Biomedical subjects

A P Soldatkin

Publications and source records attributed to A P Soldatkin.

At least 19 recordsLinked to original sources

PubMed2005

Carbon fibre-based microbiosensors for in vivo measurements of acetylcholine and choline.

This report describes technical improvements to the manufacture of a carbon fibre electrode for the stable and sensitive detection of H2O2 (detection limit at 0.5 microM). This electrode was also modified through the co-immobilisation of acetylcholinesterase (AChE) and/or choline oxidase (ChOx) in a bovine serum albumin (BSA) membrane for the development of a sensor for in vivo measurements of acetylcholine and choline. Amperometric measurements were performed using a conventional three-electrode system forming part of a flow-injection set-up at an applied potential of 800-1100 mV relative to an Ag/AgCl reference electrode. The optimised biosensor obtained was reproducible and stable, and exhibited a detection limit of 1 microM for both acetylcholine and choline. However, due to the high operating potential used, the biosensor was prone to substantial interference from other electroactive compounds, such as ascorbic acid. Therefore, in a further step, a mediated electron transfer approach was used that incorporated horseradish peroxidase into an osmium-based redox hydrogel layered onto the active surface of the electrode. Afterwards, a Nafion layer and a coating containing AChE and/or ChOx co-immobilised in a BSA membrane were successively deposited. This procedure further increased the selectivity of the biosensor, when operated in the same flow-injection system but at an applied potential of -50 mV relative to an Ag/AgCl reference electrode. The sensor exhibited good selectivity and a high sensitivity over a concentration range (0.3-100 microM) suitable for the measurement of choline and acetylcholine in vivo.

Acetylcholine↗

PubMed2003

A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs.

A novel urea biosensor based on immobilised recombinant urease as sensitive element and ion sensitive field effect transistor as transducer was developed. Recombinant urease from E. coli with an increased Km was photoimmobilised in PVA/SbQ (poly(vinyl alcohol) containing styrylpyridinium) membrane and has demonstrated quite good performance as biosensitive element. Enzymatic field effect transistors based on such a bioselective element were studied in model buffer solutions. This biosensor demonstrated an extended dynamic range up to 80 mM, a quite good reproducibility (standard deviation of the sensor responses was approximately 2.5%, n= 20 for urea concentration 10 mM) and a high stability. Such characteristics fit with the analytical requirements needed for urea control in plasma and liquids used during renal dialysis.

Biosensing Techniques↗

PubMed2003

Use of competitive inhibition for driving sensitivity and dynamic range of urea ENFETs.

An urea biosensor based on urease-BSA (bovine serum albumin) membrane immobilised on the surface of an ion-sensitive field effect transistor (ISFET) has been studied in a mix buffer solution composed of potassium phosphate, Tris, citric acid and sodium tetraborate. In this mix buffer, the biosensor showed a dynamic larger than the one observed in a phosphate or Tris buffer. Investigation of the individual effect of each component of the buffer solution on the biosensor response has shown that tetraborate anion acts as a strong competitive inhibitor for the hydrolysis reaction of urea catalysed by urease. The biosensor response was investigated in a phosphate buffer with different concentrations of tetraborate anion. The results showed that the apparent constant of Michaelis-Menten, K(m(app)), increases from 4.3 to 79.3 mM, for experiments realised without and with 0.5 mM sodium tetraborate, respectively. The mean value, determined graphically, for the inhibition constant, K(i), was 29 microM. The graphical representation of biosensor calibration curves in semilogarithmic co-ordinates showed that the linear range of the biosensor can be extended up to three orders of magnitude, allowing an urea detection in a concentration range 0-100 mM.

Biosensing Techniques↗

PubMed2002

A novel enzyme biosensor for steroidal glycoalkaloids detection based on pH-sensitive field effect transistors.

For the design of a biosensor sensitive to steroidal glycoalkaloids, pH-Sensitive Field Effect Transistors as transducers and immobilised butyrylcholinesterase as a biorecognition element have been used. The total potato glycoalcaloids can be measured by this biosensor in the concentration range 0.5-100 microM with detection limits of 0.5 microM for alpha-chaconine and of 2.0 microM for alpha-solanine and solanidine, respectively. The responses of the developed biosensors were reproducible with a relative standard deviation of about 1.5% and 5% for intra- and inter-sensor responses (both cases, n=10, for an alkaloid concentration of 5 microM), respectively. Moreover, due to the reversibility of the enzyme inhibition, the same sensor chip with immobilised butyrylcholinesterase can be used several times (for at least 100 measurements) after a simple washing by a buffer solution and can be stored at 4 degrees C for at least 3 months without any significant loss of the enzymatic activity.

Alkaloids↗

PubMed2002

Development of enzyme biosensor based on pH-sensitive field-effect transistors for detection of phenolic compounds.

This article describes a biosensor based on pH-sensitive field-effect transistors (pH-FETs) as transducer, and immobilised enzyme tyrosinase as biorecognition element, which was used for the determination of phenolic compounds in water solutions. The biologically active membrane was formed by cross-linking of tyrosinase with bovine serum albumin (BSA) in saturated glutaraldehyde (GA) vapours on the sensitive transducer surface. The main analytical characteristics were studied under different conditions as well as the possibility to optimise these working parameters. Different factors such as the pH of immobilisation, the enzyme loading, the time of exposition to glutaraldehyde vapours were investigated in regards to the influence on sensitivity, limit of detection, dynamic range, and operational and storage stability.

Biosensing Techniques↗

PubMed2000

Development of highly selective and stable potentiometric sensors for formaldehyde determination.

Two types of biosensors selective to formaldehyde have been developed on the basis of pH-sensitive field effect transistor as a transducer. Highly or partially purified alcohol oxidase (AOX) and the permeabilised cells of methylotrophic yeast Hansenula polymorpha (as a source of AOX) have been used as sensitive elements. The response time in steady-state measurement mode is in the range of 10-60 s for the enzyme-based sensors and 60-120 s for the cell-based sensor. When measured in kinetic mode the response time of all biosensors developed was less than 5 s. The linear dynamic range of the sensor output signals corresponds to 5-200 mM formaldehyde for highly and partially purified alcohol oxidase, and 5-50 mM formaldehyde for the cells. The operational stability of the biosensors is not less than 7 h, and the relative standard deviation of intra-sensor response is approximately 2 and 5% for the enzyme- and cell-based sensors, respectively. When stored at 4 degrees C, the enzyme and cell sensor responses have been found stable for more than 60 and 30 days, respectively. Both types of biosensors demonstrate a high selectivity to formaldehyde with no potentiometric response to primary alcohols, including methanol, or glycerol and glucose. The possible reasons of such unexpected high selectivity of AOX-based FET-sensors to formaldehyde are discussed. The influence of the biomembrane composition and the effect of different buffers on the sensor response to formaldehyde are also discussed.

Alcohol Oxidoreductases↗

PubMed1999

Application of enzyme field-effect transistors for determination of glucose concentrations in blood serum.

Glucose-sensitive enzyme field effect transistors (ENFETs) modified by an additional Nafion membrane have been developed and used for diluted blood samples analysis. The ENFET was used in the linear portion of the calibration curve up to 1.5 mM glucose in a model solution, which corresponds with up to 60 mM glucose in the undiluted samples (dilution 1:40). The high linearity of the Grans curve (factor of linearity is 1.03) obtained by the method of standard additions indicates the high precision of analysis. Glucose concentrations in different blood serum samples determined by ENFETs were compared with those measured by the commercial analyzer 'Eksan-G' and colorimetric method ('Diagluc' enzymatic kit), and good correlation between these methods was revealed. The high reproducibility and operational stability of the biosensor developed were demonstrated.

Animals↗

PubMed1994

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.

Biosensing Techniques↗

PubMed1992

Cell-free translation systems from different eukaryotes differ in their sensitivity to a template sugar-phosphate backbone.

To study the role of a messenger sugar-phosphate backbone in the ribosomal decoding process, poly(U) and poly(dT) template activity in different eukaryotic systems has been compared. 80S ribosomes from Saccharomyces cerevisiae appeared to be able to translate poly(dT) both in the presence and in the absence of elongation factors, contrary to poly(U). However, ribosomes from higher eukaryotes (wheat germ, rabbit liver) are completely inefficient in poly(dT) translation. Moreover, rabbit liver ribosomes fail to bind effectively phenylalanyl-tRNA in the presence of poly(dT) although the polynucleotide seems to interact with the ribosomal decoding center. It is also of particular interest that hybrid ribosomes formed from the yeast and rabbit liver subunits can translate poly(dT) only when the large ribosomal subunit from yeast is used.

Animals↗

PubMed1988

The role of a template sugar-phosphate backbone in the ribosomal decoding mechanism. Comparative study of poly(U) and poly(dT) template activity.

To study the role of a template sugar-phosphate backbone in the ribosomal decoding process, poly(U), poly(dT) and poly(dU)-directed cell-free amino acid incorporation was investigated under the influence of neomycin and high concentrations of Mg2+. The specificity of a factor-dependent translation system of Escherichia coli was shown to change according to the principle: "either ribo- or deoxyribopolynucleotide messenger". Poly(dT) is shown to be effectively translated in the absence of elongation factors, both at low (2 degrees C) and high (37 degrees C) temperature. Neomycin inhibits factor-free poly(dT) translation. Little or no poly(U) translation is observed in this system. A chromatographic analysis of the oligophenylalanine residues synthesized seems to show that translocation is the main step responsible for ribosome specificity to the ribo- or deoxyribopolynucleotide template in both factor-dependent and factor-free translation systems.

Cell-Free System↗

PubMed1983

The accuracy of poly(U) translation by different eukaryotic tRNAs.

In this work we present comparative data on rates of phenylalanine and leucine incorporation into the poly(U) dependent product of cell-free translation by different eukaryotic tRNAs at high Mg2+ concentration. The frequency of translation errors has been found to depend upon the value of the tRNAPhe:tRNALeu ratio and the peculiarities of isoacceptor tRNAsLeu of different origin.

Animals↗

PubMedDate not supplied

[Cellular microbiosensors for methanol and ethanol determination on the basis of pH-sensitive field transistors].

Cellular sensors for methanol and ethanol determination were developed using immobilized mutant cells of methylotrophic yeasts Hansenula polymorpha and Pichia pinus (able to extrude protons in the presence of alcohol) and pH-sensitive field effect transistors (pH-SFETs). The intact cells of yeasts were immobilized in Ca-alginate gel to obtain a biomembrane. The minimal detectable response was obtained to approximately 0.5 mM of methanol and ethanol, a linear dependence of biosensor's response on the logarithmic alcohol concentration was observed in the range from 5 to 100 mM for both types of alcohol. The prospects for application of biosensors to determine alcohols in the analytical practice are discussed.

Biosensing Techniques↗

PubMedDate not supplied

[Study of immobilization and properties of urease for creation of a biosensor based on semiconductor structures].

Many-sided investigations of urease immobilization methods were carried out to create the biosensor devices on the base of semiconductor structures. Special attention was concentrated on the biomembrane formation by means of urease and bovine serum albumin (BSA) cross-linking by gaseous glutaraldehyde. Optimal conditions for the formation process were selected which preserve about 20% of total urease activity after the cross-linking. The properties of enzyme immobilized by the above-mentioned method have been comprehensively studied. They included the urease activity dependence on pH, ionic strength, incubation buffer capacity as well as the enzyme stability during its functioning, storing and thermoinactivation. As was shown, for immobilized ureas Km value for urea at pH 7.0 and 20 degrees C is 1.65 time less than for free enzyme. In the presence of EDTA (1 mM) the enzyme activity in the biomembrane is practically unchanged under a month storing. Biomembrane possesses good adhesion to silicon surface and its swelling level under different conditions does not exceed 35%. The conclusion is made about the prospects of the used method of biomembrane formation for biosensor technology based on semiconductor structures.

Enzyme Stability↗

PubMedDate not supplied

[The basis of high-fidelity translation].

The structural, thermodynamic and kinetic bases of high accuracy of translation are reviewed. Particular attention is given to the structural features of tRNA which are essential for cognate specificity.

Amino Acyl-tRNA Synthetases↗

PubMedDate not supplied

[Ambiguous polyuridylic acid translation by transport RNA from various eukaryotic substances].

Various eucaryotic tRNAs were compared from the standpoint of their ability to misread poly-U in the tRNA-dependent cell-free system of wheat germs. The highest level of 14C-leucine incorporation into the product of translation is observed for tRNA from differentiated (lactating) mammary gland under conditions of the increased Mg2+ concentration. Possible reasons may be connected with different tRNALeu/tRNAPhe ratios in these tissues and with the appearance of specific isoacceptory tRNA4Leu in the total set of tRNA from the lactating gland.

Animals↗

PubMedDate not supplied

[Code correspondence of tRNK Leu 1-6 in the mammary gland of cows].

Preparations of ribosome subunits from the rabbit liver are completely free from the endogenic mRNA admixture and high-active in a system of codon-dependent binding of tRNA. Leucine codons of uridyl-uridyl-adenine (UUA) and uridyl-uridyl-guanine (UUG) are synthesized by the triester method. The correspondence of six leucine isoacceptor tRNA from mammary gland of cows to cytidyl-uridyl-uridine (CUU), UUA and UUG codons is studied. The code response of tRNA is compared with their ability to erroneously translate polyuridylic acid.

Animals↗

PubMedDate not supplied

[Use of conductometric microsensors for studying kinetic parameters of enzymes].

An alternative method based on thin-film conductometric microsensor is suggested for studying enzyme kinetics. It is established that the immobilized enzyme as well as soluble one is described by the classic lows of enzyme kinetics. Major parameters (Km and Vmax) are identical to those measured by the widely used methods and their amounts are 1.33 mM and 9.04 microS/min for soluble urease and 3.73 mM and 13.8 microS/min for immobilized urease; 5.26 mM and 13.9 microS/min for immobilized glucose oxidase; 13.6 mM and 122.0 microS/min for immobilized acetyl cholinesterase; 10.8 mM and 129.9 microS/min for immobilized butyryl cholinesterase. The possibility of conductometric analyzer application for detection of toxin concentration, in particular pesticides, is shown. Advantages and disadvantages of the system suggested are discussed.

Biosensing Techniques↗

PubMedDate not supplied

[Optimal conditions for the functioning of a urease-based biosensor and pH-sensitive field transistors. Determination of urea in solution].

The article deals with the optimal conditions of urea determination in solution by the enzyme sensor based on pH-sensitive field effect transistor. The dependence of response level on temperature, pH, ionic strength, buffer capacity were investigated. 10 mM phosphate buffer, pH 7.4 containing 200 mM NaCl is optimal for possible measurements of urea in diluted blood. Temperature change in the measured samples from 15 degrees to 30 degrees C does not affect the sensor signal value. Stability of immobilized ureas was investigated at storage under different conditions. In the presence of 1 mM EDTA activity of immobilized enzyme does not change during one month storage at 4 degrees C.

Biosensing Techniques↗