Elena Domínguez

Amperometric biosensors based on an apparent direct electron transfer between electrodes and immobilized peroxidases. Plenary lecture

An apparent direct electron transfer between various electrode materials and peroxidases immobilized on the surface of the electrode has been reported in the last few years. An electrocatalytic reduction of hydrogen peroxide stars at about +600 mV versus a saturated calomel (reference) electrode (SCE) at neutral pH. The efficiency of the electrocatalytic current increases as the applied potential is made more negative and starts to level off at about –200 mV versus SCE. Amperometric biosensors for hydrogen peroxide can be constructed with these types of peroxidase modified electrodes. By co-immobilizing a hydrogen peroxide-producing oxidase with the peroxidase, amperometric biosensors can be made that respond to the substrate of the oxidase within a potential range essentially free of interfering electrochemical reactions. Examples of glucose, alcohol and amino acid sensors are shown.

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.

Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors

The developments in the techniques of NADH catalytic oxidation relevant for incorporation in amperometric biosensors with dehydrogenase enzymes are reviewed with special emphasis in the years following 1990. The review stresses the direct electro-catalytic methods of NAD+ recycling as opposed to enzymatic regeneration of the coenzyme. These developments are viewed and evaluated from a mechanistic perspective of recycling of NADH to enzymatically active NAD+, and from the point of view of development of technologically useful reagentless dehydrogenase biosensors. An effort is made to propose a method for the standardization of evaluation of new mediating and direct coenzyme recycling schemes. A perspective is given for the requirements that have to be met for successful biosensor development incorporating dehydrogenase enzymes that open the analytical possibilities to a number of new analytes. The intrinsic limitations of the system are finally discussed and a view of the future of the field is presented.

Tyrosinase graphite-epoxy based composite electrodes for detection of phenols

The characterization and analytical performance of a tyrosinase graphite-epoxy electrode for the detection of phenolic compounds are described. The biocomposite configuration is based on the entrapment of commercially available tyrosinase in a graphite-epoxy matrix, and the mixing of the resulting conductive epoxy resin with a hardener. The enzyme electrode is mounted as a working electrode in an amperometric flow cell of the confined wall-jet type and studied in the flow injection mode. The bioprobe is electrochemically characterized by hydrodynamic and cyclic voltammetry for catechol and phenol. An applied potential of −100 mV vs. Ag/AgCl is found to be optimal for electrochemical reduction of the enzyme products (quinone forms) for the biocomposite electrode. The dependence of the response of the biocomposite on the flow rate, the amount of loaded enzyme, the buffer composition, pH, and oxygen is investigated. The response of the biosensor to different phenolic compounds is also evaluated. The limits of detection (S/N = 3) for phenol and catechol were 1·0 μM and 0·04 μM, respectively. No loss in response could be detected after 100 injections of catechol (R.S.D. <2%). Stability of the biocomposite depends on storage conditions. Theoretical advantages described in the literature for biocomposite electrodes, for example, repolishing and bulk modification, are empirically studied in this work.

Amperometric biosensor for the determination of phenolic compounds using a tyrosinase graphite electrode in a flow injection system

Selective and sensitive devices for the monitoring of phenol and phenolic compounds are required in clinical and environmental analysis. This paper describes a biosensor for the analysis of phenolic compounds in a flow injection system. The enzyme electrode is based on the use of immobilized tyrosinase and the amperometric detection of the enzymatic product at -50 mV vs. SCE. The enzyme is covalently immobilized on the surface of a carbodiimide-activated graphite electrode. The biosensor responds to a variety of phenolic substrates with different conversion efficiencies. The detection limit for phenol is 0.003 microM (S/N = 3), a quantification limit of 0.01 microM (rsd 3.7%), and an extended dynamic range up to 5 microM is achieved with a sample frequency of 110 samples per hour.

Reagentless chemically modified carbon paste electrode based on a phenothiazine polymer derivative and yeast alcohol dehydrogenase for the analysis of ethanol

Amperometric biosensors for ethanol were constructed by immobilizing yeast alcohol dehydrogenase (ADH) on carbon paste (graphite powder: paraffin oil) chemically modified with a polymer to which a necessary mediator for electrocatalytic NADH oxidation had been covalently attached. Three different approaches were tested: adsorption of ADH on the electrode surface and adding NAD+ to the contacting buffer; having both ADH and NAD+ mixed into the paste; and co-immobilizing ADH and NAD+ in the paste by adding polyethylenimine to the reaction mixture. The last approach resulted in the highest and fastest response. When inserted into a flow injection system, the biosensor responded linearly to ethanol between 2 μm and 3 mm. The biosensor operates at +100 mV vs. Ag/AgCl.

Phenol oxidase-based biosensors as selective detection units in column liquid chromatography for the determination of phenolic compounds

Abstract Amperometric biosensors of two types based on the phenol oxidase tyrosinase (EC 1.18.14.1, monophenol monooxygenase) are presented. The enzyme was immobilised either on solid graphite electrodes or in carbon paste electrodes. The performance of the two biosensors was investigated with respect to immobilisation technique, pH, flow-rate and oxygen dependence. The use of detergents in the mobile phase was shown to greatly influence activity, selectivity, and operational stability of the biosensors. One of the developed biosensors was further used as a selective and sensitive detector in a column liquid chromatographic system for the determination of phenolic compounds in a spiked wastewater sample.

Reagentless biosensors based on self-deposited redox polyelectrolyte-oxidoreductases architectures

Reagentless fructose and alcohol biosensors have been produced with a versatile enzyme immobilisation technique which mimics natural interactions and flexibility of living systems. The electrode architecture is built up on electrostatic interactions by the sequential adsorption of redox polyelectrolytes and redox enzymes giving rise to the efficient transformation of substrate fluxes into electrocatalytic currents. All investigated multilayer structures were self-deposited on 3-mercapto-1-propanesulfonic acid monolayers self-assembled on gold electrodes. Fructose dehydrogenase, horseradish peroxidase (HRP) and the couple HRP-alcohol oxidase were electrochemically connected with a cationic poly[(vinylpyridine)Os(bpy)2Cl] redox polymer (RP) interface in a layer-by-layer self-deposited architecture. The dependence of the distance on the electrochemical response of this interface was also studied showing a clear decrease in the Faradaic current when the distance to the electrode surface was increased. The sensitivities obtained for each biosensor were 19.3, 58.1 and 10.6 mA M(-1) cm(-1) for fructose, H2O2 and methanol, respectively. The sensitivity values can be easily controlled by a rational deposition and manipulation of the charge in the catalytic layers. The electrostatic assembly of the electrochemical interface and the catalytic layers resulted in integrated biochemical systems in which mass transfer diffusion and heterogeneous catalytic and electron transfer steps are efficiently coupled and can be easily manipulated.

Electrochemical detection of DNA hybridization based on DNA-templated assembly of silver cluster

Abstract The growth of metals on DNA templates has generated considerable interest in connection to the design of metallic nanostructures. Here we exploit the DNA-induced generation of metal clusters for developing an electrical biosensing protocol. The new hybridization assay employs a probe-modified gold surface, and is based on the electrostatic ‘collection’ of silver cations along the DNA duplex, the reductive formation of silver nanoclusters along the DNA backbone, dissolution of the silver aggregate and stripping potentiometric detection of the dissolved silver at a thick-film carbon electrode. The new protocol thus combines the inherent signal amplification of stripping analysis with effective discrimination against nonhybridized DNA.

Rate-Limiting Steps of Tyrosinase-Modified Electrodes for the Detection of Catechol

The response currents obtained for tyrosinase-modified Teflon/graphite, carbon paste, and solid graphite electrodes in the presence of catechol are analyzed primarily using rotating disk electrode experiments. The rate-limiting steps, such as the electrochemical reduction of o-quinones and the enzymatic reduction of oxygen as well as the enzymatic oxidation of catechol, are theoretically considered and experimentally demonstrated for the different electrode configurations.