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Cytochrom P450-Elektrochemie
(2001)
Protein multilayers, consisting of cytochrome c (cyt c) and poly(aniline sulfonic acid) (PASA), are investigated by electrochemical quartz crystal microbalance with dissipation monitoring (E-QCM-D). This technique reveals that a four-bilayer assembly has rather rigid properties. A thickness of 16.3 +/- 0.8 nm is calculated with the Sauerbrey equation and is found to be in good agreement with a viscoelastic model. The electroactive amount of cyt c is estimated by the deposited mass under the assumption of 50% coupled water. Temperature-induced stabilization of the multilayer assembly has been investigated in the temperature range between 30 and 45 degrees C. The treatment results in a loss of material and a contraction of the film. The electroactive amount of cyt c also decreases during heating and remains constant after the cooling period. The contraction of the film is accompanied by the enhanced stability of the assembly. In addition, it is found that cyt c and PASA can be assembled at higher temperatures, resulting in the formation of multilayer systems with less dissipation.
Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active site. Since their discovery and the elucidation of their structure, they have attracted the interest of scientist for many years, particularly due to their catalytic abilities. Since the late 1970s attempts have concentrated on the construction and development of electrochemical sensors. Although sensors based on mediated electron transfer have also been constructed, the direct electron transfer approach has attracted most of the interest. This has enabled the investigation of the electrochemical properties of the various isoforms of CYP. Furthermore, CYP utilized to construct biosensors for the determination of substrates important in environmental monitoring, pharmaceutical industry and clinical practice. (c) 2004 Elsevier B. V. All rights reserved
Dendritic polyglycerol-poly(ethylene glycol)-based polymer networks for biosensing application
(2014)
This work describes the formation of a new dendritic polyglycerol-poly(ethylene glycol)-based 3D polymer network as a matrix for immobilization of the redox enzyme periplasmatic aldehyde oxidoreductase to create an electrochemical biosensor. The novel network is built directly on the gold surface, where it simultaneously stabilizes the enzyme for up to 4 days. The prepared biosensors can be used for amperometric detection of benzaldehyde in the range of 0.8-400 mu M.
Determination of phenolic compounds using recombinant tyrosinanse from Streptomyces antibioticus
(2001)
The development of an electrochemical piezoelectric sensor for the detection of glycated hemoglobin is presented. The total hemoglobin (Hb) content is monitored with a mass-sensitive quartz crystal modified with surfactants, and the glycated fraction of the immobilized Hb is determined by subsequent voltarnmetric measurement of the coupled ferroceneboronic acid. Different modifications of the sensor were tested for their hemoglobin binding ability. Deoxycholate (DOCA) was found to be the most suitable among the examined modifiers. Piezoelectric quartz crystals with gold electrodes were modified with DOCA by covalent binding to a pre-formatted 4-aminothiophenol monolayer. The properties of the Hb binding to DOCA and the pH effect on this interaction were studied. In the proposed assay for glycated hemoglobin at first an Hb sample is incubated with ferroceneboronic acid (FcBA), which binds to the fructosyl residue of the glycated Hb. Then this preincubated Hb sample is allowed to interact with the DOCA-modified piezoelectric quartz crystal. The binding is monitored by quartz crystal nanobalance QCN). The amount of FcBA present on the sensor surface is determined by square wave voltammetry. The binding of FcBA results in well-defined peaks with an EO' of +200 mV versus Ag/AgC1 (1 M KC1). The peak height depends on the degree of glycated Hb in the sample ranging from 0% to 20% of total Hb. The regeneration of the sensing surface is achieved by pepsin digestion of the deposited Hb. Thus the sensor can be re-used more than 30 times.
CYP106A2 is one of only a few known steroid hydroxylases of bacterial origin, which might be interesting for biotechnological applications. Despite the enzyme having been studied for more than 30 years, its physiological function remains elusive. To date, there have been no reports of the redox potential of CYP106A2, which was supposed to be unusually low for a cytochrome P450. In this work we show that cyclic voltammetry is not only suitable to determine the redox potential of challenging proteins such as CYP106A2, measured at - 128 mV vs. NHE, but also to study molecular interactions of the enzyme with different interaction partners via the respective electrochemical responses. The effect of small ligands, such as carbon monoxide and cyanide, was observed on the cyclic voltammograms of CYP106A2. Furthermore, we found that Tween 80 caused a positive shift of the redox potential of immobilised CYP106A2 indicative for water expulsion from the haem environment. Moreover, electron transfer mediation phenomena with biological redox partners (e.g. ferredoxins) were studied. Finally, the influence of two different kinds of substrates on the electrochemical response of CYP106A2 was assessed, aligning observations from spectral and electrochemical studies.
Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase
(2009)
In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples (OsHRP)-H-III/(OsHRP)-H-IV, (OsHRP)-H-IV/(OsHRP)-H-V and (OsHRP)-H-V/ (OsHRP)-H-VI. The midpoint potentials differ dependent on the electrode material used with E-1/2 (Os-III/IV) of -0.4 V (ITO) and -0.25 V (GC), E-1/2 (Os-IV/V) of -0.16 V (ITO) and +0.10 V (GC), and E-1/2 (Os-V/VI)of +018 V (ITO), respectively Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.
A nanohybrid consisting of poly(3-aminobenzenesulfonic acid-co-aniline) and multiwalled carbon nanotubes [MWCNT-P(ABS-A)]) on a gold electrode was used to immobilize the hexameric tyrosine-coordinated heme protein (HTHP). The enzyme showed direct electron transfer between the heme group of the protein and the nanostructured surface. Desorption of the noncovalently bound heme from the protein could be excluded by control measurements with adsorbed hemin on aminohexanthiol-modified electrodes. The nanostructuring and the optimised charge characteristics resulted in a higher protein coverage as compared with MUA/MU modified electrodes. The adsorbed enzyme shows catalytic activity for the cathodic H2O2 reduction and oxidation of NADH.
Three different sizes of chitosan-capped Au nanoparticles were synthesized and were used to incorporate Agrocybe aegerita peroxygenase (AaeAPO) onto the surface of glassy carbon electrode. The direct electron transfer of AaeAPO was achieved in all films. The highest amount of electroactive enzyme and highest electron transfer rate constant k(s) of AaeAPO were obtained in the film with the smallest size of chitosan-capped Au nanoparticles.
In anaerobic solutions, quasi-reversible oxidation and reduction are obtained with a formal potential of -0.280V vs. Ag/AgCl 1 M KCl in 100 mM (pH 7.0) PBS at scan rate of 1 V s(-1). Bioelectrocatalytic reduction currents can be obtained with the AaeAPO-modified electrode on addition of hydrogen peroxide. This reaction was suppressed when sodium azide, an inhibitor of AaeAPO, was present. Furthermore, the peroxide-dependent conversion of aniline was characterized and it was found that a polymer product via p-aminophenol is formed. And the AaeAPO biosensor was applied to determine aniline and p-aminophenol.
A method for construction of biosensors with membranous cytochrome P450 isoenzymes was developed based on clay/ detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated cytochrome P450 2134 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bio-electrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay- modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between -0.292 and - 0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bio-electrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of product formation also indicates the bioelectrocatialysis by CYP2B4
The bioelectrocatalytic sulfite oxidation by human sulfite oxidase (hSO) on indium tin oxide (ITO) is reported, which is facilitated by functionalizing of the electrode surface with polyethylenimine (PEI)-entrapped CdS nanoparticles and enzyme. hSO was assembled onto the electrode with a high surface loading of electroactive enzyme. In the presence of sulfite but without additional mediators, a high bioelectrocatalytic current was generated. Reference experiments with only PEI showed direct electron transfer and catalytic activity of hSO, but these were less pronounced. The application of the polyelectrolyte-entrapped quantum dots (QDs) on ITO electrodes provides a compatible surface for enzyme binding with promotion of electron transfer. Variations of the buffer solution conditions, e.g., ionic strength, pH, viscosity, and the effect of oxygen, were studied in order to understand intramolecular and heterogeneous electron transfer from hSO to the electrode. The results are consistent with a model derived for the enzyme by using flash photolysis in solution and spectroelectrochemistry and molecular dynamic simulations of hSO on monolayer-modified gold electrodes. Moreover, for the first time a photoelectrochemical electrode involving immobilized hSO is demonstrated where photoexcitation of the CdS/hSO-modified electrode lead to an enhanced generation of bioelectrocatalytic currents upon sulfite addition. Oxidation starts already at the redox potential of the electron transfer domain of hSO and is greatly increased by application of a small overpotential to the CdS/hSO-modified ITO.