Amperometric Cholesterol Biosensor Using Layer-by-Layer Adsorption Technique on Polyaniline-coated Polyester Films

An amperometric cholesterol biosensor was fabricated using polyaniline-coated polyester films. Polyaniline was dissolved in chloroform with camphorsulfonic acid, and polystyrene was added to this solution. Using this mixed solution, the coating was placed onto polyester films. Cholesterol oxidase was immobilized onto these films using an electrostatic layer-by-layer adsorption technique. Poly(diallyldimethylammonium chloride) was used as the counter ion source. The level of adsorption was examined and evidence of layer-by-layer adsorption was investigated using a quartz crystal microbalance (QCM). A cholesterol biosensor was fabricated from these films as a working electrode, and it was used to measure the cholesterol concentration.     

Evaluation of amperometric glucose biosensors based on glucose oxidase encapsulated within enzymatically synthesized polyaniline and polypyrrole

In this article potential and suitability of enzymatically synthesized conducting polymers polyaniline (PANI) and polypyrrole (PPY) for fabrication of enzymatic amperometric glucose biosensors were evaluated. The polymerisation of these polymers was induced by catalytic activity of glucose oxidase (GOx) from Penicillium vitale cross-linked by glutaraldehyde (GA) on the graphite rod electrode (GOx-electrode) surface. The main precursors for initiation of polymerisation reactions were hydrogen peroxide as an initiator of polymerisation reaction and β-d-gluconic acid as a medium, which reduced the pH towards acidic one is the most suitable for the formation of PANI and PPY. During the polymerisation reactions the immobilized GOx was self-encapsulated within formed PANI or PPY layers in order to form GOx/PANI- and GOx/PPY-modified electrodes (GOx/PANI-electrode and GOx/PPY-electrode, respectively). Kinetic properties of GOx, which is acting as a biocatalyst in GOx/PANI- and GOx/PPY-electrodes, were studied and results were compared with GOx-electrode. The results show that in both GOx/PANI- and GOx/PPY-electrodes self-encapsulated GOx exhibited different parameters of catalysed reaction kinetics due to increasing diffusion limitations if compared with that of the GOx-electrode and it allowed the detection of glucose in a wider concentration interval. Moreover, both GOx/PANI- and GOx/PPY-electrodes exhibited good operational stability and reproducibility of analytical signal. The electrochemical characteristics of formed PANI and PPY in the GOx/PANI- and GOx/PPY-electrodes were also determined. In addition, the influence of temperature, pH and common interfering compounds on the steady-state current response of modified electrodes were investigated and discussed.     

Research on the Repeated Use of Novel Ferrocene-Tagged Nanomaterial for Determination of Glucose

A good novel double ferrocene-tagged nanomaterial is designed for glucose determination and its recycling stability. Double Ferrocene-tagged nanomaterial displays higher catalytic activity toward the glucose oxidation than non–ferrocene-tagged nanomaterial. In this study novel nanoparticles including double ferrocene, N-{2-[Bis(2-aminoethyl)amino]ethyl}aminomethyl-polystyrene (2AEPS) and ferrocene aldehyde (Fc) have been synthesized by means of condensation and investigated the enzymatic properties of glucose oxidase enzyme (GOD) immobilized on there. Double ferrocene-tagged nanomaterial-GOD shows high reusability and storage capacity and fast incubation time determination of glucose. 2AEPSFc-GOD retains more than 15% of the initial activity after forty five successive cycles, which is a perfect performance.     

Copper adaptation and methylotrophic metabolism in Candida boidinii

The importance of the antioxidant enzyme superoxide dismutase (CuZnSOD) in the metabolic switch from normotrophic to methylotrophic conditions was studied in the facultative methylotrophic yeast Candida boidinii. Copper adaptation was performed to qualify C. boidinii as a suitable cellular system to study the effect of induction of CuZnSOD, and other biochemical components along the copper detoxification system, on methanol adaptation. Copper adaptation results in the induction of CuZnSOD peroxidase activity as well as of glutathione. The effects at the metabolic level of exposure to both copper and methanol were also studied: the results suggest that the effect on antioxidant enzyme levels as a function of the change of trophic condition are predominant with respect to the effects of copper administration. Thus, the methanol-dependent induction of such enzymes is likely to provide a sufficient protection for the cells against toxic effects depending on copper administration. Administration of copper under methylotrophic conditions decreases the growth rate in spite of the high levels of antioxidant enzymes that are elicited by copper treatment. The adaptation to methanol metabolism was studied alsoafter methanol-independent induction of CuZnSOD, glutathione and catalase levels, obtained by exposure to high copper concentrations in glucose-containing medium. The metabolic changes induced by copper are persistent over several re-inoculations in normo-cupric glucose medium, thus allowing the study of the glucose-to-methanol switch on cells exhibiting high levels of antioxidant enzyme activities. Under such conditions the lag time observed during the transition from normotrophic to methylotrophic conditions is strongly reduced.     

Reagentless recycling of pyruvate oxidase on a conducting polymer-modified electrode

The enzyme pyruvate oxidase (PyOD) covalently immobilized on an original conducting copolymer poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-thioacetic-1,4-naphthoquinone acid) can be recycled under anaerobic conditions, at +0.1 V versus SCE. It is first demonstrated that the quinone group is an efficient co-substrate for PyOD in homogeneous conditions, then this efficiency is preserved when the quinone group is embedded in the polymer structure. The copolymer remains efficient even in aerated media. The low working potential avoids side-oxidations of interfering species as ascorbic acid or salycilate.     

GLUCOSE OXIDASE REACTION FOR ESTIMATION OF GLUCOSE WITHOUT HORSERADISH PEROXIDASE. SOME MICROBIOLOGICAL AND FERMENTATION APPLICATIONS

An enzymic method has been developed for analysis of glucose. Glucose oxidase acts on glucose to produce hydrogen peroxide which acts to directly reduce the green Cu(II) 2–2′-bicinchoninate complex to a violet complex without horseradish peroxidase. A concentration range of 20–200 μM glucose was used but the reaction shows a linear range of 20–800 μM glucose. Interference is controlled by using a blank determination which has not been treated with glucose oxidase. The reaction has been used to estimate glucose levels in Saccharomyces cerevisiae fermentation and α-amylase, invertase and β-galactosidase reactions and coloured corn-steep fermentation media.     

The molecular mechanism of membrane proteins probed by evanescent infrared waves

The catalytic action of membrane proteins is vital to many cellular processes. Yet the molecular mechanisms remain poorly understood. We describe here the technique of evanescent infrared difference spectroscopy as a tool to decipher the structural changes associated with the enzymatic action of membrane proteins. Functional changes as minute as the protonation state of individual amino acid side chains can be observed and linked to interactions with a ligand, agonist, effector, or redox partner.     

Development of a screen-printed cholesterol biosensor: Comparing the performance of gold and platinum as the working electrode material and fabrication using a self-assembly approach

Gold (Au) and platinum (Pt) were used as the working electrode material to detect cholesterol in solution through enzymatically generated hydrogen peroxide (H₂O₂). Both gold and platinum were capable of detecting cholesterol through the electrochemical oxidation of H₂O₂, and could be used as the working electrode material. By comparison, however, Au was preferable over Pt in terms of higher response current and better sensitivity. Therefore, Au was chosen as the working electrode material for the fabrication of a thick-film screen-printed cholesterol biosensor consisting of three electrodes on an alumina substrate (working: Au, reference: Ag/AgCl, and counter: Au). The immobilization of the enzyme cholesterol oxidase (ChOx, E.C. 1.1.3.6) on the Au working electrode was achieved using a self-assembly approach. A thiol, 3-mercaptopropionic acid (MPA), was self-assembled onto the gold working electrode forming a thin organic layer that served as the anchor for the enzyme immobilization. 1-Ethyl-3(3-dimethylamino propyl)carbodiimide methiodide (EDC) was then used to immobilize the enzyme ChOx covalently on the gold working electrode through the carbodiimide coupling between the carboxyl (–COOH) groups of the self-assembled MPA layer and the amino (–NH₂) groups of the enzyme. Electrochemical measurements showed that this biosensor responded well to cholesterol, confirming that the self-assembly immobilization method was effective. The reproducibility, the interference, and the storage stability of the biosensor were studied and assessed.