Arkadi V. Eremenko

Organic-phase biosensing of enzyme inhibitors

Abstract Organic-phase biosensors, suitable for monitoring low levels of enzyme inhibitors in non-aqueous media, are described. The inhibition of tyrosinase and horseradish peroxidase by thiourea, benzoic acid, diethyldithiocarbamate, hydroxylammonium sulphate and mercaptoethanol is exploited for highly sensitive amperometric measurements in organic media. Fast on-line monitoring of various inhibitors is illustrated in a flow-injection operation, with an enzyme inhibition based detector and an acetonitrile carrier solution. Repetitive injections of a 1 × 10 −4 M diethyldithiocarbamate solution (at a rate of 60 samples per hour) yielded a relative standard deviation of 3.3%. Sensing advantages accrue from such operation, particularly applicability to inhibitors with poor water solubility and solvent-induced changes in the mechanism of inhibition, are discussed.

A laccase electrode for organic-phase enzymatic assays

The biocatalytic activity of catalase in non-aqueous environments is exploited for developing an organic-phase amperometric biosensor. The catalase is immobilized within the Eastman-AQ film on the glassy carbon surface. Various experimental variables influencing the response of the t-butyl hydroperoxide substrate are explored for optimum performance.

Monomolecular enzyme films stabilized by amphiphilic polyelectrolytes for biosensor devices

Abstract This paper describes the use of new amphiphilic polyelectrolytes for protein immobilization. Monomolecular films of glucose oxidase (GO) and monoamine oxidase (MAO) stabilized by amphiphilic polyelectrolytes (polyethyleneimine and poly-4-vinylpyridine derivatives modified by lauryl chains) were formed on a water surface in a Langmuir-Blodgett trough. The compressed films containing the enzymes were transferred onto the surface of a polypropylene membrane of a Clark electrode according to the Langmuir-Schaefer method. The analytical responses of the resulting biosensors were linear over the range 1–10 mM of glucose and 8–100 μM of tyramine. Furthermore, direct functional coupling of GO and ferrocenecarboxylic acid in multilayer films stabilized by amphiphilic polyelectrolytes was demonstrated. The amperometric response of such a sensor was linear over the range 1–20 mM of glucose. The dependence of the kinetic parameters of the enzymes on the amphiphilic polyelectrolyte structures is discussed.

Biosensor analysis of blood esterases for organophosphorus compounds exposure assessment: approaches to simultaneous determination of several esterases.

This paper reviews our previously published data and presents new results on biosensor assay of blood esterases. Tyrosinase and choline oxidase biosensors based on nanostructured polyelectrolyte films were developed for these purposes. Experiments were performed on the quantitative determination of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CaE), and neuropathy target esterase (NTE) in samples of whole blood of rats, mice, and humans. Good agreement was found between biosensor and spectrophotometric assays for AChE, BChE, and CaE. No direct comparison could be made for NTE because its activity cannot be measured spectrophotometrically in whole blood. A new method of simultaneous quantitative determination of AChE and BChE in test mixtures is also described. This method represents a bifunctional biosensor for the simultaneous analysis of choline and phenol based on integration of individual sensors. Algorithms for calculation of separate concentrations of AChE and BChE in the mixture were developed. The mean error of calculated component concentrations was approximately 6% for binary test mixtures. The present work provides a foundation for building multiplexed systems for the simultaneous determination of multiple esterases with applications to biomonitoring for exposures to organophosphorus compounds.