Enzyme electrodes and their application

Starting from the state of the art, principles for improving the analytical characteristics of enzyme electrodes are discussed. Coupling of appropriate amperometric electrode processes with enzyme systems, e.g. urease or aminopeptidases, results in a simplification of operation. Optimal sample frequencies are realized on the basis of enzyme membranes, with both a small characteristic diffusion time and a high enzyme activity, applied in a well-designed sample-processing system. Coupled enzyme reactions of the sequence or competition type are successfully used for extension to new analytes, e.g. inhibitors, cofactors or alternative substrates. Cyclization of the analyte enhances the sensitivity of enzyme electrodes to the nanomolar concentration range. Enzymic anti-interference layers are a tool for improving the sensor specificity. The operational characteristics of enzyme electrodes are thus adaptable to any given analytical problem.     

Physical mapping of the Mycoplasma gallisepticum S6 genome with localization of selected genes.

We report the construction of a physical map of the Mycoplasma gallisepticum S6 genome by field-inversion gel electrophoresis of DNA fragments generated by digestion of genomic DNA with rare-cutting restriction endonucleases. The size of the M. gallisepticum S6 genome was calculated to be approximately 1,054 kb. The loci of several genes have been assigned to the map by Southern hybridization utilizing specific gene probes.     

A reagentless amperometric electrode based on carbon paste, chemically modified with D-lactate dehydrogenase, NAD(+), and mediator containing polymer for D-lactic acid analysis. II. On-line monitoring of fermentation process

The production of D-lactic acid by Lactobacillus delbrueckii (ATCC 9649) during fermentation was monitored on-line with a reagentless D-lactate dehydrogenase modified carbon paste electrode in a flow injection system integrated with a filtration sampling device. The time delay between sampling and detection was approximately 6 min. The use of an electropolymerized ortho-phenylenediamine membrane on the elctrode resulted in a very selective sensor response with acceptable stability and sensitivity. The D-lactate concentrations determined on-line agreed well with those determined by a standard method, suggesting that this sensor system is suitable for on-line monitoring of fermentation processes. (c) 1995 John Wiley & Sons, Inc.     

Nucleic Acid Tools for Invasive Fungal Disease Diagnosis

Current Fungal Infection Reports - This review has incorporated the knowledge and experience of the leads of each of the laboratory working parties of the fungal PCR initiative in order to provide...     

Direct heterogeneous electron transfer of theophylline oxidase

Direct electron transfer (DET) was shown between the heme containing enzyme theophylline oxidase (ThO) and the surface of both graphite and gold electrodes. As proof on graphite a steady state current for theophylline was recorded using the electrode modified with adsorbed ThO. The electrode showed a Michaelis-Menten-like response to theophylline with a detection limit of 0.2 mM and a Michaelis-Menten constant equal to 3.2 mM. These initial results open up a possibility for the development of reagentless third generation biosensor based on heterogeneous DET between ThO and an electrode. On gold DET between ThO and the surface of aldrithiol modified gold was studied with spectroelectrochemical measurements. DET was observed for soluble ThO as a change of its spectrum in a gold capillary responding to a change in the applied potential. It was shown that the redox conversion of the heme domain of the enzyme is directly (mediatorlessly) driven by the potential applied at the gold electrode. The measurements enabled an estimation of the formal potential (E degrees ') of the redox process equal to -275 +/- 50 mV versus Ag|AgClsat at pH 7.0. The experimentally determined number of the electrons involved in this heterogeneous electron transfer process was estimated to be equal to 0.53. The low precision in determination of the E degrees ' and the value of the number of electrons lower than one indicate that kinetic restrictions disturbed the evaluation of the true thermodynamic values from relatively fast spectroelectrochemical measurements.     

Electrocatalytic oxidation of NAD(P) H at mediator-modified electrodes

A review is presented dealing with electrocatalytic NADH oxidation at mediator-modified electrodes, summarising the history of the topic, as well as the present state of the art.     

Characterization of two new multiforms of Trametes pubescens laccase

Electrochemical properties of two multiforms of laccase from Trametes pubescens basidiomycete (LAC1 and LAC2) have been studied. The standard redox potentials of the T1 sites of the enzymes were found to be 746 and 738 mV vs. NHE for LAC1 and LAC2, respectively. Bioelectroreduction of oxygen based on direct electron transfer between each of the two forms of Trametes pubescens laccase and spectrographic graphite electrodes has been demonstrated and studied. It is concluded that the T1 site of laccase is the first electron acceptor, both in solution (homogeneous case) and when the enzymes are adsorbed on the surface of the graphite electrode (heterogeneous case). Thus, the previously proposed mechanism of oxygen bioelectroreduction by adsorbed fungal laccase was additionally confirmed using two forms of the enzyme. Moreover, the assumed need for extracellular laccase to communicate directly and electronically with a solid matrix (lignin) in the course of lignin degradation is discussed. In summary, the possible roles of multiforms of the enzyme based on their electrochemical, biochemical, spectral, and kinetic properties have been suggested to consist in broadening of the substrate specificity of the enzyme, in turn yielding the possibility to dynamically regulate the process of lignin degradation according to the real-time survival needs of the organism.     

Bioelectrocatalytic detection of theophylline at theophylline oxidase electrodes

Bioelectrocatalytic oxidation of theophylline was studied at gold and graphite electrodes modified with microbial theophylline oxidase (ThOx), a multi-cofactor redox enzyme capable of selective oxidation of theophylline. Gold electrodes were additionally modified with self-assembled monolayers (SAMs) of (-OH)- and (-NH(2))-terminated alkanethiols of different chain lengths, to achieve compatibility between ThOx and the electrode surface. On graphite, ThOx was either physically co-adsorbed with a surfactant didodecyldimethylammonium bromide (DDAB), or entrapped within an Os-redox-polymer film. At all electrodes, ThOx was bioelectrocatalytically active; direct electrochemistry of ThOx in the absence of theophylline was followed only at the SAM-modified gold electrodes. Direct electrochemistry of ThOx correlated with redox transformations of the heme domain of ThOx, with a E(o/)of -110+/-2 mV versus Ag|AgCl, at pH 7. Bioelectrocatalytic oxidation of theophylline was optimal at mixed (-OH)/(-NH(2))-terminated SAMs; co-adsorption of ThOx with DDAB improved the bioelectrocatalytic performance of the ThOx-electrode. In both cases, the response to theophylline was within the mM range. Alternatively, a reagentless ThOx-electrode based on ThOx cross-linked within the Os-redox-polymer matrix demonstrated a linear response to theophylline within the physiologically important 0.02-0.6mM (3.6-72 mg l(-1)) concentration range with a sensitivity of 52.1+/-7.8 mA cm(-2)M(-1).     

Redox potentials of the blue copper sites of bilirubin oxidases

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was >650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.     

New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 1. Investigation of the influence of structural parameters on the extent of degradation

Six methyl celluloses (MCs), one with a degree of substitution (DS) of 1.32 and five with DS between 1.83 and 1.88, were thoroughly investigated. Monomer composition and methyl distribution in the polymer chain were analyzed after total or partial random hydrolysis and appropriate derivatization with gas chromatography (GC) and mass spectrometry (MS), respectively, and used as reference data. The same MCs were then hydrolyzed with an enzyme preparation of Trichoderma longibrachiatum and further investigated with size-exclusion chromatography with multiangle light scattering and refractive index detection (SEC-MALS/RI) and MS. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) in combination with various MS analyzers were compared with respect to quantification of the degradation products directly and after perdeuteriomethylation. The methyl group distribution in the oligomeric fractions and the average DS as a function of chain length were calculated from ESI mass spectra. With help of the reference analysis, patterns could be corrected for the unspecific contribution of end groups. By labeling and ESI tandem MS, our knowledge about the tolerance of the enzymes' sub-sites with respect to the number of methyl groups could be improved.