A. V. El'skaya

Molecularly imprinted self-assembled films with specificity to cholesterol

Abstract Molecularly imprinted hydrophobic monolayers have been fabricated by spontaneous self-assembly of hexadecyl mercaptan in the presence of cholesterol on gold surfaces. The lypophilic layer of long chain hexadecan have been formed around the template molecules adsorbed on gold surface. The extraction of cholesterol has resulted in formation of sites in the layer of hexadecyl mercaptan which could selectively re-bind the template molecules. The imprinting cavities have been used as channels for potassium ferricyanide which was reduced at the electrode surface. It has been shown, that the molecular recognition process running inside of these channels diminished the mass-transport of potassium ferricyanide to the electrode surface and consequently reduced an electrochemical signal. The change of potassium ferricyanide reduction peak has been related to the template concentration. The formation of the cholesterol-specific monolayers, their specificity and stability has been studied and the possibility of its application for sensor development has been discussed.

Application of urease conductometric biosensor for heavy-metal ion determination

Abstract Urease conductometric biosensors consisting of interdigitated gold electrodes and enzyme membranes formed on their sensitive parts have been used for a quantitative estimation of general water pollution with heavy-metal ions. The measurements of the urease residual activity have been carried out in Tris-HNO3 buffer after preincubation in model metal-salt solution. The detection limits, depending on preincubation time and dynamic ranges, have been determined in model solutions of heavy-metal ions. The sequence of metals ions relative to their toxicity toward urease is: Hg2+ > Cu2+ > Cd2+ > Co2+ > Pb2+ > Sr2+ > . The conditions for practical applications of the biosensors have been investigated and critically evaluated for optimization. Urease reactivation by EDTA after inhibition by heavy-metal ions has been demonstrated. The performance characteristics of the conductometric biosensor are discussed.

Functional Interaction of Mammalian Valyl-tRNA Synthetase with Elongation Factor EF-1α in the Complex with EF-1H

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high M r complex with the four subunits of elongation factor EF-1H. The β, γ, and δ subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1α could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1α and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAValsynthesis catalyzed by the valyl-tRNA synthetase component of the ValRS·EF-1H complex. This effect is not observed in the presence of EF-1α and GDP or EF-Tu·GTP and requires association of valyl-tRNA synthetase within the ValRS·EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1α catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1α·GTP·Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1α·GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.

Thin-film conductometric biosensors for glucose and urea determination☆

The characteristics of the developed conductometric biosensors for urea and glucose determination are described. Conductometric transducers based on thin-film interdigitated metal (Au, Cr, Cu, Ni) electrodes were studied, and enzymes urease and glucose oxidase were used for the selective membranes formation on the chips having gold electrodes. The influence of ionic strength and buffer capacity of the samples on the biosensors response in kinetic and steady-state modes of measurements was thoroughly tested. It was shown that the kinetic response of the sensors does not depend on the buffer capacity of the analyzed sample. In basic features the performance of the developed biosensors is rather close to that of respective enzyme field effect transistor, though the former are much superior when the technological complexity of the transducer itself is considered and taking into account that conductometric sensors require no reference electrode.

Multibiosensor based on enzyme inhibition analysis for determination of different toxic substances

An original concept of an enzyme multibiosensor for determination of toxic substances based on enzyme inhibition analysis has been proposed and its main performances have been analysed. For the development of this multibiosensor, two types of transducers such as potentiometric pH-sensitive field-effect transistors and conductometric thin-films interdigitated electrodes, and three enzymes, namely urease, acetylcholinesterase and butyrylcholinesterase have been used. The experimental data have been treated by multivariate correspondence analysis. A complete procedure for a simultaneous determination of some heavy metal ions and pesticides has been proposed and its advantages have been discussed.

Polyaniline label-based conductometric sensor for IgG detection

Abstract A new method based on a biochemical binding system is developed for detecting the presence and measuring the concentration of a definite analyte in fluids. The conductometric scheme of detection has been employed for detection of the binding reaction of immunoglobulins. Two identical pairs of gold interdigitated electrodes deposited on the insulating solid support have been used as a detector. An electrically conducting polymer, namely polyaniline, has been proposed as a label for immunosensor development. A set of water-soluble forms of polyaniline with different molecular weights and oxidation levels have been prepared by chemical oxidation of aniline and characterized using gel-permeation chromatography. The acceptability of different kinds of polyaniline, and teh selectivity and sensitivity of sensor response have been studied. The method of antibody detection in competitive electroimmunoassay has been developed. The lowest antigen concentration which may be detected in the competitive mode was found to be about 500 nm/ml. The results of impedance spectroscopy measurements obtained for different state of the electrodes are analyzed in order to clarify the mechanism of the sensor response.

Novel conductometric biosensor based on three-enzyme system for selective determination of heavy metal ions.

A differential pair of planar thin-film interdigitated electrodes, deposited on a ceramic pad, was used as a conductometric transducer. The three-enzyme system (invertase, mutarotase, glucose oxidase), immobilized on the transducer surface, was used as a bioselective element. The ratio between enzymes in the membrane was found experimentally considering the highest biosensor sensitivity to substrate (sucrose) and heavy metal ions. Optimal concentration of sucrose for inhibitory analysis was 1.25 mM and incubation time in the investigated solution amounted to 10-20 min. The developed biosensor demonstrated the best sensitivity toward ions Hg(2+) and Ag(+). A principal possibility of the biosensor reactivation either by EDTA solution after inhibition with silver ions or by cysteine solution after inhibition with mercury ions was shown.

Glucose-sensitive ion-sensitive field-effect transistor-based biosensor with additional positively charged membrane. Dynamic range extension and reduction of buffer concentration influence on the sensor response

Glucose-sensitive sensors based on ion-sensitive field-effect transistors were prepared by cross-linking a mixture of glucose oxidase and bovine serum albumin in saturated glutaraldehyde vapour on the sensitive area of the transducer. After this procedure, 4-vinylpyridine-styrene copolymer membranes were deposited on top of the sensors by drop-coating of 0.5% polymer solution in ethanol. The use of this additional membrane resulted in substantial extension of the dynamic range of the sensor (up to 20 mM of glucose) and reduction of buffer concentration dependence of the sensor response. Also the effects of pH and ionic strength were examined for the sensors with and without additional membranes.

Glucose sensitive conductometric biosensor with additional Nafion membrane: reduction of influence of buffer capacity on the sensor response and extension of its dynamic range

Abstract Glucose-sensitive enzyme conductometric biosensors based on interdigitated gold electrodes were prepared by cross-linking glucose oxidase with bovine serum albumin in a saturated glutaraldehyde vapour on the sensor chips. Nafion membranes were deposited on top of the glucose sensor by a spin-coating procedure. The effects of buffer concentration and ionic strength were examined for the glucose sensors with and without Nafion membranes. Additional Nafion membranes resulted in a substantial reduction of the effect of buffer concentration on the sensor response and in an extension of the dynamic range of the sensor up to a glucose concentration of more than 10 mM Moreover the comparison of the properties of the developed conductometric glucose biosensors (operational and storage stability, reproducibility of measurements) demonstrates better performance of the sensor with an additional Nafion membrane.

Overall characterization of ISFET-based glucose biosensor

Abstract Two types of glucose-sensitive enzyme field-effect transistor (ENFET) with glucose oxidase immobilized in polyacrylamide gel (PAAG) and by crosslinking with bovine serum albumin (BSA) have been investigated. These ENFETs with biomembranes demonstrate remarkable versatility in their properties and it is possible to fabricate sensors with the required characteristics. The highest sensitivity to glucose is obtained for an ENFET with a PAAG membrane; it reaches 150 mV/decade in 0.2 mM phosphate buffer, pH 7.3. Enzyme layers of different thicknesses have been produced and the sensor response time greatly depends on this thickness, ranging from about 5 min for thick (⩾ 30 μm) to less than 10 s for thin (⩽ 5 μm) BSA membranes. Conditions for ENFET-based glucose determination in blood have been examined and a procedure for a related glucose test proposed and verified. The effect of such sample characteristics as pH and concentration of working buffer, ionic strength, proteins and oxygen contents have been examined and taken into account.