.     

Simultaneous monitoring of glucose and lactate by an interference and cross-talk free dual electrode amperometric biosensor based on electropolymerized thin films.

An interference and cross-talk free dual electrode amperometric biosensor integrated with a microdialysis sampling system is described, for simultaneous monitoring of glucose and lactate by flow injection analysis. The biosensor is based on a conventional thin layer flow-through cell equipped with a Pt dual electrode (parallel configuration). Each Pt disk was modified by a composite bilayer consisting of an electrosynthesised overoxidized polypyrrole (PPYox) anti-interference membrane covered by an enzyme entrapping gel, obtained by glutaraldehyde co-crosslinking of glucose oxidase or lactate oxidase with bovine serum albumin. The advantages of covalent immobilization techniques were coupled with the excellent interference-rejection capabilities of PPYox. Ascorbate, cysteine, urate and paracetamol produced lactate or glucose bias in the low micromolar range; their responses were, however, completely suppressed when the sample was injected through the microdialysis unit. Under these operational conditions the flow injection responses for glucose and lactate were linear up to 100 and 20 mM with typical sensitivities of 9.9 (+/- 0.1) and 7.2 (+/- 0.1) nA/mM. respectively. The shelf-lifetime of the biosensor was at least 2 months. The potential of the described biosensor was demonstrated by the simultaneous determination of lactate and glucose in untreated tomato juice samples; results were in good agreement with those of a reference method.     

A new set up for multi-analyte sensing: at-line bio-process monitoring

A multi-analyte sensing device is described, for simultaneous at-line monitoring of glucose, ethanol, pO?-value and cell density. It consists of a dual biosensor, a modified microscope and a fiber optical pO?-sensor that are integrated into a flow analysis (FA) system. The biosensor is based on a conventional thin layer flow-through cell equipped with a gold (Au) dual electrode (serial configuration). The biosensors with no cross-talking were produced by modifying the electrochemical transducers. Each Au surface was initially modified by self-assembled monolayer (SAM) of cysteamine. Alcohol oxidase (AOx) and pyranose oxidase (PyOx) were immobilized each onto a gold surface by means of PAMAM (polyamidoamine) dendrimer via glutaraldehyde cross-linking. The responses for glucose and ethanol were linear up to 0.5 mM. The operational stability of the biosensors was very promising, after 11 h continuous operation, only 6.0% of the initial activity was lost. The potential of the described biosensor was demonstrated by parallel determination of ethanol and glucose in yeast fermentation process. Simultaneously the cell density of the culture was monitored with an in situ microscope (ISM), which was integrated into the FA system. Both the used in situ microscope and the image processing algorithm used for the analysis of the acquired image data are described. Furthermore the pO?-value was monitored using a fiber optical sensor, which was embedded in a flow cell. The multi-sensor device allows the at-line monitoring of several process values without the need for further sampling or time consuming offline measurements.     

Fabrication of microband glucose biosensors using a screen-printing water-based carbon ink and their application in serum analysis

Microband glucose biosensors were fabricated by screen-printing a water-based carbon ink formulation containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, then insulating and sectioning through the thick (20mum) film to expose a 3mm-long working electrode edge. The performance of these biosensors for glucose analysis was investigated at 25 degrees C. Voltammetry in glucose-containing buffer solutions established that an operating potential of +0.4V vs. Ag/AgCl was suitable for analysis under both stirring and quiescent conditions. The influence of pH on biosensor performance was established and an operational pH of 8.0 was selected. Steady-state responses were obtained under quiescent conditions, suggesting a mixed mechanism predominated by radial diffusion, indicative of microelectrode behaviour. Calibration studies obtained with these biosensors showed steady-state currents that were linearly dependent on glucose concentration from the limit of detection (0.27mM) up to 2.0mM, with a precision for replicate biosensors of 6.2-10.7%. When applied to the determination of glucose in human serum, the concentration compared favourably to that determined by a spectroscopic method. These results have demonstrated a simple means of fabricating biosensors for glucose measurement and determination in situations where low-current real-time monitoring under quiescent conditions would be desirable.     

Study on Different Molecular Weights of Chitosan as an Immobilization Matrix for a Glucose Biosensor

Two chitosan samples (medium molecular weight (MMCHI) and low molecular weight (LMCHI)) were investigated as an enzyme immobilization matrix for the fabrication of a glucose biosensor. Chitosan membranes prepared from acetic acid were flexible, transparent, smooth and quick-drying. The FTIR spectra showed the existence of intermolecular interactions between chitosan and glucose oxidase (GOD). Higher catalytic activities were observed on for GOD-MMCHI than GOD-LMCHI and for those crosslinked with glutaraldehyde than using the adsorption technique. Enzyme loading greater than 0.6 mg decreased the activity. Under optimum conditions (pH 6.0, 35°C and applied potential of 0.6 V) response times of 85 s and 65 s were observed for medium molecular weight chitosan glucose biosensor (GOD-MMCHI/PT) and low molecular weight chitosan glucose biosensor (GOD-LMCHI/PT), respectively. The apparent Michaelis-Menten constant () was found to be 12.737 mM for GOD-MMCHI/PT and 17.692 mM for GOD-LMCHI/PT. This indicated that GOD-MMCHI/PT had greater affinity for the enzyme. Moreover, GOD-MMCHI/PT showed higher sensitivity (52.3666 nA/mM glucose) when compared with GOD-LMCHI/PT (9.8579 nA/mM glucose) at S/N>3. Better repeatability and reproducibility were achieved with GOD-MMCHI/PT than GOD-LMCHI/PT regarding glucose measurement. GOD-MMCHI/PT was found to give the highest enzymatic activity among the electrodes under investigation. The extent of interference encountered by GOD-MMCHI/PT and GOD-LMCHI/PT was not significantly different. Although the Nafion coated biosensor significantly reduced the signal due to the interferents under study, it also significantly reduced the response to glucose. The performance of the biosensors in the determination of glucose in rat serum was evaluated. Comparatively better accuracy and recovery results were obtained for GOD-MMCHI/PT. Hence, GOD-MMCHI/PT showed a better performance when compared with GOD-LMCHI/PT. In conclusion, chitosan membranes shave the potential to be a suitable matrix for the development of glucose biosensors.     

Fabrication of glucose oxidase/polypyrrole biosensor by galvanostatic method in various pH aqueous solutions

The pH effect of pyrrole electropolymerization in the presence of glucose oxidase (GODx) on the performance and characteristic of galvanostatically fabricated glucose oxidase/polypyrrole (Ppy) biosensor is reported. Preparing the GODx/Ppy biosensors in 0.1 M KCl saline solution with various pH containing 0.05 M pyrrole monomer and 0.5 mg/ml GODx at 382 microA/cm2 current density for 100 mC/cm2 film thickness, both the galvanostatic responses and characteristics of these resulted biosensors were obtained. The results revealed that the galvanostatic glucose biosensor fabricated at neutral pH condition exhibited much higher sensitivity than those fabricated at lower or higher pH conditions, and had a good linearity form zero to 10 mM glucose with the sensitivity of 7 nA/mM. Finally, the long-term stability and the kinetic parameters, Michaelis constant and maximum current, of this biosensor were also reported.     

Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect

Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose fluctuations were characterized at the single cell level and tend to decrease the induction of GFP. Simulations run on the basis of a stochastic hydrodynamic model have shown the variability and the frequencies at which biosensors are exposed to glucose gradient in the SDR. GFP leakage was observed to a great extent in the case of a culture operated in well-mixed fed-batch mode, by comparison with those operated in SDR. GFP leakage seems to be correlated to a higher membrane permeability, confirming previous studies highlighting a better cell viability in cultures operated in a fluctuating environment. Our results suggest that GFP leakage could be used in parallel to the normal GFP biosensor function in order to assess microbial viability in process conditions.     

Offline glucose biomonitoring in yeast culture by polyamidoamine/cysteamine-modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.     

Novel planar glucose biosensors for continuous monitoring use

Novel planar glucose biosensors to be used for continuous monitoring have been developed. The electrodes are produced with the "screen printing" technique, and present a high degree of reproducibility together with a low cost and the possibility of mass production. Prior to enzyme immobilisation, electrodes are chemically modified with ferric hexacyanoferrate (Prussian Blue). This allows the detection of the hydrogen peroxide produced by the enzymatic reaction catalysed by GOD, at low applied potential (ca. 0.0 V versus Ag/AgCl), highly limiting any electrochemical interferences. The layer of Prussian Blue (PB) showed a high stability at the working conditions (pH 7.4) and also after 1 year of storage dry at RT, no loss of activity was observed. The assembled glucose biosensors, showed high sensitivity towards glucose together with a long-term operational and storage stability. In a continuous flow system, with all the analytical parameters optimised, the glucose biosensors detected glucose concentration as low as 0.025 mM with a linear range up to 1.0mM. These probes were also tested over 50-60 h in a continuous flow mode to evaluate their operational stability. A 0.5 mM concentration of glucose was continuously fluxed into a biosensor wall-jet cell and the current due to the hydrogen peroxide reduction was continuously monitored. After 50-60 h, the drift of the signal observed was around 30%. Because of their high stability, these sensors suggest the possibility of using such biosensors, in conjunction with a microdialysis probe, for a continuous monitoring of glucose for clinical purposes.     

Effect of glucose on embryo quality and post-thaw viability of in-vitro-produced bovine embryos

The present study was carried out to evaluate the effect of glucose absence during the first 24 h of culture on blastocyst quality and survival after freezing and thawing. In Experiment 1, IVM/TVF bovine zygotes from a slaughterhouse were cultured for 24 h in SOFm, either in the absence or in the presence of 1.5 mM glucose and then further cultured for 7 d in SOFm with 1.5 mM glucose. Absence of glucose during the first 24 h of culture increased (P < 0.001) the percentage of embryos that developed to the morula and blastocyst stages. In Experiment 2, presumptive zygotes were incubated for 24 h in the absence of glucose and were then cultured for 7 d in the presence of 1.5, 3 or 5 mM glucose. There were no differences in the percentages of embryos developing to morula or blastocyst stages at 1.5 or 3 mM glucose, whereas the 5 mM concentration appeared to be detrimental (P < 0.001). Blastocysts from Experiments 1 and 2 were assessed for freezing resistance by means of the ability of frozen-thawed embryos to re-expand their blastocoelic cavity and hatch after culture for 72 h in vitro. For Grade 1 and 2 blastocysts, the post-freezing survival rate was unaffected when glucose was omitted during the first 24 h of culture, provided that the glucose was subsequently maintained between 1.5 and 3 mM. At 5 mM glucose, blastocoelic re-expansion was inhibited (P < 0.03). Addition of 1.5 or 3 mM glucose to the culture medium following 24 h of culture without glucose did not affect embryo cell number, whereas 5 mM significantly decreased it (P < 0.01). These results indicate that the first 24 h of culture without glucose do not affect embryo quality or post-thaw viability, but an increase in blastocyst yield was observed. After 24 h of culture addition of glucose in the range 1.5 to 3 mM was beneficial, while as higher concentrations decreased the efficacy of this in vitro production technique.     

A novel, disposable, screen-printed amperometric biosensor for glucose in serum fabricated using a water-based carbon ink

Screen-printed amperometric glucose biosensors have been fabricated using a water-based carbon ink. The enzyme glucose oxidase (GOD) and the electro-catalyst cobalt phthalocyanine were mixed with the carbon ink prior to the screen-printing process; therefore, biosensors are prepared in a one-step fabrication procedure. Optimisation of the biosensor performance was achieved by studying the effects of pH, buffer strength, and applied potential on the analytical response. Calibration studies were performed under optimum conditions, using amperometry in stirred solution, with an operating potential of +500 mV versus SCE. The sensitivity was found to be 1170 nA mM(-1), with a linear range of 0.025-2 mM; the former represents the detection limit. The disposable amperometric biosensor was evaluated by carrying out replicate determinations on a sample of bovine serum. This was achieved by the method of multiple standard additions and included a correction for background currents arising from oxidizable serum components. The mean serum concentration was calculated to be 8.63 mM and compared well with the supplier's value of 8.3 mM; the coefficient of variation was calculated to be 3.3% (n=6).     

Mediator-free electrochemical biosensor based on buckypaper with enhanced stability and sensitivity for glucose detection

Here we report on a novel platform based on buckypaper for the design of high-performance electrochemical biosensors. Using glucose oxidase as a model enzyme, we constructed a biocompatible mediator-free biosensor and studied the potential effect of the buckypaper on the stability of the biosensor with both amperometry and FTIR spectroscopy. The results showed that the biosensor responses sensitively and selectively to glucose with a considerable functional lifetime of over 80 days. The fabricated enzymatic sensor detects glucose with a dynamic linear range of over 9 mM and a detection limit of 0.01 mM. To examine the efficiency of enzyme immobilization, the Michaelis–Menten constant was calculated to be 4.67 mM. In addition, the fabricated electrochemical biosensor shows high selectivity; no amperometric response to the common interference species such as ascorbic acid, uric acid and acetamidophenol was observed. The facile and robust buckypaper-based platform proposed in this study opens the door for the design of high-performance electrochemical biosensors for medical diagnostics and environmental monitoring.     

Synthesis and secretion of lecithin-cholesterol acyltransferase by the human hepatoma cell line HepG2

The human liver cell line HepG2 was investigated for its synthesis and secretion of lecithin-cholesterol acyltransferase. The cells were grown to confluency in Eagle's minimal essential medium plus 10% fetal bovine serum. At the onset of the study, fetal bovine serum was removed and cells were grown in minimal essential medium only. At 6, 12, 24, and 48 h the cells were harvested, and the culture medium collected at each time point was assayed for lecithin-cholesterol acyltransferase mass and activity, cholesterol esterification rate, and apolipoprotein A-I mass. The rate of the enzyme secretion measured by both mass and activity was linear over 24 h of culture. The enzyme mass by radioimmunoassay was 1.7, 4.1, 7.9 and 13.7 ng/ml culture medium (or 8.3, 19.9, 38.5 and 66.7 ng/mg cell protein), respectively, and enzyme activity using an exogenous source of phosphatidylcholine/cholesterol liposomes containing apolipoprotein A-I as substrate was 85, 170, 315, and 402 pmol cholesterol esterified/h per ml culture medium (or 414, 828, 1534 and 1957 pmol cholesterol esterified/h per mg cell protein) for 6, 12, 24, and 48 h of culture, respectively. The endogenous cholesterol esterification rate of the culture medium was 47, 104, 224 and 330 pmol/h per ml and apolipoprotein A-I mass was 305, 720, 2400 and 3940 ng/ml culture medium over the same time frame. In contrast to culture medium, low levels of enzyme activity (approximately 10% of that in culture medium at 24 and 48 h) were observed in the extracts of HepG2 cells. The enzyme secreted by HepG2 was found to be similarly activated by apolipoprotein A-I, apolipoprotein E, or apolipoprotein A-IV, and was similarly inhibited by phenylmethylsulfonyl fluoride, dithiobisnitrobenzoate, p-hydroxymercuribenzoate, or iodoacetate as compared to human plasma enzyme. High-performance gel filtration of the culture medium revealed that the HepG2-secreted enzyme was associated with a fraction having a mean apparent molecular weight of approximately 200,000. We concluded that human hepatoma HepG2 cells synthesize and secrete lecithin-cholesterol acyltransferase, which is functionally homologous to the human plasma enzyme.     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.     

Recombinant Microdochium nivale carbohydrate oxidase and its application in an amperometric glucose sensor

Biosensors containing recombinant carbohydrate oxidase from Microdochium nivale (rMnO) were developed by means of either chemically modified carbon paste or graphite electrode. 1-(N,N-dimethylamine)-4-(4-morpholine)benzene (AMB) and 1,1'-dimethylferrocene (DMFc) have been used as mediators. The biosensors showed a linear calibration graph up to 18 mM of glucose when operated at 0.04-0.36 V versus a saturated calomel electrode. Almost no change was detected in the sensitivity of the biosensors at pH 7.2-8.1. The biosensors responded to other aldoses in the D-configuration, however, maximal sensitivity of the biosensor was towards D-glucose. The biosensor did not response to polyhydroxylic compounds such as D-mannitol, D-sorbitol and inositol. The advantages of the biosensors based on rMnO in comparison to Aspergillus niger glucose oxidase is a wider linear range, low sensitivity to oxygen and (in some cases) broad specificity.     

Energy substrates and the completion of spontaneous meiotic maturation

This study was carried out to examine how different combinations of pyruvate and glucose affect spontaneous meiotic maturation of cumulus-cell-enclosed mouse oocytes (CEO) to metaphase II (MII). Most experiments used an open system in which oocytes were cultured in 1 ml medium in plastic tubes. Initial experiments examined the dose response effects of pyruvate or glucose alone in the presence or absence of 2 mM glutamine. When medium lacked both pyruvate and glucose, more than 91% of the oocytes died in glutamine-free medium during 15 h of culture; viability was restored with the addition of glutamine, but only 11% of the CEO reached MII. In the absence of glutamine, 62-68% of oocytes completed maturation in 0.23-2.3 mM pyruvate, while 44-60% MII was observed in 0.55-27.8 mM glucose. The addition of glutamine to these cultures had a general suppressive effect on the completion of maturation. When glucose was added to pyruvate-containing cultures, the combination of 1 mM pyruvate/5.5 mM glucose was most effective in supporting maturation (about 90% MII), with little effect of glutamine. No further increase in maturation was observed when glucose was increased five-fold (to 27.8 mM). The positive effect of glucose was in part attributed to stimulation of glycolysis and increased production of pyruvate, since a reduced culture volume (8 microl), which allows the accumulation of secreted pyruvate, improved maturation in glucose-containing, but not pyruvate-containing, medium, and FSH, which stimulates glycolysis, increased progression to MII in glucose-containing, but not pyruvate-containing, medium. Yet these results also suggest that glucose has a beneficial effect on maturation apart from simple provision of pyruvate. The pyruvate effect was directly on the oocyte, because denuded oocytes responded more effectively than CEO to this energy substrate. The highest percentage of MII oocytes (96-97%) occurred in microdrop cultures containing glucose but lacking glutamine. These results indicate that glutamine supports oocyte viability but is not an adequate energy source for the completion of spontaneous meiotic maturation and may be detrimental. In addition, while pyruvate and glucose alone can each support meiotic progression of CEO to MII, optimal maturation requires the provision of both substrates to the culture medium when a large volume (1 ml) is used. It is concluded that careful attention to specific energy substrate supplementation and culture volume is important to optimise spontaneous meiotic maturation in vitro.     

Micropropagation of Alnus cordata (Loisel.) Loisel.

Procedures were developed for micropropagation of Alnus cordata through in vitro axillary shoot multiplication of axillary bud explants cultured in Murashige & Skoog (MS) medium. Establishment of cultures from plants grown in the field was very difficult due to bacterial contamination and phenolic oxidation in explants causing severe browning. Explants were first cultured on an MS medium containing 4.4 M 6-benzyladenine and 87.6 mM sucrose (initiation medium) for 7 days and then transferred to an MS medium containing 1.1 M 6-benzyladenine and 333 mM glucose (multiplication medium) for a further 20–25 days. It was necessary to transfer cultures from initiation medium to multiplication medium after 7 days to minimize excessive callus growth, abnormally thick and brittle leaves, inhibition of shoot elongation, and senescence. Shoot multiplication comparable to the above method was achieved by culture of axillary bud explants in MS medium supplemented with 1.1–4.4 M 6-benzyladenine and 333 mM glucose 4–5 weeks after culture. Shoots rooted in MS medium (1/2 x macro-nutrients) supplemented with 1.2–4.9 M indolebutyric acid. Also, 98% rooting was achieved when cultures were treated with 625 mgl^-1 indolebutyric acid for 24 h at the end of the shoot production stage and rooted in vivo as mini-cuttings. Plantlets established well in soil.     

Amperometric determination of glucose,based on the direct electron transfer between glucose oxidase and tin oxide

An amperometric glucose biosensor was designed for the detection of glucose in blood, urine, beverages, and fermentation systems. In typical glucose biosensors that employ enzymes, mediators are used for efficient electron transfer between the enzymes and the electrode. However, some of these mediators are known to be toxic to the enzymes and also must be immobilized on the surface of the electrode. We propose a mediator-free glucose biosensor that uses a glucose oxidase immobilized on a tin oxide electrode. Direct electron transfer is possible in this system because the tin oxide has redox properties similar to those of mediators. The method for immobilization of the glucose oxidase onto the tin oxide is also very simple. Tin oxide was prepared by the anodization and annealing of pure tin, and this provides a large surface area for the immobilization step because of its porosity. Glucose oxidase was immobilized onto the tin oxide using the membrane entrapment method. The proposed method provides a simple process for fabricating the enzyme electrode. Glucose oxidase immobilized onto the tin oxide, prepared in accordance with this method, has a relatively large current response when comparedto those of other glucose biosensors. The sensitivity of the biosensor was 19.55 μA/mM, and a linear response was observed between 0∼3 mM glucose. This biosensor demonstrated good reproducibility and stability.     

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.