Alcohol-FET sensor based on a complex cell membrane enzyme system

An alcohol -FET sensor was developed by use of a complex enzyme system in a cell membrane and an ion-sensitive field effect transistor (ISFET). The cell membrane of Gluconobacter suboxydans IFO 12528, which converts ethanol to acetic acid, was immobilized on the gate of an ISFET with calcium alginate gel coated with nitrocellulose. This ISFET (1), a reference ISFET without the cell membrane (ISFET 2) and an Ag/AgCl reference electrode were placed in 5 mM Trismalate buffer (pH 5.5, 25°C), and the differential output between ISFETS 1 and 2 was measured. The output of the sensor was stabilized by adding pyrroloquinoline quinone. The response time was ca. 10 min., and there was a linear relationship between the differential output voltage and the ethanol concentration up to 20 mg l^?1. The output of the sensor was stable for 40 h below 30°C. The sensor responded to ethanol, propan- 1-ol and butan- 1-ol, but not to methanol, propan-2-ol and butan-2-ol. The sensor was used to determine blood ethanol.     

Alcohol,lactate and glutamate sensors based on oxidoreductases with regeneration of nicotinamide adenine dinucleotide

Flowthrough enzyme electrodes are reported for determinations of alcohol, lactate and glutamate. Oxidoreductases mixed with immobilized NAD⁺ cofactor are held between a suitable platinum electrode and a semipermeable membrane. The coenzyme is readily regenerated either directly by electrochemical oxidation or by using phenazine methosulphate (PMS⁺) as intermediate. Continuous flow conditions are used. The sensitivity obtained with the alcohol dehydrogenase electrode was 50, 620 or 810 nA mol^-1 of ethanol, respectively, when regeneration was done electrochemically or with 0.1 or 0.5 mM PMS⁺. The sensitivities for the lactate and glutamate sensors in the presence of 0.5 mM PMS⁺, were 14 and 50 nA mmol^-1 for D,L-lactate and L-glutamate, respectively. The calibration curves were linear for concentrations up to 0.5, 1.5 and 100 mM of glutamate, lactate and ethanol, respectively. The sensitivity of the alcohol and lactate sensors decreased by 50–55% within 60 h and that of the glutamate sensor within 6 h.     

Development of a hypoxanthine biosensor based on immobilized xanthine oxidase chemically modified electrode

An amperometric biosensor for hypoxanthine determination has been developed. The sensor uses a Nafion-paraquat chemically modified glassy-carbon electrode. It detects the oxygen consumed by the enzymatic reaction catalyzed by xanthine oxidase which is immobilized on the electrode surface. The sensor responds linearly to hypoxanthine over the concentration range of 1 × 10⁻⁶ M −2 × 10⁻⁴ M. The biosensor can be reused for more than 100 times without significant deterioration in performance. After 32 days storage at 3–5°C, the sensor response remains at 68% of the initial level. The high sensitivity, selectivity and stability of this biosensor demonstrates its practical applicability for a simple, rapid and economical determination of hypoxanthine in fish samples.     

Amperometric determination of sulfite with sulfite oxidase immobilized at a platinum electrode surface

Sulfite oxidase is immobilized on collagen membrane at the surface of a platinum electrode and catalyzes the oxidation of sulfite to sulfate with stoichiometric production of hydrogen peroxide. The hydrogen peroxide is detected amperometically at the platinum electrode at an applied potential of 700 mV. The system responds linearly to sulfite in the range 1–150 μM, with a detection limit of 0.2 μM. The enzyme retains over 95% of its activity for three weeks if stored at ?20° C when the probe is not in use.     

A Bioanode Using Thermostable Alcohol Dehydrogenase for an Ethanol Biofuel Cell Operating at High Temperatures

To extend the range of biofuel cell applications, we wish to increase their maximum operational temperatures. Using a thermostable alcohol dehydrogenase as a biocatalyst, we prepared an enzyme‐immobilized bioanode that can operate at high temperatures. The catalytic current for ethanol oxidation was increased using this electrode at temperatures up to 80 °C.     

Glucose sensors based on enzyme immobilization onto biocompatible membranes obtained by radiation-induced polymerization

Amperometric glucose biosensors based on glucose oxidase immobilized onto poly(2-hydroxyethylmethacrylate) membranes obtained by γ radiation-induced polymerization were constructed. In a threeelectrode configuration, smooth or platinized platinum electrodes with different shapes were used, in order to detect the amount of hydrogen peroxide produced in the glucose oxidation. A saturated calomel electrode and a platinum foil were used as a reference and counterelectrode, respectively. The biocompatible obtained sensors were characterized as regards the temperature effect, the response, and lifetime. The determination of glucose in standard solutions was carried out, and linear calibration curves were obtained. Depending on the electrode configuration, the sensor had a response time of 1–4 min, and the measuring range extended from 5 × 10^?5 to 4 × 10^?3M.     

mga Genosensor for Early Detection of Human Rheumatic Heart Disease

The 5′ amino-labeled DNA probe complementary to mga gene of Streptococcus pyogenes was immobilized on carboxylated multiwall carbon nanotubes electrode and hybridized with 0.1–100 ng/6 μl single-stranded genomic DNA (ssG-DNA) of S. pyogenes from throat swab of suspected rheumatic heart disease (RHD) patients. Electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance (EI). The sensitivity of the sensor was 106.03?(μA/cm²)/ng and limit of detection (LOD) was found 0.014 ng/6 μl with regression coefficient (R ²) of 0.921 using DPV. The genosensor was characterized by FTIR and SEM, and electrode was found stable for 6 months on storage at 4 °C with 5–6 % loss in initial DPV current. mga genosensor is the first report on RHD sensor which can save life of several suspected patients by early diagnosis in 30 min.     

Development,Investigation of Parameters and Estimation of Possibility of Adaptation of Pichia angusta Based Microbial Sensor for Ethanol Detection

Abstract

The analytical parameters of recognizing element of a biosensor for ethanol detection have been estimated in three wild type strains of Pichia angusta. The possibility of increasing sensor selectivity through induction and inhibition of intracellular enzymes in the course of biomass cultivation has been studied. A biosensor based on the cells of strain P. angusta VKM Y‐2518 grown on 1% methanol proved to be the most prospective for ethanol detection. This sensor was insensitive to carbohydrates and organic acids; the interfering compound at ethanol detection was methanol. The lower limit of ethanol detection was 0.012 mM. The optimum of sensor response dependence on pH and ion force of the buffer solution was in the range of pH 7.2–7.6 and around 30 mM, respectively. Indications of the sensor were stable for 5 days. The stability of cells was studied at storage as wet centrifuged biomass at 4°C and in immobilized state at 20, 4, and ?10°C. The storage of centrifuged cells at 4°C proved to be optimal. The sensor based on P. angusta VKM Y‐2518 was used for ethanol detection in alcoholic beverage; besides, it can be used for ethanol detection in biological fluids and at optimization of enzymatic processes.     

Impact of High Temperature on Ethanol Fermentation by Kluyveromyces marxianus Immobilized on Banana Leaf Sheath Pieces

Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C). Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared. Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30–40 °C. Temperature of 45 °C restricted yeast growth and lengthened the fermentation. The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures. Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress. The free cells of K. marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane. For fermentation at 40 °C with immobilized cells of K. marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level.     

Impedance-based total-NOx sensor using stabilized zirconia and ZnCr2O4 sensing electrode operating at high temperature

We report here a new-type zirconia-based sensor that can detect total NO_x content at high temperatures such as 700 °C. A closed-one-end yttria-stabilized zirconia (YSZ) tube was used as a base sensor material. An oxide sensing electrode (SE) and a Pt counter electrode (CE) were formed on the outer and inner surfaces of the YSZ tube, respectively. The complex impedance of the device using a ZnCr₂O₄-sensing electrode was measured with an impedance analyzer in the frequency and the temperature ranges 0.1 Hz–100 kHz and 600–700 °C, respectively. A large semicircular arc was observed in complex impedance plots (Cole–Cole plots) in the lower frequency range examined and it seemed to correspond to the electrolyte/electrode interface. The impedance value at 1 Hz of the present device was found to vary almost linearly with the concentration of NO (or NO₂) from 50 to 400 ppm in the sample gas at 600–700 °C. Furthermore, it is noted that the sensitivity of NO is almost equal to that of NO₂. This means that the present device can detect the total NO_x at higher temperatures.     

Development of a disposable miniature l-lysine sensor

A hybrid l-lysine sensor consisting of an immobilized l-lysine decarboxylase and a miniature bacterial CO₂ sensor was fabricated using semiconductor techniques. The bacteria was immobilized in a calcium alginate gel in a miniature oxygen electrode cell together with the electrolyte. The enzyme was immobilized in a bovine serum albumin matrix on a gas-permeable membrane. The cell was formed on a silicon substrate by anisotropic etching and had a two-gold-electrode configuration. The response time of the l-lysine sensor was 1–3 min. The optimum pH was 6.0 and the optimum temperature was 33°C. The response to l-lysine concentration was linear from 25 to 400 μM. Reproducible responses were obtained by adding more than 1 μM pyridoxal-5′-phosphate. The sensor had excellent selectivity for l-lysine and a stable response for more than 25 repetitive operations.     

Carbon Dioxide Sensor Using Thermophilic Bacteria

Abstract

The amperometric carbon dioxide sensor we developed uses chemoautotrophic thermophilic bacteria and an oxygen-sensing electrode. The sensor was fabricated by packing the sensitive area of the bicarbonate sensor in a cell with a gas-permeable membrane on one side. The membrane was constantly supplied with an oxygen-saturated buffer solution. The sensor's operating range was 34°C to 58°C. A linear relationship was obtained in a 1-mM to 8-mM NaHCO₃ concentration in a buffer solution (pH 5.5) and a 3% to 12% CO₂ concentration in air. The effect of the sensor's low sensitivity to sodium acetate and ethanol was greatly improved by supplying fresh, oxygen-saturated buffer solution. The sensor had a life of more than a month.     

An interference-free first generation alcohol biosensor based on a gold electrode modified by an overoxidised non-conducting polypyrrole film

An interference-free first generation alcohol biosensor based on alcohol oxidase immobilized, by glutaraldehyde co-crosslinking with bovine serum albumin, on a perm-selective electropolymerized film modified gold electrode is described. The use, in place of platinum, of a gold electrode, properly conditioned by an electrochemical pre-treatment, allowed the elimination of the electrode fouling and of the interference caused by the direct alcohol oxidation at the electrode surface. The efficiency of overoxidized polypyrrole and poly-ortho-phenylendiamine as anti-interferent perm-selective films towards the most common electroactive interferents was also investigated.The low response time and the fabrication procedure employed in this study allowed the use of the biosensor in a flow injection system equipped with a conventional thin-layer electrochemical cell.The biosensor showed a high sensitivity, a good stability and a complete suppression of electroactive interferences in operational conditions. Biosensor performances were tested by the rapid determination of the alcohol content in just diluted wine samples and the results compared well with those obtained with the classical method of distillation.     

A study of cryostructuring of polymer systems. 46. physicochemical properties and microstructure of poly(vinyl alcohol) cryogels formed from polymer solutions in mixtures of dimethyl sulfoxide with low-molecular-mass alcohols

Poly(vinyl alcohol) (PVA) cryogels (PVACGs) are obtained and studied. The PVACGs are formed by freezing–defrosting of polymer solutions in dimethyl sulfoxide (DMSO) or its mixtures with one of the first members of the series low-molecular-mass aliphatic alcohols (methanol, ethanol, n-propanol, and n-butanol). PVA content in these solutions is 100 g/L, while the concentration of an aliphatic alcohol is varied in a range of 0.44–2.55 mol/L depending on its nature. The polymer solutions are subjected to the cryogenic treatment at temperatures 30, 40, or 50°C lower than the crystallization temperature of DMSO (+18.4°C). The frozen samples are defrosted at a heating rate of 0.03°C/min. It is shown that, in a certain range of lowmolecular-mass alcohol content in an initial system, its cryogenic treatment yields coarse-pored heterophase cryogels that have higher rigidity and heat endurance than those of DMSO–PVA cryogels. It has been shown that polymer cryoconcentration and phase separation play important roles in the formation of a cellular microstructure and an increase in the rigidity and heat endurance of PVACGs obtained in the presence of low-molecular-mass alcohols.     

A microsensor for adenosine-5′-triphosphate pH-sensitive field effect transistors

The sensor for adenosine-5′-triphosphate (ATP) is based on H⁺-ATPase immobilized via a polyvinylbutyral resin on a pH-sensitive field effect transistor. A linear relationship was obtained between the initial rate of change of the differential gate output voltage and the logarithm of the ATP concentration over the range 0.2–1.0 mM ATP. The optimum pH was 9.0 at 40°C but pH 7.0 was preferred for routine measurements. Only slight responses were obtained for 1 mM glucose, creatinine or urea. The ATP-sensing system exhibited a response to 1 mM ATP for at least 18 days.     

Sensitive phenol determination based on co-modifying tyrosinase and palygorskite on glassy carbon electrode

A sensitive tyrosinase biosensor, based on co-modifying tyrosinase and palygorskite on glassy carbon electrode, was developed for phenol analysis. Palygorskite, a kind of natural one-dimensional clay with good biocompatibility, high specific surface area and porous morphology, works as a perfect matrix of enzyme. Tyrosinase retains its inherent bioactivity when immobilized in palygorskite, which leads to a high sensitivity of 1.897 A mol^?1 L. The sensor response achieves 95% of steady-state-current in no more than 3 s, and the linear range of the bioelectrode spans the concentration of phenol from 5?×?10^?8 to 1?×?10^?4 mol L^?1 with a correlation coefficient of 0.9992. The results show no apparent decrease in the response over 2 weeks, and about 80% of the response was retained after 2 months when the electrode was stored at 4–5 °C.     

Kinetics of ethanol production from carob pods extract by immobilizedSaccharomyces cerevisiae cells

Kinetics of ethanol production from carob pods extract by immobilizedS. cerevisiae cells in static and shake flask fermentation have been investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The optimum values of ethanol concentration, ethanol productivity, ethanol yield, and fermentation efficiency were obtained at pH range 3.5–6.5 and temperature between 30–35°C. A maximum ethanol concentration (65 g/L), ethanol productivity (8.3 g/Lh), ethanol yield (0.44 g/g), and fermentation efficiency (95%) was achieved at an initial sugar concentration of 200, 150, 100, and 200 g/L, respectively. The highest values of specific ethanol production rate and specific sugar uptake rate were obtained at pH 6.5, temperature 40°C, and initial sugar concentration of 100 g/L. Other kinetic parameters, biomass concentration, biomass yield, and specific biomass production rate were maximum at pH 5.5, temperature 30°C, and initial sugar concentration 150 g/L. Under the same fermentation conditions non-sterilized carob pod extract gave higher ethanol concentration than sterilized medium. In repeated batch fermentations, the immobilizedS. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 5 d.     

A New Amperometric Carbon Paste Enzyme Electrode for Ethanol Determination

Abstract

In this study, a new amperometric carbon paste enzyme electrode for determination of ethanol was developed. The carbon paste was prepared by mixing alcohol dehydrogenase, its coenzyme nicotinamide adenine dinucleotide (oxidized form, NAD⁺), poly(vinylferrocene) (PVF) that was used as a mediator, graphite powder and paraffin oil, then the paste was placed into cavity of a glass electrode body. Determination of ethanol was performed by oxidation of nicotinamide adenine dinucleotide (reduced form, NADH) generated enzymatically at +0.7 V. The effects of enzyme, coenzyme and PVF amounts; pH; buffer concentration and temperature were investigated. The linear working range of the enzyme electrode was 4.0×10^?4–4.5×10^?3 M, determination limit was 3.9×10^?4 M and response time was 50 s. The optimum pH, buffer concentration, temperature, and amounts of enzyme, NAD⁺ and PVF for enzyme electrode were found to be 8.5, 0.10 M, 37°C, 2.0, 6.0, and 12.0 mg, respectively. The storage stability of enzyme electrode at +4°C was 7 days. Enzyme electrode was used for determination of ethanol in two different wine samples and results were in good agreement with those obtained by gas chromatography.     

Semiconductor gas sensors in bioreactor control

A simple semiconductor gas sensor (TGS 812) is used for the on-line measurement and control of indole during the production of l-tryptophan from indole and l-serine with immobilized E. coli cells. Indole is estimated in the reactor gas space. In combination with an automatic indole supply system, a feed-batch process became possible. The indole concentration was monitored and kept within the optimal range (300–600 mg l^?1). A simple gas-sensing electrode dipped in the reaction medium provides direct measurement of organic solvents and gases in the liquid. Such a system is suitable for on-line determination of ethanol (10–70 g l^?1) during continuous production of ethanol with immobilized yeast cells.     

Production of ethanol from waste paper using immobilized yeasts

This work is aimed at a selection of yeast strains suitable for simultaneous saccharification and fermentation of waste paper. The waste paper, as a lignocellulosic material, represents an unconventional source for the production of ethanol which is a promising alternative fuel. The yeast strains Saccharomyces cerevisiae and Pichia kudriavzevii produced the highest amounts of ethanol at 30 °C and were also resistant at 40 °C during the first 92 h of fermentation. These two strains were immobilized by entrapment into poly(vinyl alcohol) hydrogel lens-shaped particles LentiKats^®. The immobilized S. cerevisiae was a better ethanol producer and retained higher metabolic activity in repeated batch fermentations than P. kudriavzevii. The immobilized S. cerevisiae was also suitable for a long-term storage, with 23% decrease in the ethanol production ability after 1-year storage of yeast cells.