An amperometric microbial biosensor development based on Candida tropicalis yeast cells for sensitive determination of ethanol

Different branchs of industry need to use ethanol in their production and some progress and not only the industry also to determine ethanol sensitively, accurately, fast and economical is very important. For the sensitive determination of ethanol a new amperometric biosensor based on Candida tropicalis cells, which contains alcohol oxidase enzyme, immobilized in gelatin by using glutaraldehyde was developed. In the study, before the microbial biosensor construction C. tropicalis cells were activated and cultured in a culture medium. By using gelatine and glutaraldehyde (0.1%) C. tropicalis cells obtained in logarithmic phase were immobilized and fixed on a pretreated teflon membrane of a dissolved oxygen probe. Ethanol determination is based on the assay of the differences on the respiration activity of the cells on the oxygenmeter in the absence and the presence of ethanol. The microbial biosensor response was depend linearly on ethanol concentration between 0.5 and 7.5 mM with 2 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount were found as 4.42 mg cm(-2) and also phosphate buffer (pH:7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the biosensor response, operational and storage stability were carried out.     

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase‐modified gold electrode

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine‐modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at ?0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4°C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A novel biosensor based on activation effect of thiamine on the activity of pyruvate oxidase

A biosensor based on pyruvate oxidase (POX) enzyme was developed for the investigation of the effect of thiamine (vitamin B(1)) molecule on the activity of the enzyme. The biosensor was prepared with a chemical covalent immobilization method on the dissolved oxygen (DO) probe by using gelatin and cross-linking agent, glutaraldehyde. POX catalyzes the degradation of pyruvate to acetylphosphate, CO(2) and H(2)O(2) in the presence of phosphate and oxygen. Thiamine is an activator for POX enzyme and determination method of the biosensor was based on this effect of thiamine on the activity of the enzyme. The biosensor responses showed increases in the presence of thiamine. Increases in the biosensor responses were related to thiamine concentration. Thiamine determination is based on the assay of the differences on the biosensor responses on the oxygenmeter in the absence and the presence of thiamine. The biosensor response depend linearly on thiamine concentration between 0.025 and 0.5 microM with 2 min response time. In the optimization studies of the biosensor the most suitable enzyme amount was found as 2.5 U cm(-2) and also phosphate buffer (pH 7.0; 50 mM) and 35 degrees C were obtained as the optimum working conditions. In the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response and reproducibility were carried out.     

Biofunctional nanocomposite of carbon nanofiber with water-soluble porphyrin for highly sensitive ethanol biosensing

A biofunctional hybrid nanocomposite of carbon nanofiber (CNF) with water-soluble iron(III) meso-tetrakis(N-methylpyridinum-4-yl) porphyrin (FeTMPyP) was designed via non-covalent interaction for preparation of highly sensitive ethanol biosensor. The prepared nanocomposite showed good dispersion in water and was characterized with steady-state electronic absorption spectroscopy and scanning electron microscope. The nanocomposite combined the good conductivity of CNF and the excellent catalytic activity of both CNF and FeTMPyP toward the reduction of dissolved oxygen, producing a method for amperometric detection of oxygen ranging from 6.5 nM to 6.4 microM at a low overpotential. The nanocomposite modified electrode was further used for assembly of alcohol oxidase to construct an amperometric biosensor for ethanol. The biosensor showed rapid and highly sensitive response to ethanol with a linear range from 2.0 microM to 112 microM. The immobilized alcohol oxidase also showed its direct electrochemistry. The biofunctional nanocomposite provides a new way to not only construct the highly sensitive biosensors but also mimic the catalytic activity of enzyme in the life process.     

A novel nitrite biosensor based on conductometric electrode modified with cytochrome c nitrite reductase composite membrane

A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples.     

Urate oxidase electrode based on dissolved oxygen probe for urine uric acid determination

A biosensor for the specific determination of uric acid in urine was developed using urate oxidase (EC 1.7.3.3) in combination with a dissolved oxygen probe. Urate oxidase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane to serve as enzyme electrode. The electrode response was maximum when 50 mM glycine buffer was used at pH 9.2 and 35 degrees C. The enzyme electrode response depends linearly on uric acid concentration between 5-40 microM with a response time of 5 min. The enzyme electrode is stable for more than 2 weeks and during this period over 35 assays were performed.     

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol

Abstract

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8?s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM–50?mM and 0.0001?µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190?days) at 4°C. The sensitivity of the sensor was found to be 155?µA mM^?1?cm^?2. A good correlation (R²?=?0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.     

An amperometric cholesterol biosensor based on multiwalled carbon nanotubes and organically modified sol-gel/chitosan hybrid composite film

A new type of amperometric cholesterol biosensor based on sol-gel chitosan/silica and multiwalled carbon nanotubes (MWCNTs) organic-inorganic hybrid composite material was developed. The hybrid composite film was used to immobilize cholesterol oxidase on the surface of Prussian blue-modified glass carbon electrode. Effects of some experimental variables such as enzyme loading, concentration of Triton X-100, pH, temperature, and applied potential on the current response of the biosensor were investigated. Analytical characteristics and dynamic parameters of the biosensors with and without MWCNTs in the hybrid film were compared, and the results show that analytical performance of the biosensor can be improved greatly after introduction of the MWCNTs. Response time, sensitivity, linear range, limit of detection (S/N=3), and apparent Michaelis-Menten constant Km are 25s, 0.54 microA mM(-1), 8.0 x 10(-6) to 4.5 x 10(-4) M, 4.0 x 10(-6) M, and 0.41 mM for the biosensor without MWCNTs and 13 s, 1.55 microA mM(-1), 4.0 x 10(-6) to 7.0 x 10(-4) M, 1.0 x 10(-6) M, and 0.24 mM for the biosensor with MWCNTs, respectively. The activation energy of the enzyme-catalyzed reaction was measured to be 42.6 kJ mol(-1). This method has been used to determine the free cholesterol concentration in real human blood samples.     

Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation

A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 microM (S/N=3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l(-1).     

Development and analytical application of an uric acid biosensor using an uricase-immobilized eggshell membrane

An uric acid biosensor fabricated from a uricase-immobilized eggshell membrane and an oxygen electrode was presented. The detection schemes involve the enzymatic reactions of the uricase leading to the depletion of dissolved oxygen level upon exposure to uric acid solution. The decrease in oxygen level was monitored and related to the uric acid concentration. The scanning electron micrographs show the microstructure of the eggshell membrane within which the uricase is successfully immobilized. The effects of enzyme loading, pH, temperature, and phosphate buffer concentration on the response of the biosensor were investigated in detail. The uric acid biosensor has a linear response range of 4.0-640 microM with a detection limit of 2.0 microM (S/N=3). The response time was less than 100 s. The biosensor exhibited good repeatable response to a 0.10mM uric acid solution with a relative standard deviation of 3.1% (n=7). The reproducibility of fabrication of the biosensors using four different membranes was good with a R.S.D. of 3.2%. The biosensor showed extremely good stability with a shelf-life of at least 3 months. Some common potential interferents in samples such as glucose, urea, ascorbic acid, lactic acid, glycine, DL-alpha-alanine, DL-cysteine, KCl, NaCl, CaCl2, MgSO4, and NH4Cl showed no interferences on the response of the uric acid biosensor. The biosensor was successfully applied to determine the uric acid level in some human serum and urine samples, and the results agreed well with those obtained by a commercial colorimetric assay kit.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

A biosensor based on catalase for determination of highly toxic chemical azide in fruit juices

In this work, an amperometric biosensor based on catalase enzyme was developed for the determination of azide. The principle of the measurements was based on the determination of the decrease in the differentiation of oxygen level which had been caused by the inhibition of catalase in the bioactive layer of the biosensor by azide. Firstly, the optimum conditions for the inhibitor biosensor were established. In the optimization studies of the biosensor, the most suitable catalase and gelatin amounts and glutaraldehyde ratio were determined. Optimum catalase activity, optimum gelatin amount and glutaraldehyde percentage were 5000 Ucm(-2), 5.94 mgcm(-2) and 2.5%, respectively. Characterization studies of the biosensor such as optimum pH and optimum temperature were carried out. The repeatability experiments were done and the average value (x), standard deviation (S.D.) and variation coefficient (C.V.) were calculated as 98.6 microM, +/-4.16 microM and 4.23%, respectively. A good linear relationship with a correlation coefficient of 0.9902 was obtained over the concentration range of 25 microM to 300 microM azide. After the optimization and characterization studies the proposed biosensor was applied to the determination of azide in certain fruit juices.     

Development of a xylitol biosensor composed of xylitol dehydrogenase and diaphorase

In preparation for the development of a xylitol biosensor, the xylitol dehydrogenase of Candida tropicalis IFO 0618 was partially purified and characterized. The optimal pH and temperature of the xylitol dehydrogenase were pH 8.0 and 50 degrees C, respectively. Of the various alcohols tested, xylitol was the most rapidly oxidized, with sorbitol and ribitol being reduced at 65% and 58% of the xylitol rate. The enzyme was completely inactive on arabitol, xylose, glucose, glycerol, and ethanol. The enzyme's xylitol oxidation favored the use of NAD+ (7.9 U/mg) over NADP+ (0.2 U/mg) as electron acceptor, while the reverse reaction, D-xylulose reduction, favored NADPH (7.7 U/mg) over NADH (0.2 U/mg) as electron donor. The K(m) values for xylitol and NAD+ were 49.8 mM and 38.2 microM, respectively. For the generation of the xylitol biosensor, the above xylitol dehydrogenase and a diaphorase were immobilized on bromocyan-activated sephallose. The gel was then attached on a dissolved oxygen electrode. In the presence of vitamin K3, NAD+ and phosphate buffer, the biosensor recorded a linear response to xylitol concentration up to 3 mM. The reaction was stable after 15 min. When the biosensor was applied to a flow injection system, optimal operation pH and temperature were 8.0 and 30 degrees C, respectively. The strengths and limitations of the xylitol biosensor are its high affinity for NAD+, slow reaction time, narrow linear range of detection, and moderate affinity for xylitol.     

Whole cell immobilized amperometric biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1 (thiamine)

A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated.     

Immobilization of urease on nanostructured polymer membrane and preparation of urea amperometric biosensor

A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.     

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.     

A glucose biosensor based on chitosan-glucose oxidase-gold nanoparticles biocomposite formed by one-step electrodeposition

An amperometric biosensor for the quantitative measurement of glucose is reported. The biosensor is based on a biocomposite that is homogeneous and easily prepared. This biocomposite is made of chitosan hydrogel, glucose oxidase, and gold nanoparticles by a direct and facile electrochemical deposition method under enzyme-friendly conditions. The resulting biocomposite provided a shelter for the enzyme to retain its bioactivity at considerably extreme conditions, and the decorated gold nanoparticles in the biocomposite offer excellent affinity to enzyme. The biosensor exhibited a rapid response (within 7s) and a linear calibration range from 5.0 microM to 2.4 mM with a detection limit of 2.7 microM for the detection of glucose. The combination of gold nanoparticles affinity and the promising feature of the biocomposite with the onestep nonmanual technique favor the sensitive determination of glucose with improved analytical capabilities.     

Sensitive determination of L-lysine with a new amperometric microbial biosensor based on Saccharomyces cerevisiae yeast cells

A new amperometric microbial biosensor based on Saccharomyces cerevisiae NRRL-12632 cells, which had been induced for lysine oxidase enzyme and immobilized in gelatin by a cross-linking agent was developed for the sensitive determination of L-lysine amino acid. To construct the microbial biosensor S. cerevisiae cells were activated and cultured in a suitable culture medium. By using gelatine (8.43 mg cm(-2)) and glutaraldehyde (0.25%), cells obtained in the logarithmic phase of the growth curve at the end of a 14 h period were immobilized and fixed on a pretreated oxygen sensitive Teflon membrane of a dissolved oxygen probe. The assay procedure of the microbial biosensor is based on the determination of the differences of the respiration activity of the cells on the oxygenmeter in the absence and the presence of L-lysine. According to the end point measurement technique used in the experiments it was determined that the microbial biosensor response depended linearly on L-lysine concentrations between 1.0 and 10.0 microM with a 1 min response time. In optimization studies of the microbial biosensor, the most suitable microorganism quantities were found to be 0.97x10(5)CFU cm(-2). In addition phosphate buffer (pH 7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the microbial biosensor responses, reproducibility of the biosensor and operational and storage stability were investigated.     

On-line monitoring of methanol in n-hexane by an organic-phase alcohol biosensor

An organic-phase alcohol biosensor has been developed by co-entrapping alcohol oxidase and horseradish peroxidase within an ionotropy polymer hydrogel matrix fabricated from silica gel particles, hydroxyethyl carboxymethylcellulose, an adduct of 3-methoxy-4-ethoxybenzaldehyde and 4-tert-butylpyridinium acetohydrazone, and octadecylsilica particles. The viability of the immobilised enzymes for the biocatalytic reaction of methanol in n-hexane was comparatively studied by using a bulk cell or a volume-changeable flow-through cell coupled with an oxygen optical transducer. It was found that the microenvironment around the enzyme, the deterioration property of the enzyme, the substrate throughput and the mass transfer process of the reactant in the bioreactor were the crucial parameters affecting the performance of the alcohol organic-phase biosensor. Our optimal biosensor was constructed from a flow-through cell packed with small particles of immobilised enzymes and it could maintain the biocatalytic reaction at high and stable rate for on-line detection of methanol in n-hexane under flow operation mode. The biosensor had an analytical working range of 2.3-90 mM methanol in n-hexane. The response times (t95) were 4.5 and 7.5 min for 60 and 10 mM methanol, respectively. The operational lifetime of the biosensor was more than 45 assays and the shelf lifetime was longer than 2 weeks. The biosensor has been successfully applied to determine the methanol content in a commercial gasoline-methanol blend sample with good recovery.     

An enzyme electrode for on-line determination of ethanol and methanol

Since a stable alcohol oxidase with a high specific activity is not commercially available, we propose to produce and purify this enzyme from a strain of the yeast Hansenula polymorpha. This alcohol oxidase was immobilized into a gelatin matrix and its activity was estimated by a pO(2) sensor. The enzyme electrode obtained was then used in a continuous flow system to measure methanol or ethanol concentrations. The sample oxygen content dependence of the signal was minimized by the support properties. Measuring time for each sample were less than two minutes including response data treatment and rinsing step. The enzyme electrode response was set for ethanol from 0.5mM to 15mM and for methanol from 10mM to 300mM. On repeated use, the electrode signal for 10mM of ethanol was stable for at least 500 assays. Analysis have been performed in different beverages such as wine and beer, and the results compared to those obtained with classical methods of analysis.