Biosensoren auf der Basis von Redoxhydrogel-integrierter Quinohemoprotein Alkoholdehydrogenase für die On-Line-Detektion von Ethanol in Wein

Weine aus der Tokaj-Region in Ungarn stellen eine komplizierte Matrix für die Analytik mit Hilfe von Biosensoren dar. Sie zeichnen sich durch gleichzeitig hohe Alkohol- und Glucosekonzentrationen aus. Die geographische Lage des Anbaugebietes bedingt ein regionales Mikroklima, das den Befall der Trauben durch die Edelfäule Botrytis cinerea fördert. Der Metabolismus dieses Pilzes erhöht den Glyceringehalt der erhaltenen Weine um ein Vielfaches und führt zur Bildung von weiteren, die Analytik störenden Stoffen. Amperometrische Biosensoren auf der Basis von Oxidasen als biologischer Erkennungskomponente erfordern ein hohes Arbeitspotential von 600 mV [vs. Ag/AgCl] zur Oxidation des katalytisch gebildeten H2O2. Dies führt zur direkten Oxidation von Inhaltsstoffen der komplexen Probenmatrix an der Elektrode und so zu einer geringen Spezifität dieser Sensoren. Darüber hinaus verfügen die kommerziell erhältlichen Alkoholoxidasen über eine breite Gruppenselektivität und Methanol ist das bevorzugte Substrat. Folglich muß die Entwicklung von Biosensoren für die Bestimmung von Ethanol bzw. Glucose in Wein eine erhebliche Verbesserung der Selektivität zu Folge haben, wobei zum einen die Wahl eines geeigneten Enzyms, zum anderen eine Elektrodenarchitektur mit verringertem Arbeitspotential wesentlich ist

Tobacco peroxidase as a new reagent for amperometric biosensors

The results of testing a new enzyme, anionic tobacco peroxidase (TOP), in various amperometric biosensors are summarized. The biochemical and electrochemical properties of the enzyme are briefly characterized. As compared to the commonly used cationic peroxidase from horseradish roots, TOP exhibits a wider optimum stability pH range, higher stability to inactivation with hydrogen peroxide, and higher efficiency in direct electron-transfer processes. The enzyme immobilized by adsorption on graphite is effective in determining aminophenols and aromatic diamines under flow conditions with a detection limit of 10 nM. Upon immobilization on graphite by incorporation into a get of a redox-active polymer (crosslinked polyvinylimidazole with osmium 4,4'-dimethylbipyridinium chloride), TOP exhibited sensitivity and stability comparable to those of horseradish peroxidase and a wider linearity range. Upon immobilization on a self-assembled thiol monolayer at a gold electrode, TOP was much superior to horseradish peroxidase in the sensitivity of determining hydrogen peroxide, regardless of the charge of the monolayer. Prospects for the further use of the native enzyme and its genetically engineered unglycosylated form are considered

Glycerol dehydrogenase based amperometric biosensor for monitoring of glycerol in alcoholic beverages

Biosensors for measurement of glycerol in FIA were constructed using NAD(+)-dependent glycerol dehydrogenase (GlDH) either co-immobilized with phenazine methosulphate (PMS) or cross-linked to an Os-complex-modified poly(vinylimidazole) redox polymer (PVI(13)dmeOs) using poly(ethyleneglycole) diglycidilether (PEGDGE). The GlDH/PMS sensor was characterized by a linear range of 0.01-1mM glycerol, a sensitivity of 1.83mA/Mcm(2), a detection limit (calculated as three times the signal-to-noise ratio) of 0.9muM, and with 50% residual activity kept after 15h of continuous operation at a sample throughput of 30 injections/h. The redox hydrogel-based biosensors showed the same dynamic range, but improved biosensors characteristics, i.e., a sensitivity of 4.79 mA/Mcm, a detection limit of 0.1 muM, and a signal loss of only 20% after 15h of operation in the same conditions. The optimized biosensor configurations were further used for analysis of glycerol in wine and the obtained results were compared with the ones obtained by spectrophotometrical experiments

Amperometric Biosensor For Ethanol Based On A Phenothiazine Derivative Modified Carbon Paste Electrode

A new amperometric biosensor for ethanol, based on carbon paste electrode modified with alcohol dehydrogenase (ADH), polyethylenimine (PEI) and using a phenothiazine derivative (DDDP; 16H,18H-dibenzo[c,1]7,9-dithia-16,18-diazapentacene) as redox mediator for NADH recycling, was developed. The biosensor response is the result of mediated oxidation of NADH, generated in the enzymatic reaction between ADH and ethanol (in the presence of NAD(+)). The biosensor sensitivity (calculated as the ratio I-max/K-M(app)) was 0.035 mA M-1 and the detection limit was 0.26 mM, while the linear response range was from 0.1 to 20 mM ethanol

Synthesis, properties and biosensor application of cycloruthenated 2-phenylimidazoles

The cycloruthenation of 2-phenylimidazole (phim) by [Ru(6-C6H6)(-Cl)Cl]2 in acetonitrile in the presence of NaOH has been carried out. The unstable intermediate [Ru(phim)(MeCN)4]PF6 formed has been converted into the complexes [Ru(phim)(4,4′-Me2bpy)(MeCN)2]PF6 (2) and [Ru(phim)(LL)2]PF6 (3, LL=phen (a), bpy, 4,4′-Me2bpy), which were characterized by the mass-spectrometry, 1H-NMR spectroscopy, UV–vis spectrophotometry, and cyclic voltammetry. The RuII/III redox potentials of complexes 3 equal 130–250 mV (vs. Ag–AgCl) at pH 7 (0.01 M phosphate). Such potential range is favorable for fast exchange of electrons with the active sites of redox enzymes. In fact, the second-order rate constant for the oxidation of reduced glucose oxidase (GO) from Aspergillus niger by the electrochemically generated RuIII derivative of complex 3a equals (8.1×106 M−1 s−1). The second-order rate constant for the oxidation of 3a by the Compound II of horseradish peroxidase is 9.3×107 M−1 s−1. Complexes 3 were used as mediators for the fabrication of enzyme electrodes by simple co-adsorbing with GO or horseradish peroxidase on graphite electrodes. These electrodes were tested in flow-injection systems and showed linear responses in the range of -glucose and H2O2 concentrations 0.1–30 mM and 1–200 M, respectively. The new mediators reported herein seem promising for the construction of amperometric biosensors based on GO, horseradish peroxidase, and similar enzymes

Reagentless Amperometric Biosensor For Nadh Detection

A sensitive and selective amperometric biosensor, based on NADH-oxidase (NADOx) working in tandem with horseradish peroxidase (HRP), which is electrically connected to graphite electrode via Os-redox polymer (RP), was developed. Two designs were considered for the biosensor optimization: (i) the first one, [G/HRP-NADOx], exploited the direct electron transfer between HRP and the graphite electrode (G); (ii) the second one, [G/RP-HR-P-NADOx], used R-P as mediator. In both cases the enzyme matrix was cross-linked using poly(ethylenglycol) diglycidyl ether. The biosensors sensitivities, estimated as the slopes of linear ranges from the calibration curves, recorded in a single line flow injection setup, showed that G/RP-HRP-NADOx electrode (1.62 +/- 0.05 mA M-1) presents a higher efficiency than G/HRP-NADOx electrode (0.30 +/- 0.03 mA M-1)

Simultaneous detection of ethanol, glucose and glycerol in wines using PQQ - dependent dehydrogenase based biosensors

Amperometric biosensors based on corresponding dehydrogenases have been developed for the determination of ethanol, glucose and glycerol. The enzymes have been integrated in redox hydrogels using an Os complex-modified non-conducting polymer employed as the electrochemical mediator and poly(ethyleneglycol)-diglycidyl ether (PEGDGE) as the cross-linking agent. The developed biosensors showed a sensitive response to ethanol, glucose and glycerol within the concentration range 2.5–250, 20–800, and 1–200 M, detection limits of 1.2, 9 and 1 M, and sensitivities of 220, 87 and 32 mA M−1 cm−2, respectively. The ethanol, glucose and glycerol content of several types of wine was determined with these biosensors, and the results were compared with those obtained by spectrophotometric methods. The developed biosensors have been successfully employed for simultaneous determination of all these substrates at the required sensitivity

Adaptive responses to static conditions in nutrient-rich cultures of luminous Ralstonia eutropha

The lux-gene fused Ralstonia eutropha, when adapting to static conditions, causes stratification of air-exposed and nutrient-rich cultures at above 0.15 mg biomass ml(-1). The O-2 respiring biofilm (luminous neuston) phase, along with the dark sub-neustonic suspension phase, develops within 5-60 min. The instability of the biphasic static culture was identified as a reason for occasionally observable oscillatory bioluminescence

Design, viualisation and utilisation of enzyme microstructures built on solid surfaces

Abstract in Undetermined Immobilized bioelements offer additional sensitivity and/or selectivity in analytical techniques, in biotechnological devices, and in different medical applications. Great research interest has recently been focused in all these fields on miniaturization techniques, special attention being accorded to the patterning of biomolecules (e.g. enzymes) on solid surfaces with micrometer resolution. Novel microanalysis systems consider the use of enzyme microstructures as e.g. part of mu- Total Analysis System platforms as biochemical microreactors or detection units. This mini-review highlights recent advances in the creation of enzyme microstructures by indirect methods (photolithography, microcontact printing. etc.) and active placement methods (electrospraying, microdispensing, etc.). Some key visualization techniques of enzyme microstructures (fluorescence microscopy, scanning electrochemical microscopy, etc.) are also mentioned together with examples of their applications or application possibilities

Amperometric determination of acetate with a tri-enzyme based sensor

The present work describes a flow injection analysis system incorporating amperometric biosensors for acetate monitoring. Tri-enzyme-modified electrodes were fabricated by immobilizing acetate kinase (AK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) into a poly(ethyleneglycole) diglycidyl ether (PEGDGE) film also containing Brilliant Cresyl Blue (BCB) as an electrochemical mediator. The concentration of acetate could be determined from the decrease in the amperometric current at +50 mV versus Ag/AgCl. Parameters, such as composition of the biosensing film, operational conditions (e.g., applied potential, flow rate) were optimized. The optimized biosensor showed a linear response to acetate in the range of 0.2-8 mM with a detection limit of 0.13 mM (calculated as 3 SIN). The acetate content of different samples (wine and vinegar) could be reliably quantified with the described biosensor