O. O. Soldatkin

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.

Enzyme biosensor systems based on porous silicon photoluminescence for detection of glucose, urea and heavy metals

A phenomenon of changes in photoluminescence of porous silicon at variations in medium pH is proposed to be used as a basis for the biosensor system development. The method of conversion of a biochemical signal into an optical one is applied for direct determination of glucose and urea as well as for inhibitory analysis of heavy metal ions. Changes in the quantum yield of porous silicon photoluminescence occur at varying pH of the tested solution due to the enzyme-substrate reaction. When creating the biosensor systems, the enzymes urease and glucose oxidase (GOD) were used as a bioselective material; their optimal concentrations were experimentally determined. It was shown that the photoluminescence intensity of porous silicon increased by 1.7 times when increasing glucose concentration in the GOD-containing reaction medium from 0 to 3.0mM, and decreased by 1.45 times at the same increase in the urea concentration in the urease-containing reaction medium. The calibration curves of dependence of the biosensor system responses on the substrate concentrations are presented. It is shown that the presence of heavy metal ions (Cu(2+), Pb(2+), and Cd(2+)) in the tested solution causes an inhibition of the enzymatic reactions catalyzed by glucose oxidase and urease, which results in a restoration of the photoluminescence quantum yield of porous silicon. It is proposed to use this effect for the inhibitory analysis of heavy metal ions.

A novel urea conductometric biosensor based on zeolite immobilized urease

A new approach was developed for urea determination where a thin film of silicalite and zeolite Beta deposited onto gold electrodes of a conductometric biosensor was used to immobilize the enzyme. Biosensor responses, operational and storage stabilities were compared with results obtained from the standard membrane methods for the same measurements. For this purpose, different surface modification techniques, which are simply named as Zeolite Membrane Transducers (ZMTs) and Zeolite Coated Transducers (ZCTs) were compared with Standard Membrane Transducers (SMTs). Silicalite and zeolite Beta with Si/Al ratios 40, 50 and 60 were used to modify the conductometric electrodes and to study the biosensor responses as a function of changing zeolitic parameters. During the measurements using ZCT electrodes, there was no need for any cross-linker to immobilize urease, which allowed the direct evaluation of the effect of changing Si/Al ratio for the same type of zeolite on the biosensor responses for the first time. It was seen that silicalite and zeolite Beta added electrodes in all cases lead to increased responses with respect to SMTs. The responses obtained from ZCTs were always higher than ZMTs as well. The responses obtained from zeolite Beta modified ZMTs and ZCTs increased as a function of increasing Si/Al ratio, which might be due to the increased hydrophobicity and/or the acid strength of the medium.

Development of conductometric biosensor array for simultaneous determination of maltose, lactose, sucrose and glucose.

The aim of this work was to develop an array of biosensors for simultaneous determination of four carbohydrates in solution. Several enzyme systems selective to lactose, maltose, sucrose and glucose were immobilised on the surface of four conductometric transducers and served as bio-recognition elements of the biosensor array. Direct enzyme analysis carried out by the developed biosensors was highly sensitive to the corresponding substrates. The analysis lasted 2 min. The dynamic range of substrate determination extended from 0.001 mM to 1.0-3.0mM, and strongly depended on the enzyme system used. An effect of the solution pH, ionic strength and buffer capacity on the biosensors responses was investigated; the conditions of simultaneous operation of all biosensors were optimised. The data on cross-impact of the substrates of all biosensors were obtained; the biosensor selectivity towards possible interfering carbohydrates was tested. The developed biosensor array showed good signal reproducibility and storage stability. The biosensor array is suited for simultaneous, quick, simple, and selective determination of maltose, lactose, sucrose and glucose.

Monitoring of the velocity of high-affinity glutamate uptake by isolated brain nerve terminals using amperometric glutamate biosensor.

Glutamate is the major excitatory neurotransmitter in the central nervous system, which is involved in the main aspects of normal brain functioning. High-affinity Na(+)-dependent glutamate transporters is key proteins, which transport extracellular glutamate to the cytoplasm of nerve cells, thereby preventing continuous activation of glutamate receptors, and thus the development of neurotoxicity. Disturbance in glutamate uptake is involved in the pathogenesis of major neurological disorders. Amperometric biosensors are the most promising and successful among electrochemical biosensors. In this study, we developed (1) amperometric glutamate biosensor, (2) methodological approach for the analysis of glutamate uptake in liquid samples of isolated rat brain nerve terminals (synaptosomes). The basal level of glutamate, the initial velocity of glutamate uptake and time-dependent accumulation of glutamate by synaptosomes were determined using developed glutamate biosensor. Comparative analysis of the data with those obtained by radioactive analysis, spectrofluorimetry and ion exchange chromatography was performed. Therefore, the methodological approach for monitoring of the velocity of glutamate uptake, which takes into consideration the definite level of endogenous glutamate in nerve terminals, was developed using glutamate biosensor.

Microbiosensor based on glucose oxidase and hexokinase co-immobilised on platinum microelectrode for selective ATP detection

ATP determination is of great importance since this compound is involved in a number of vital biological processes. To monitor ATP concentration levels, we have developed a microbiosensor based on cylindrical platinum microelectrode, covered with a layer of poly-m-phenylendiamine (PPD), and layer of co-immobilised glucose oxidase and hexokinase. Conditions for biosensor measurement of ATP (pH, Mg(2+) and substrates concentration) in vitro and microbiosensor characteristics such as sensitivity, selectivity, reproducibility, storage stability were studied and optimized. Under optimal conditions the microbiosensor can measure ATP concentrations down to a 2.5 microM detection limit with response time about 15 s. Interferences by electroactive compounds like biogenic amines and their metabolites, ascorbic acid, uric acid and L-cystein are rejected in general by the PPD layer. The microbiosensor developed is insensitive to ATP analogues (or substances with similar structure), such as ADP, AMP, GTP and UTP, too. It can be used for ATP analysis in vitro in the reactions consuming or producing macroergic triphosphate molecules to study kinetics of the process and in drug design concerning development of inhibitors specific to target kinases and others target enzymes.

Development of novel enzyme potentiometric biosensor based on pH-sensitive field-effect transistors for aflatoxin B1 analysis in real samples

This study aimed at the development and optimization of a potentiometric biosensor based on pH-sensitive field-effect transistors and acetylcholinesterase for aflatoxin B1 determination in real samples. Optimal conditions for bioselective elements operation were defined and analytical characteristics of the proposed biosensor were studied. The proposed biosensor characterized high operational stability and reproducibility of signal. Selectivity of acetylcholinesterase-biosensor to aflatoxins in relation to other groups of toxic substances was analyzed. The developed biosensor was applied to the determination of aflatoxin B1 in real samples (sesame, walnut and pea).

Application of enzyme/zeolite sensor for urea analysis in serum.

Urea biosensor based on zeolite-adsorbed urease was applied for analysis of blood serum samples. It should be noted, that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, high reproducibility and repeatability (RSD=9% and 4%, respectively). The linear range of urea determination by using the biosensor was 0.003-0.75 mM, and the limit of urea detection was 3 μM. The method of standard addition was used for analysis of serum samples with 500-fold dilution. Total time of analysis was 10 min. Good reproducibility of urea determination in real samples was demonstrated (RSD=10%). Biosensor results were verified by using a common method of urea determination (diacetyl monoxime reaction). It was shown that by using this biosensor distinguishing healthy people from people with renal dysfunction becomes easier.

Study of zeolite influence on analytical characteristics of urea biosensor based on ion-selective field-effect transistors

A possibility of the creation of potentiometric biosensor by adsorption of enzyme urease on zeolite was investigated. Several variants of zeolites (nano beta, calcinated nano beta, silicalite, and nano L) were chosen for experiments. The surface of pH-sensitive field-effect transistors was modified with particles of zeolites, and then the enzyme was adsorbed. As a control, we used the method of enzyme immobilization in glutaraldehyde vapour (without zeolites). It was shown that all used zeolites can serve as adsorbents (with different effectiveness). The biosensors obtained by urease adsorption on zeolites were characterized by good analytical parameters (signal reproducibility, linear range, detection limit and the minimal drift factor of a baseline). In this work, it was shown that modification of the surface of pH-sensitive field-effect transistors with zeolites can improve some characteristics of biosensors.

A novel biosensor method for surfactant determination based on acetylcholinesterase inhibition

A novel enzyme biosensor based on acetylcholinesterase inhibition for the determination of surfactants in aqueous solutions is described. Acetylcholinesterase-based bioselective element was deposited via glutaraldehyde on the surface of conductometric transducers. Different variants of inhibitory analysis of surfactants were tested, and finally surfactant’s concentration was evaluated by measuring initial rate of acetylcholinesterase inhibition. Besides, we studied the effect of solution characteristics on working parameters of the biosensor for direct measurement of acetylcholine and for inhibitory determination of surfactants. The biosensor’s sensitivity to anionic and cationic surfactants (0.35 mg l −1 ) was tested. The high operational stability of the biosensor during determination of acetylcholine (RSD 2%) and surfactants (RSD 11%) was shown. Finally, we discussed the selectivity of the biosensor toward surfactants and other AChE inhibitors. The proposed biosensor can be used as a component of the multibiosensor for ecological monitoring of toxicants.