High-yield enzymatic bioconversion of hydroquinone to α-arbutin, a powerful skin lightening agent, by amylosucrase

α-Arbutin (α-Ab) is a powerful skin whitening agent that blocks epidermal melanin biosynthesis by inhibiting the enzymatic oxidation of tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA). α-Ab was effectively synthesized from hydroquinone (HQ) by enzymatic biotransformation using amylosucrase (ASase). The ASase gene from Deinococcus geothermalis (DGAS) was expressed and efficiently purified from Escherichia coli using a constitutive expression system. The expressed DGAS was functional and performed a glycosyltransferase reaction using sucrose as a donor and HQ as an acceptor. The presence of a single HQ bioconversion product was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The HQ bioconversion product was isolated by silica gel open column chromatography and its chemical structure determined by 1H and 13C nuclear magnetic resonance (NMR). The product was determined to be hydroquinone-O-α-D-glucopyranoside with a glucose molecule linked to HQ through an α-glycosidic bond. However, the production yield of the transfer reaction was significantly low (1.3%) due to the instability of HQ in the reaction mixture. The instability of HQ was considerably improved by antioxidant agents, particularly ascorbic acid, implying that HQ is labile to oxidation. A maximum yield of HQ transfer product of 90% was obtained at a 10:1 molar ratio of donor (sucrose) and acceptor (HQ) molecules in the presence of 0.2 mM ascorbic acid.     

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.     

Tyrosinase-catalyzed hydroxylation of hydroquinone,a depigmenting agent,to hydroxyhydroquinone: A kinetic study

Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone (HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex met-tyrosinase-HHQ, which can evolve in two different ways, forming deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone or on the other way generating met-tyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot close the enzyme catalytic cycle for the lack of reductant (HHQ). However, in the presence of hydrogen peroxide, met-tyrosinase (inactive on hydroquinone) is transformed into oxy-tyrosinase, which is active on HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into 2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which after binding to oxygen, originates oxy-tyrosinase. This enzymatic form is now capable of reacting with HQ to generate p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of HHQ during the action of tyrosinase on HQ is demonstrated by means of high performance liquid chromatography mass spectrometry (HPLC–MS) by using hydrogen peroxide and high ascorbic acid concentrations. We propose a kinetic mechanism for the tyrosinase oxidation of HQ which allows us the kinetic characterization of the process. A possible explanation of the cytotoxic effect of HQ is discussed.     

Construction of d-amino acid biosensor based on d-amino acid oxidase immobilized onto poly (indole-5-carboxylic acid)/zinc sulfide nanoparticles hybrid film

d-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto poly indole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodeposited on surface of an Au electrode. A highly sensitive d-amino acid biosensor was constructed using this enzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5 and 35 °C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor response (mA) and d-alanine concentration in the range 0.001–2.0 mM. The sensitivity of the biosensor was 58.85 μA cm^?2 mM^?1 with a detection limit of 0.001 mM (S/N = 3). The enzyme electrode was used 120 times over a period of 2 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor was evaluated and employed for measurement of d-amino acid level in fruits and vegetables.     

Chemiluminescence flow-through biosensor for glucose with eggshell membrane as enzyme immobilization platform

In this study, a new chemiluminescence (CL) flow-through biosensor for glucose was developed by immobilizing glucose oxidase (GOD) and horseradish peroxidase (HRP) on the eggshell membrane with glutaraldehyde as a cross-linker. The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and hydrogen peroxide (H2O2) and then H2O2 oxidizing luminol to produce CL emission in the presence of HRP. The immobilization condition (e.g., immobilization time, GOD/HRP ratio, glutaraldehyde concentration) was studied in detail. It showed good storage stability at 4 degrees C over a 5-month period. The proposed biosensor exhibited short response time, high sensitivity, easy operation, and simple sensor assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

Fiber optic monooxygenase biosensor for toluene concentration measurement in aqueous samples

Measurements of pollutants such as toluene are critical for the characterization of contaminated sites and for the monitoring of remediation processes and wastewater treatment effluents. Fiber optic enzymatic biosensors have the potential to provide cost-effective, real time, continuous, in situ measurements. In this study, a fiber optic enzymatic biosensor was constructed and characterized for the measurement of toluene concentrations in aqueous solutions. The biological recognition element was toluene ortho-monooxygenase (TOM), expressed by Escherichia coli TG1 carrying pBS(Kan)TOM, while an optical fiber coated with an oxygen-sensitive ruthenium-based phosphorescent dye served as the transducer. Toluene was detected based on the enzymatic reaction catalyzed by TOM, which resulted in the consumption of oxygen and changes in the phosphorescence intensity. The biosensor was found to have a limit of detection of 3 μM, a linear signal range up to 100 μM, and a response time of 1 h. The performance was reproducible with different biosensors (RSD=7.4%, n=8). The biosensor activity declined with each measurement and with storage time, particularly at elevated temperatures. This activity loss could be partially reversed by exposure to formate, suggesting that NADH consumption was the primary factor limiting lifetime. This is the first report of an enzymatic toluene sensor and of an oxygenase-based biosensor. Since many oxygenases have been reported, the design concept of this oxygenase-based biosensor has the potential to broaden biosensor applications in environmental monitoring.     

An amperometric bilirubin biosensor based on a conductive poly-terthiophene-Mn(II) complex

An amperometric bilirubin biosensor was fabricated by complexing the Mn(II) ion with a conducting polymer and the final biosensor surface was coated with a thin polyethyleneimine (PEI) film containing an enzyme, ascorbate oxidase (AsOx). The complexation between poly-5,2'-5',2'-terthiophene-3-carboxylic acid (PolyTTCA) and Mn(II) through the formation of Mn-O bond was confirmed by XPS. The PolyTTCA-Mn(II) complex was also characterized using cyclic voltammetry. The PolyTTCA-Mn(II)/PEI-AsOx biosensor specifically detect bilirubin through the mediated electron transfer by the Mn(II) ion. To optimize the experimental condition, various experimental parameters such as pH, temperature, and applied potential were examined. A linear calibration plot for bilirubin was obtained between 0.1 microM and 50 microM with the detection limit of 40+/-3.8 nM. Interferences from other biological compounds, especially ascorbate and dopamine were efficiently minimized by coating the biosensor surface with PEI-AsOx. The bilirubin sensor exhibited good stability and fast response time (<5s). The applicability of this bilirubin sensor was tested in a human serum sample.     

Novel Pathway for Conversion of Chlorohydroxyquinol to Maleylacetate in Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.     

Detection of pesticides using an amperometric biosensor based on ferophthalocyanine chemically modified carbon paste electrode and immobilized bienzymatic system

A new highly sensitive amperometric method for the detection of organophosphorus compounds has been developed. The method is based on a ferophthalocyanine chemically modified carbon paste electrode coupled with acetylcholinesterase and choline oxidase co-immobilized onto the surface of a dialysis membrane. The activity of cholinesterase is non-competitively inhibited in the presence of pesticides. The highest sensitivity to inhibitors was found for a membrane containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetylcholine as substrate; choline produced by hydrolysis in the enzymatic layer was oxidized by choline-oxidase and subsequently H(2)O(2) produced was electrochemically detected at +0.35 V vs. Ag/AgCl. The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, up to 10(-10) M of paraoxon and carbofuran can be detected.     

Determination of glutamic acid decarboxylase activity and inhibition by an H2O2-sensing glutamic acid oxidase biosensor.

The catalytic activity of the enzyme L-glutamic acid decarboxylase (GAD) is determined by an amperometric method based on a recently developed glutamate-selective biosensor. The biosensor is composed of an amperometric H2O2 electrode and a biocatalytic membrane containing the enzyme glutamic acid oxidase (GAO). The biosensor allows the direct and continuous measurement of GA levels by monitoring the H2O2 produced at the electrode interface as a coproduct of the GAO-catalyzed GA oxidation to alpha-ketoglutaric acid. Since GA is transformed to gamma-aminobutyric acid and CO2 under the catalytic activity of GAD, the rate of GA consumption in solution, monitored by the GAO biosensor, represents a reliable measure of GAD catalytic activity. Additional experiments performed in the presence of different concentrations of the GAD inhibitor valproic acid have shown the suitability of the proposed approach for the study of GAD inhibitors also. Discussion of the main experimental characteristics of this new analytical method is given in terms of sensitivity, reproducibility, and reliability of the experimental results and ease, time, and cost of operation.     

A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 μM to 30 × 10^?5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.     

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.     

An acetylcholinesterase biosensor for determination of low concentrations of Paraoxon and Dichlorvos

The characterization of an economic and ease-to-use carbon paste acetylcholinesterase (AChE) based biosensor to determine the concentration of pesticides Paraoxon and Dichlorvos is discussed. AChE hydrolyses acetylthiocholine (ATCh) in thiocoline (TC) and acetic acid (AA). When AChE is immobilized into a paste carbon working electrode kept at +410 mV vs. Ag/AgCl electrode, the enzyme reaction rate using acetylthiocholine chloride (ATCl) as substrate is monitored as a current intensity. Because Paraoxon and Dichlorvos inhibit the AChE reaction, the decrease of the current intensity, at fixed ATCl concentration, is a measure of their concentration. Linear calibration curves for Paraoxon and Dichlorvos determination have been obtained. The detection limits resulted to be 0.86 ppb and 4.2 ppb for Paraoxon and Dichlorvos, respectively, while the extension of the linear range was up 23 ppb for the former pesticide and up to 33 ppb for the latter. Because the inhibited enzyme can be reactivated when immediately treated with an oxime, the biosensor reactivation has been studied when 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were used. TMB-4 resulted more effective. The comparison with the behavior of similar AChE based biosensors is also presented.     

Immobilized enzymatic fluorescence capillary biosensor for determination of sulfated bile acid in urine

The authors have proposed an immobilized enzymatic fluorescence capillary biosensor (SBAs-IE-FCBS) for the determination of sulfated bile acids (SBAs). The reaction principle of the biosensor is that under the catalysis of the bile acid sulfate sulfatase (BSS) and beta-hydroxysteroid dehydrogenase (beta-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3beta-hydroxyl bile acids, then the latter reacts with nicotinamide adenine dinucleotide (NAD(+)), and is converted into 3-ketosteroid; meanwhile, NAD(+) is converted to reduced nicotinamide adenine dinucleotide (NADH). NADH continuously reacts with 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS) and is converted into NAD(+) circularly and 1-MPMSH(2). Finally resazurin is reduced into resorufin by 1-MPMSH(2), the formed resorufin (lambda(ex)/lambda(em): 540 nm/580 nm) is used for quantifying the concentration of SBAs. Optimized conditions being suitable with the biosensor are as follows: the concentrations of BSS and beta-HSD used for the immobilization all are 5 kUL(-1); the concentrations of 1-MPMS and resazurin all are 25 micromolL(-1); the concentrations of Tris-HCl buffer and NAD(+) are 100 and 400 micromolL(-1), respectively; total volume of the enzyme, reagent and sample is only 18 microL per time for determining; the reaction temperature is 37 degrees C; the reaction time is 15min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 micromolL(-1). The relative standard deviation is less than 3.4%, and the detection limit was 0.16 micromolL(-1). The recoveries are in the range 95.5-106%. This SBA-IE-FCBS can be used for quantifying SBAs in urine to diagnose and judge hepatobiliary diseases, etc.     

Development of biosensor based on immobilized L-glutamate oxidase for determination of monosodium glutamate in food

A monosodium glutamate (MSG) biosensor with immobilized L-glutamate oxidase (L-GLOD) has been developed and studied for analysis of MSG in sauces, soup etc. The immobilized enzymatic membrane was attached with oxygen electrode with a push cap system. The detection limit of the sensor was 1 mg/dl and the standard curve was found to be linear upto 20 mg/dl. Response time of the sensor was 2 min. Cross-linking with glutaraldehyde in presence of Bovine Serum Albumin (BSA) as a spacer molecule has been used for immobilization. Optimization of the sensor was done with an increase in L-GLOD concentration (6.3-31.5 IU) and also with increase in loading volume of enzyme solution (5-20 microl). Optimization of pH and temperature was also studied. The permeability of O2 through different membrane was studied with and without immobilized L-GLOD. The enzymatic membrane was used for over 20 measurements and stability of the membrane was observed.     

An amperometric uric acid biosensor based on modified Ir-C electrode

The level of uric acid (UA) has a high relationship with gout, hyperuricemia and Lesch-Nyan syndrome. The determination of UA is an important indicator for clinics and diagnoses of kidney failure. An amperometric UA biosensor based on an Ir-modified carbon (Ir-C) working electrode with immobilizing uricase (EC 1.7.3.3) was developed by thick film screen printing technique. This is the first time to report the utilization of an uricase/Ir-C electrode for the determination of UA by using chronoamperometric (CA) method. The high selectivity of UA biosensor was achieved due to the reduction of H(2)O(2) oxidation potential based on Ir-C electrode. Using uricase/Ir-C as the sensing electrode, the interference from the electroactive biological species, such as ascorbic acid (AA) and UA (might be directly oxidized on the sensing electrode) was slight at the sensing potential of 0.25 V (versus Ag/AgCl). UA was detected amperometrically based on uricase/Ir-C electrode with a sensitivity of 16.60 microAmM(-1) over the concentration range of 0.1-0.8 mMUA, which was within the normal range in blood. The detection limit of UA biosensor was 0.01 mM (S/N=6.18) in pH 7 phosphate buffer solution (PBS) at 37 degrees C. The effects of pH, temperature, and enzymatic loading on the sensing characteristics of the UA biosensor were also investigated in this study.     

Modelling amperometric enzyme electrode with substrate cyclic conversion

A mathematical model of amperometric enzyme electrodes in which chemical amplification by cyclic substrate conversion takes place in a single enzyme membrane has been developed. The model is based on non-stationary diffusion equations containing a non-linear term related to Michaelis-Menten kinetic of the enzymatic reaction. The digital simulation was carried out using the finite difference technique. The influence of the substrate concentration, the maximal enzymatic rate as well as the membrane thickness on the biosensor response was investigated. The numerical experiments demonstrate significant (up to dozens of times) gain in biosensor sensitivity at low concentrations of substrate when the biosensor response is under diffusion control.     

Stereoselectivity of 1-aminocyclopropanecarboxylate malonyltransferase toward stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid

A malonyltransferase isolated from mungbean (Vigna radiata L.) hypocotyls catalyzed the malonylation of both 1-aminocyclopropane-1-carboxylic acid (ACC) and D-amino acids. The possibility that ACC was recognized by the enzyme as a D-amino acid was investigated by examining the efficiencies of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) serving as substrates of malonyltransferase and as inhibitors of ACC malonyltransferase. Although all four isomers were malonylated by the enzyme and competitively inhibited the malonylation of ACC to N-malonyl-ACC, (1R,2S)-AEC and (1R,2R)-AEC, both of which have an R-configuration as a D-amino acid, had lower Km and Ki values (0.1 to 0.2 mM) than their enantiomers, (1S,2R)-AEC (Km and Ki values were about 1 mM) and (1S,2S)-AEC (Km and Ki values were higher than 10 mM), which have an S-configuration as an L-amino acid. Similarly, (R)-isovaline (2-amino-2-methylbutanoic acid), which has an R-configuration as a D-amino acid, inhibited more effectively the enzymatic conversion of ACC to malonyl-ACC than did (S)-isovaline, which has an S-configuration as an L-amino acid. In mungbean hypocotyls (1R,2S)-AEC and (1R,2R)-AEC were also more efficiently converted into malonyl conjugates and more efficiently inhibited the conversion of radioactive ACC into malonyl-ACC than their enantiomers, although the differences in efficiency among stereoisomers were smaller in hypocotyls than in enzymatic reactions. These results suggest that ACC is recognized by the enzyme as a D-amino acid.     

Amperometric hydrogen peroxide biosensor with sol-gel/chitosan network-like film as immobilization matrix

A new type of sol-gel/organic hybrid composite material based on the cross-linking of natural polymer chitosan with (3-aoryloxypropyl) dimethoxymethylsilane was developed for the fabrication of an amperometric H(2)O(2) biosensor. The composite film was used to immobilize horseradish peroxidase (HRP) on a gold disk electrode. The properties of sol-gel/chitosan and sol-gel/chitosan-HRP films have been carefully characterized by atomic force microscopy and Fourier transform infrared. By using fluorescent label, a protein density on sol-gel/chitosan has been calculated to be 3.14 x 10(12) moleculescm(-2). With the aid of catechol mediator, the biosensor had a fast response of less than 2 s with linear range of 5.0 x 10(-9)-1.0 x 10(-7) mol l(-1) and a detection limit of 2 x 10(-9) mol l(-1). Its current response shows a typical Michaelis-Menten mechanism. The apparent Michaelis-Menten constant K(M)(app) is found to be 1.30 micromol l(-1). The activation energy for enzymatic reaction is calculated to be 8.22 kJ mol(-1). The biosensor retained approximately 75% of its original activity after about 60 days of storage in a phosphate buffer at 4 degrees C.     

Fabrication of ultra-thin polypyrrole-glucose oxidase film from supporting electrolyte-free monomer solution for potentiometric biosensing of glucose

A simple electropolymerisation process is described for the fabrication of an ultra-thin ( approximately 55 nm) polypyrrole (PPy)-glucose oxidase (GOD) film in a supporting electrolyte-free monomer solution for potentiometric biosensing of glucose. The optimum conditions for growing the ultra-thin film include 0.1 M pyrrole, 55-110 U/ml GOD, an applied current density of 0.05 mA/cm(2) and an electrical charge of 25 mC/cm(2). Long-term storage of the biosensor in acetate buffer improved the sensitivity of the biosensor by a factor of approximately two. The biosensor can also be used repeatedly for over 2 months with little or no loss in sensitivity. The interference effect of ascorbic acid was successfully reduced by inclusion of an outer PPy-Cl layer.