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     

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).     

Analysis of various sugars by means of immobilized enzyme coupled flow injection analysis

Glucose, maltose, sucrose, lactose, xylose, sorbose, galactose, fructose and gluconolactone were analyzed by means of immobilized pyranose oxidase as well as by the combination of immobilized glucose oxidase with immobilized glycoamylase, invertase, mutarotase, maltase (α-glucosidase) and glucose isomerase by flow injection analysis (FIA). For the simultaneous analysis of glucose and other sugars three different flow-injection configurations were applied and compared. The average error of prediction of the analyses were better than 3% in model media and better than 6% in yeast extract containing media.     

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

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 The culture sample was peristaltically withdrawn from the bioreactor and after cell separation by a steam sterilizable ceramic microfilter, the filtrate was continuously fed to the FIA mediated-biosensor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

Application of a biosensor for monitoring of ethanol

An alcohol biosensor for the measurement of ethanol has been developed. It comprises an alcohol oxidase/chitosan immobilized eggshell membrane and a commercial oxygen sensor. Ethanol determination is based on the depletion of dissolved oxygen content upon exposure to ethanol solution. The decrease in oxygen level was monitored and related to the ethanol concentration. The biosensor response depends linearly on ethanol concentration between 60 microM and 0.80 mM with a detection limit of 30 microM (S/N=3) and 1 min response time. In the optimization studies of the enzyme biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30% (w/v), respectively. The phosphate buffer (pH 7.4, 25 mM) and room temperature (20-25 degrees C) were chosen as the optimum working conditions. In the characterization studies of the ethanol biosensor some parameters such as interference effects, operational and storage stability were studied in detail. The biosensor was also tested with various wine samples. The results of this newly developed biosensor were comparable to the results obtained by a gas chromatographic method.     

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

An FIA biosensor system for the determination of phosphate.

A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at 0.7 V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-1-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100 microM phosphate, with a minimum detectable concentration of 1.25 microM phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.     

On-line monitoring of monoclonal antibodies in animal cell culture using a grating coupler

A grating coupler was used for the on-line determination of monoclonal antibodies produced in perfused animal cell bioreactor. The device was connected with the culture vessel via a flow-injection analysis (FIA) system, which was controlled automatically. Specific antimouse lgG antibodies were immobilized on the surface of the sensor-chip. After injection of the sample, the binding of mouse lgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the on-line detection of produced monoclonal antibodies in the range of 10 to 150 mug/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off-line) and a competitive immunochemical assay using the grating coupler (off-line). (c) 1993 John Wiley & Sons, Inc.     

Determination of l-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode

A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of l-glutamate using l-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5ml/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine l-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatographic assay using an amino acid analyser. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of l-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatographic analysis.     

Hydrogen peroxide as an oxygen source for immobilized Gluconobacter oxydans converting glycerol to dihydroxyacetone

Summary A flow injection analysis (FIA) system with amperometric detection was developed for measuring hydrogen peroxide which was used as an oxygen source for immobilized cells. A constant concentration of peroxide in the reactor was maintained by processing the analytical signal in a computer programmed as a PI-regulator. The concentration of dissolved oxygen was followed using a commercial Clark-electrode. The simultaneous measurements of hydrogen peroxide and dissolved oxygen are discussed with respect to process control.Conversion of glycerol to dihydroxyacetone by Gluconobacter oxydans immobilized in calcium alginate was used as a model system.Initial specific productivity increased with increasing hydrogen peroxide concentration. However, decreases in viable counts, enzymatic activities and overall productivities were noted. Various techniques for improving operational stability are discussed.     

Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes

An on-line assay for a thermostable pullulanase and antithrombin III (AT III) is described. The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein. Assay automation was achieved by utilizing the flow injection analysis (FIA) principles. The apparatus, a stopped-flow, merging-zone manifold, is described in detail. Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min. A method for simulating cultivation conditions was developed for assay optimization. Using this method, a detection limit of I mg l⁻¹ together with a standard deviation of 1.5 was found. A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase. Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined. The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.     

Optical bio-sniffer for ethanol vapor using an oxygen-sensitive optical fiber

An optical bio-sniffer for ethanol was constructed by immobilizing alcohol oxidase (AOD) onto a tip of a fiber optic oxygen sensor with a tube-ring, using an oxygen sensitive ruthenium organic complex (excitation, 470 nm; fluorescent, 600 nm). A reaction unit for circulating buffer solution was applied to the tip of the device. After the experiment in the liquid phase, the sniffer-device was applied for gas analysis using a gas flow measurement system with a gas generator. The optical device was applied to detect the oxygen consumption induced by AOD enzymatic reaction with alcohol application. The sensor in the liquid phase was used to measure ethanol solution from 0.50 to 9.09 mmol/l. Then, the bio-sniffer was calibrated against ethanol vapor from 0.71 to 51.49 ppm with good gas-selectivity based on the AOD substrate specificity. The bio-sniffer with the reaction unit was also used to monitor the concentration change of gaseous ethanol by rinsing and cleaning the fiber tip and the enzyme membrane with buffer solution.     

A highly sensitive and temporal visualization system for gaseous ethanol with chemiluminescence enhancer

A two‐dimensional gaseous ethanol visualization system has been developed and demonstrated using a horseradish peroxidase–luminol–hydrogen peroxide system with high‐purity luminol solution and a chemiluminescence (CL) enhancer. This system measures ethanol concentrations as intensities of CL via the luminol reaction. CL was emitted when the gaseous ethanol was injected onto an enzyme‐immobilized membrane, which was employed as a screen for two‐dimensional gas visualization. The average intensity of CL on the substrate was linearly related to the concentration of standard ethanol gas. These results were compared with the CL intensity of the CCD camera recording image in the visualization system. This system is available for gas components not only for spatial but also for temporal analysis in real time. A high‐purity sodium salt HG solution (L‐HG) instead of standard luminol solution and an enhancer, eosin Y (EY) solution, were adapted for improvement of CL intensity of the system. The visualization of gaseous ethanol was achieved at a detection limit of 3 ppm at optimized concentrations of L‐HG solution and EY. Copyright © 2011 John Wiley & Sons, Ltd.     

Prerequisites for the on-line control of microbial processes by flow injection analysis.

Problems associated with the use of biosensors in process control, e.g. difficulties of sterilization and sensor fouling, are shortly displayed, and possibilities to overcome them are outlined. The advantages of flow injection analysis (FIA) are demonstrated and examples for efficient sampling systems connected with this method are reviewed. Special emphasis is given to problem-orientated sample pretreatments, preventing fast inactivation of immobilized enzymes in the analysis system. Examples of problem-orientated sample pretreatment units are given. A proposal for a computer-controlled self-calibrating FIA system is given.     

On-line determination of glucose and lactate concentrations in animal cell culture based on fibre optic detection of oxygen in flow-injection analysis.

A flow-injection analysis (FIA) system based on fibre optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the on-line monitoring of glucose and lactate concentrations in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by molecular oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fibre optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an r.s.d. of 5% at 30 mM (five measurements) and for 0-30 mM lactate, with an r.s.d. of 5% at 30 mM (five measurements). The enzyme reactors used were stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per hour. The system has been successfully applied to the on-line monitoring of glucose and lactate concentrations of an animal cell culture designed for the production of recombinant human antithrombine III (AT-III). Results of the on-line measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyser from Yellow Springs Instrument Company (YSI).     

Detection of sulfamethoxazole by a piezoquarz immunosensor]

A mass susceptible immunosensor for FIA of sulfamethoxazole residues in liquid products was designed. The immunosensor is based on piezoelectric transducer. Hapten-protein conjugate (SMX-Diazo-BSA) immobilized on the preliminarily silanized electrode surface of piezoelectric quartz crystal was used as the bioreceptor coating. Optimization of the FIA conditions permitted to develop a simple and express procedure for one-step detection of sulfamethoxazole in a sample and further regeneration of the bioreceptor layer. The measuring ranges are 1 to 50 ng/ml and the detection limit is 0.15 ng/ml. The detection results were compared with the HPLC data. The advantages of the new procedure are its simplicity and rapid provision of the analysis results, possible direct detection of the analyte without additional label and repeated use of the bioreceptor layer. The new immunosensor was applied to testing of various milk specimens. It was shown that the quantity of sulfamethoxazole in all the specimens was lower than the recommended Euroresidue standards (100 ng/ml).     

On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product anlysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was succesfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.Abbreviations TIA Turbidimetric immunoassay - mAb Monoclonal Antibody     

Bienzymatic amperometric sensor for protein assay in milk

A new bienzymatic amperometric sensor is proposed for the assay of the protein content of milk. The sensor is based on two enzymes: carboxypeptidase A and L-amino acid oxidase. The response characteristics obtained for this sensor (detection limit of 1.5 micromol/L, linear concentration range between 1.8 and 2.8 micromol/L), as well as high selectivity over possible interferences from milk, made it applicable as a detector in flow injection analysis (FIA). The response characteristics obtained in the non-equilibrium conditions (FIA system) are: detection limit of 1.5 micromol/L and linear concentration range between 2 and 3.5 micromol/L. Without FIA, the average recovery of proteins from milk and milk products is 99.06 +/- 0.07% and, by utilization of FIA, it increased to 99.73 +/- 0.03. The sensor proved a good reliability for the assay of proteins in milk and milk products.     

Flow injection analysis of serum urea using urease covalently immobilized on 2-fluoro-1-methylpyridinium salt-activated fractogel and fluorescence detection

Serum samples were analyzed for their urea content using fluorescence flow injection analysis incorporating an immobilized urease bioreactor and a gas permeable separator. The urease was immobilized under mild and facile conditions to a hydrophilic 2-fluoro-1-methylpyridinium-activated support. The ammonia released as a result of urease-catalyzed urea hydrolysis diffused through a gas permeable membrane into a constant stream of o-phthaldehyde solution to form a highly fluorescent product with lambda ex at 340 nm and lambda em at 455 nm. Up to 25 serum samples can be analyzed per hour. The within-day coefficient of variation (CV) was 1.12% and the day-to-day CV was 1.25% for serum containing 10.50 mg urea nitrogen dl-1. The bioreactor shows excellent storage (at 4 degrees C) and operational stabilities (at 37 degrees C).