Julija Razumiene

New bioorganometallic ferrocene derivatives as efficient mediators for glucose and ethanol biosensors based on PQQ-dependent dehydrogenases

One known and two new ferrocene-containing mediators incorporating the organometallic moiety and the fragments of natural substrates of oxidative enzymes, viz. 4-ferrocenylphenol (FP), 2-ferrocenyl-4-nitrophenol (FNP), and N -(4-hydroxybenzylidene)-4ferrocenylaniline (HBFA), were studied as electron transfer mediators between the coenzyme pyrroloquinoline quinone (PQQ) of glucose (GDH) and alcohol (ADH) dehydrogenases and the carbon electrode surface. A screen-printed carbon electrode (SPCE) suitable for ADH and GDH immobilization served as a transducer. The electrodes were integrated into a flow-through amperometric cell. All data were obtained at a flow rate of 1 ml min � 1 . The maximal currents (jmax) obtained from the calibration curves for the oxidation of ethanol and D-glucose by ADH and GDH of 2.3 and 3.0 mA, respectively, were obtained when SPCE was modified with HBFA, i.e. with a mediator with a longer arm and a high degree of conjugation. The biosensors were used for ethanol and D-glucose measurements in beverages. There was a good correspondence (r � /0.978 for D-glucose and r � /0.920 for ethanol) between the data obtained by using the biosensors, on one hand, and by the refractometric or hydrometric methods, on the other. The operational stability of biosensors is determined by the inactivation of the immobilized enzymes rather than by leakage of a mediator from an electrode. # 2002 Elsevier Science B.V. All rights reserved.

4-Ferrocenylphenol as an electron transfer mediator in PQQ-dependent alcohol and glucose dehydrogenase-catalyzed reactions

Abstract Enzyme electrodes containing pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) as a biological component in combination with 4-ferrocenylphenol ( 1 ) as an electron transfer mediator between PQQ and a carbon electrode were constructed and used for measurements of ethanol and d -glucose. Analysis of the current response of the carbon electrodes modified with 1 at different pH and potentials demonstrated that 1 participates in the bioelectrocatalytic oxidation of d -glucose or ethanol. The biosensors showed the highest response at pH 5.5 and the working potentials of 0.3 and 0.4 V (versus Ag|AgCl) for ADH and GDH, respectively. The electrocatalytic processes under such conditions at these electrodes are characterized by the apparent values of the Michaelis constants K M app of 7.1 and 13 mM and the maximal current density j max 40 and 26 μA cm −2 for ethanol and d -glucose, respectively. No electrocatalysis was found when glucose oxidase from Aspergillus niger was used instead of GDH.

Direct bioelectrocatalysis at carbon electrodes modified with quinohemoprotein alcohol dehydrogenase from Gluconobacter sp. 33

A newly isolated, purified, and characterized PQQ-dependent alcohol dehydrogenase (a bacterial membrane-bound protein) was recently found to display a surprisingly large linear range and high selectivity towards ethanol when integrated into a conducting polymer network on a platinum electrode. These findings motivated us to study the enzyme when simply immobilized onto carbonaceous surfaces in order to establish its characteristics and suitability for sensor development, the sensor design being based on a direct-electron transfer pathway. Graphite rods and screen-printed electrodes were modified in two different ways, and were operated both in FIA and batch mode. The obtained biosensor characteristics were highly dependent on the sensor architecture, the highest sensitivity (179 mA M-1 cm(-2)) and lowest detection limit (1 muM) being obtained for screen-printed electrodes used in a batch mode. A mechanism of the observed direct electron transfer between the enzymes active center and the electrode is proposed. (Less)

Modified graphitized carbon black as transducing material for reagentless H2O2 and enzyme sensors

Direct electron transfer between redox enzymes and electrodes is the basis for the third generation biosensors. We established direct electron transfer between quinohemoprotein alcohol dehydrogenase (PQQ-ADH) and modified carbon black (CBs) electrodes. Furthermore, for the first time, this phenomenon was observed for pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH). Reagentless enzyme biosensors suitable for the determination of ethanol, glucose and sensors for hydrogen peroxide were designed using CB electrodes and screen-printing technique. Aiming to create an optimal transducing material for biosensors, a set of CB batches was synthesized using the matrix of Plackett-Burman experimental design. Depending on the obtained surface functional groups as well as the nano-scale carbon structures in CBs batches, the maximal direct electron transfer current of glucose and ethanol biosensors can vary from 20 to 300 nA and from 30 to 6300 nA for glucose and ethanol, respectively. Using modified CB electrodes, an electrocatalytic oxidation of H(2)O(2) takes place at more negative potentials (0.1-0.4V versus Ag/AgCl). Moreover, H(2)O(2) oxidation efficiency depends on the amount and morphology of fine fraction in the modified CBs.

An oxygen-independent ethanol sensor based on quinohemoprotein alcohol dehydrogenase covalently bound to a functionalized polypyrrole film

In the present work the characteristics of a phenazine methosulphate mediated alcohol biosensor based on a newly isolated quinohemoprotein alcohol dehydrogenase are described. The enzyme was covalently linked at a functionalized polypyrrole film which had been electrochemically deposited on the surface of a platinum-black electrode. The biosensor architecture developed was characterized with regard to sensitivity, selectivity, and long-term operational stability. Owing to the inherent properties of the new enzyme the related biosensors are oxygen-independent and exhibit improved selectivity to ethanol in contrast to alcohol biosensors based on alcohol oxidase or on cationic nicotinamide adenine dinucleotide dependent alcohol dehydrogenase.

Bioelectrochemical Conversion of Urea on Carbon Black Electrode and Application

The amperometric biosensor for urea determination is designed. The decomposition product of urea produced by urease is oxidized in an electrochemical way. The specially developed carbon black (CB) paste electrode is covered by a semipermeable membrane containing immobilized urease. Three types of the urea biosensor action are identified. At low electrode working potentials (0-0.1 V), one electron electrochemical oxidation of carbamic acid can be monitored. Cation-radical of the carbamic acid whereupon undergoes dimerization to hydrazine. At higher electrode potentials (0.2-0.5 V), the electro-oxidation of both carbamic acid and hydrazine are observed. In addition, at more high potential (> 0.6 V), electro-oxidation of ammonia and amination of the electrode surface are observed. The first type of biosensor is less serviceable because of the slow equilibrium process. However, the third type of biosensor, because of the high potential of action and irreproducible response, is also not valuable. The working potential of 0.35 V is selected for optimal urease-CB electrode operation, and the response properties of the electrode are also characterized. The biosensor possesses a linear range of response up to 5 mM of urea, a coefficient of variation equaling 3.7%, a response time of 1.5 min. The biosensor is tested for urea detection in milk.

Modelling carbon nanotubes-based mediatorless biosensor.

This paper presents a mathematical model of carbon nanotubes-based mediatorless biosensor. The developed model is based on nonlinear non-stationary reaction-diffusion equations. The model involves four layers (compartments): a layer of enzyme solution entrapped on a terylene membrane, a layer of the single walled carbon nanotubes deposited on a perforated membrane, and an outer diffusion layer. The biosensor response and sensitivity are investigated by changing the model parameters with a special emphasis on the mediatorless transfer of the electrons in the layer of the enzyme-loaded carbon nanotubes. The numerical simulation at transient and steady state conditions was carried out using the finite difference technique. The mathematical model and the numerical solution were validated by experimental data. The obtained agreement between the simulation results and the experimental data was admissible at different concentrations of the substrate.

Development of Multi-parameter Analyser based on Electrochemical Urea Biosensors and Electrolyte Electrodes for Monitoring of Hemodialysis Patients

The idea of developing multi-parameter urea analyser comprising urea, Na+ and K+ selective electrodes has been considered. For this purpose the urea biosensors based on urease and recombinant urease working in amperometric and potentiometric way were developed. The working parameters of both urea biosensors were studied and optimized. Possibilities of real samples analysis using the developed biosensors were shown by measuring urea concentrations in blood dialysate taken from patients with renal failure. Both the potentiometric and the amperometric biosensors demonstrated high degree of signal reproducibility (the relative standard deviation of responses did not exceed 5 %). Change of sodium and potassium concentrations during blood hemodialysis is dangerous life-threatening condition and their monitoring is an important feature of point-of-care analyser. For this purpose high integrity commercial Na+ and K + selective electrodes were analysed and our own signal amplification and processing system proposed.

Amperometric Urea Sensor

The prototype of amperometric biosensor for urea determination was designed. The enzyme electrode, made of a specially developed modified graphite (MG) paste, was produced by covering the electrode surface with adjustable membrane containing immobilized urease from Canavalia ensiformis (E.C. 3.5.1.5.). Simple methodology of urea determination in real time has been proposed. The experimental study and the mathematical model of the biosensor action have been performed.

Reagentless and mediator-based electrochemical biosensors for food industry and medicine

FAD containing dehydrogenases could donate electrons to Prussian Blue coated electrodes and could operate in the absence of oxygen. The same electrode could also be used for the reduction of hydrogen peroxide produced at low potentials or oxidation of hydrogen peroxide at higher potentials. These features were demonstrated using a glycerol phosphate oxidase as an example. The next group of carbon electrodes was used for the electro-oxidation of the products of urea hydrolysis catalyzed by urease. Three different mechanisms of action were proposed depending on the electrode potential. Electrochemical urea biosensors operating at low potentials (0-0.3 V) were designed. During the third approach the chemically modified carbon particles were applied for the wiring of PQQ-dependent dehydrogenases. Reagentless biosensors for glucose and other monosaccharides were created.