Development and optimization of a novel conductometric bi-enzyme biosensor for L-arginine determination.

A highly sensitive conductometric biosensor for l-arginine determination was developed by exploiting the unique biorecognition capacities of two enzymes of urea cycle - arginase (E.C. 3.5.3.1) and urease (E.C. 3.5.1.5). The enzymes were co-immobilized in a single bioselective membrane on the working sensor, while a lysine rich bovine serum albumin (BSA) membrane was immobilized on the reference sensor, allowing differential measurements. The optimum percentage ratio of arginase and urease within the bioselective membrane was determined when the biosensor sensitivity to l-arginine and urea was optimum. Analytical characteristics of the conductometric biosensor for l-arginine determination were compared for two types of enzyme immobilization (cross-linking with glutaraldehyde (GA) and entrapment in the polymeric membrane). The optimum features in terms of the sensitivity, the linear range, and the detection limit (4.2 µS/mM, 0.01-4mM, and 5.0 × 10(-7)M, respectively) were found for l-arginine biosensor based on enzyme cross-linking with GA. A quantitative determination of l-arginine in the real sample (a drinkable solution "Arginine Veyron") gave a satisfactory result compared to the data provided by the producer (a relative error was 4.6%). The developed biosensor showed high operational and storage stability.

[Kinetic properties of butyrylcholinesterases immobilised on pH-sensitive field-effect transistor surface and inhibitory action of steroidal glycoalkaloids on these enzymes].

The inhibitory action of steroid glycoalkaloids alpha-solanine, alpha-chaconine and tomatine on horse and human serum butyryl cholinesterases immobilized on the pH-sensitive field-effect transistors has been studied. Using acetyl- and butyryl choline as substrates, the optimal pH and the apparent kinetic parameters (< K(m) >, < V(max) >) of immobilized butyryl cholinesterases have been calculated in the absence of inhibitors. The affinity of each enzyme to glycoalkaloids has been estimated from calculation of apparent inhibition constants < K(i) > and inhibition coefficients i(0.5). Application of the studied cholinesterases for biosensoric determination of glycoalkaloids in the wide range of concentrations (10(-7)-10(-4) M) in different media has been discussed.

[Study of the interaction of main potato glycoalkaloids in inhibition of immobilized butyryl cholinesterase].

The interaction of main potato glycoalkaloids alpha-solanine and alpha-chaconine in inhibition of horse serum butyryl cholinesterases immobilized on the pH-sensitive field-effect transistors has been investigated. The method of isobol diagram of Loewe and Muishnek has been used for interpretation of results. It has been shown the alpha-chaconine inhibits the immobilized bytyryl cholinesterases more strongly than alpha-solanine, and their mixture has the addition effect.

[Amperometric biosensor for ethanol analysis in wines and grape must during wine fermentation].

The amperometric biosensor for ethanol determination based on alcohol oxidase immobilised by the method of electrochemical polymerization has been developed. The industrial screen-printed platinum electrodes were used as transducers for creation of amperometric alcohol biosensor. Optimal conditions for electrochemical deposition of an active membrane with alcohol oxidase has been determined. Biosensors are characterised by good reproducibility and operational stability with minimal detection limit of ethanol 8 x 10(-5) M. The good correlation of results for ethanol detection in wine and during wine fermentation by using the developed amperometric biosensor with the data obtained by the standard methods was shown (r = 0.995).

[Amperometric biosensor for lactate analysis in wines and grape must during fermentation].

The amperometric biosensor based on lactate oxidase for determination of lactate has been developed, and two methods of immobilization of lactate oxidase on the surface of industrial screen-printed platinum electrodes SensLab were compared. A sensor with immobilized in the Resydrol polymer lactate oxidase by the method of physical adsorption is characterized of narrow dynamic range and greater response value in comparison with a biosensor based on immobilised in poly(3,4-ethylenedioxythiophene) lactate oxidase by the method of electrochemical polymerization. Operational stability of the biosensor developed was studied and it was shown, that the immobilization method does not influence their stability. The analysis of the lactate in wine and during wine fermentation has been conducted. High correlation of the data obtained by means of amperometric lactate biosensor and a standard method of an ionic chromatography has been shown. The developed biosensor could be applied in the food industry for the control and optimization of the wine fermentation process, and quality control of wine.

[Development of enzyme biosensor based on trypsin and conductometric thin-film electrodes for protein and artificial substrates determination]. / Enzimosensor na osnovi tripsinu ta konduktometrichnykh planarnykh elektrodiv dlia viznacheniia vmistu bilkiv peptidnikh substrativ u vodnikh rozchinakh

Two analytical systems based on the soluble and immobilized trypsin and conductometric thin-film electrodes were developed to measure concentration of artificial substrate and protein in solution. It was shown that these systems allow one to determine concentrations of Ha-benzoyl-L-arginin-ethyl-ester in the range of 0.1-1.0 mM and concentrations of HSA: 0.1-2.0 mg/ml with soluble and 0.1-0.8 mg/ml with immobilized trypsin.

[A conductometric enzymatic glucose sensor. A search for ways to improve analytical characteristics]. / Konduktometrychnyi fermentnyi gliukosensor. Poshuk shliakhiv polipshennia analitychnykh kharakterystyk.

Characteristics of conductometric enzyme glucosensor based on thin-film interdigitated electrodes have been studied. The obtained biosensor exhibits a linear dynamic range of 0.01-2 mM for glucose and sensitivity 5 microS/min*mM for kinetic mode of measurements and 8 microS/mM for steady-state mode of measurements. Additional 2% PVB- and NAFION-membranes are suggested for the extension of the dynamic range. This permits extending the dynamic range of the sensor up to glicose concentration of 10 mM. Dependence of activity of immobilized enzyme on ionic strength and buffer capacity of the solution has been studied. It was shown that formation of additional membranes results in the substantial reduction of the buffer concentration effect on the sensor response. The obtained results are interpreted in terms of permselective properties of the additional membrane formed.

[A conductometric method of measuring enzymatic catalysis]. / Konduktometrychnyi metod u fermentatyvnomu katalizi.

Theoretical bases of the conductometric method of measurements are considered, respective formulas and equations are presented. The possibilities of application of this method for registration of the enzyme processes are shown, as well as examples of its use in conductometric classic procedure and in conductometric enzyme biosensors. Different methods of immobilization of the biological material on the surface of the transducers, schemes and methods of measurements are presented, the advantages and disadvantages of this method as compared with general biochemical methods are discussed.