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An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
An amperometric biosensor for the determination of glycated hemoglobin in human whole blood is proposed. The principle is based on the electrochemical measurement of ferroceneboronic acid (FcBA) that has been specifically bound to the glycated N-terminus. Hemoglobin is immobilized on a zirconium dioxide nanoparticle modified pyrolytic graphite electrode (PGE) in the presence of didodecyldimethylammonium bromide (DDAB). The incubation of this sensor in FcBA solution leads to the formation of an FcBA-modified surface due to the affinity interaction between boronate and the glycated sites of the hemoglobin. The binding of FcBA results in well-defined redox peaks with an E-0' of 0.299 V versus Ag/AgCl (1 M KCl). The square wave voltammetric response of the bound FcBA reflects the amount of glycated hemoglobin at the surface. This signal increases linearily with the degree of glycated hemoglobin from 6.8 to 14.0% of total immobilized hemoglobin. The scheme was applied to the determination of the fraction of glycated hemoglobin in whole blood samples.
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
Electrochemical investigations of the blood oxygen carrier protein include both mediated and direct electron transfer. The reaction of haemoglobin (Hb) with typical mediators, e.g., ferricyanide, can be quantified by measuring the produced ferrocyanide which is equivalent to the Hb concentration. Immobilization of the mediator within the electrode body allows reagentless electrochemical measuring of Hb. On the other hand, entrapment of the protein within layers of polyclectrolytes, lipids, nanoparticles of clay or gold leads to a fast heterogeneous electron exchange of the partially denatured Hb. (c) 2005 Elsevier B.V. All rights reserved
Affinity interaction betwen phenylboronic acid-carrying self-assembled monolayers and FAD or HRP
(2005)
A method is provided for the recognition of glycated molecules based on their binding affinities to boronate- carrying monolayers. The affinity interaction of flavin adenine dinucleotide (FAD) and horseradish peroxidase (HRP) with phenylboronic acid monolayers on gold was investigated by using voltammetric and microgravimetric methods. Conjugates of 3-aminopherrylboronic acid and 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) or 11-mercaptoundecanoic acid were prepared and self-assembled on gold surfaces to generate monolayers. FAD is bound to this modified sur-face and recognized by a pair of redox peaks with a formal potential of -0.433 V in a 0.1 m phosphate buffer solution, pH 6.5. Upon addition of a sugar to the buffer, the bound FAD could be replaced, indicating that the binding is reversible. Voltammetric, mass measurements, and photometric activity assays show that the HRP can also be bound to the interface. This binding is reversible, and HRP can be replaced by sorbitol or removed in acidic solution. The effects of pH, incubation time, and concentration of H2O2 were studied by comparing the catalytic reduction of H2O2 in the presence of the electron-donor thionine. The catalytic current of the HRP-loaded electrode was proportional to HRP concentrations in the incubation solution in the range between 5 mu g mL(-1) and 0.4 mg mL(-1) with a linear slope of 3.34 mu A mL mg(-1) and a correlation coefficient of 0.9945
The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4'-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4'-dithiodipyridin and dithionite modified electrodes. A formal potential (E-0') of -373 mV vs Ag/AgCl 1 M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis. (C) 2003 Elsevier Inc. All rights reserved
Electrochemical properties of beta-(10-phenoxazinyl) propylamine (APPX) and beta-(10-phenoxazinyl) propionic acid (PPX) have been studied in solution, and in immobilized state on gold electrodes modified with monolayers of cystamine and mercaptoundecanoic acid. A reversible diffusion-controlled process of APPX and PPX was observed at a bare gold electrode. The electrochemical conversion of both compounds at modified gold electrodes was a quasireversible diffusion-controlled process. The redox potential of immobilized APPX (443 mV) was similar to the potential in solution, while the value of the immobilized PPX was 131 mV higher than in solution. The immobilized mediators were electrocatalytically active in the fungal peroxidase-catalyzed hydrogen peroxide reduction