A. P. Soldatkin

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.

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.

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.

Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system.

d-Serine is an endogenous ligand for N-methyl-d-aspartate (NMDA) receptors, and alterations in its concentration have been related to several brain disorders, especially schizophrenia. It is therefore an important target neuromodulator for the pharmaceutical industry. To monitor d-serine levels in vivo, we have developed a microbiosensor based on cylindrical platinum microelectrodes, covered with a membrane of poly-m-phenylenediamine (PPD) and a layer of immobilized d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). By detecting the hydrogen peroxide produced by enzymatic degradation of d-serine, this microbiosensor shows a detection limit of 16 nM and a mean response time of 2 s. Interferences by ascorbic acid, uric acid, l-cysteine, and by biogenic amines and their metabolites are rejected at more than 97% by the PPD layer. Although several d-amino acids are potential substrates for RgDAAO, d-serine was the only endogenous substrate present in sufficient concentration to be detected by our microbiosensor in the central nervous system. When implanted in the cortex of anesthetized rats, this microbiosensor detected the increase in concentration of d-serine resulting from its diffusion across the blood-brain barrier after an intraperitoneal injection. This new device will make it possible to investigate in vivo the variations in d-serine concentrations occurring under normal and pathological conditions and to assess the pharmacological potency of new drugs designed to impact d-serine metabolism.

Creatinine sensitive biosensor based on ISFETs and creatinine deiminase immobilised in BSA membrane.

A creatinine sensitive biosensor based on ion sensitive field-effect transistors (ISFETs) with immobilised creatinine deiminase (CD) is developed. CD is immobilised on the transducer surface by classical cross-linking with bovine serum albumin (BSA) in a glutaraldehyde (GA) vapour. The linear dynamic ranges of biosensors are between 0 and 5 mM creatinine concentration, and the sensor sensitivity depends on the sample buffer concentration. Minimal detection limit for creatinine determination in model solution with 144 mM NaCl and 5% BSA, pH 7.4, is about 10 muM. Biosensor responses are reproducible and stable during continuous work at least for 8 h, and the relative standard deviation of sensor response is approximately 3% (n=48, for creatinine concentrations of 0.2 and 0.6 mM). An investigation about storage stability of creatinine sensitive ENFETs kept in dry at 4-6 degrees C shows that biosensors demonstrate an excellent storage stability for at least 6 months and more. Moreover, creatinine sensitive enzymatic field-effect transistors (ENFETs), demonstrating very good performances, are very selective and specific and well suitable for hemodialysis monitoring.

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.

Conductometric formaldehyde sensitive biosensor with specifically adapted analytical characteristics

A conductometric enzyme biosensor for determination of formaldehyde in aqueous solutions has been developed using interdigitated thin-film planar electrodes and immobilised alcohol oxidase from Hansenula polymorpha . The biosensor steady-state response was reached after about 1 min. Its dynamic range can vary from 0.05 to 500 mM formaldehyde and depends on the time of enzymatic membrane cross-linking by glutaraldehyde and on the buffer concentration used. The biosensor developed was not completely specific and selective. It demonstrated no response to primary alcohols and other substrates alone. Unfortunately, the response of this biosensor in a mixture of formaldehyde and methanol was decreased in comparison to the one observed for pure formaldehyde, even if no response was obtained with the interfering specie alone. The operational stability was not <20 h and the relative standard deviation appeared to be about 3%. Moreover, the storage stability was more than 1 month.

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.

Application of amperometric biosensors for analysis of ethanol, glucose, and lactate in wine.

This article presents the application of amperometric biosensors based on platinum printed electrodes SensLab and immobilized enzymes, alcohol oxidase, glucose oxidase, and lactate oxidase, for wine analysis. Created devices demonstrate linear response to ethanol, glucose, and lactate within the concentration range 0.3-20 mM, 0.04-2.5 mM, and 0.008-1 mM, respectively. No decrease in ethanol and glucose biosensor activity is revealed during 2 months after fabrication, and the operational stability of the lactate biosensor is sufficient only during 4 days. Developed biosensors showed high selectivity to the substrate and are successfully applied to the analysis of such complex mixtures as wine and must. Good correlation of the results of analysis of different wines and must obtained by amperometric biosensors with immobilized oxidases and traditional methods is shown. Created biosensors can be used as a basis of a commercial device for express analysis of ethanol, glucose, and lactate in wine and must during its fermentation. Application of such devices for quality control in foodstuff industry can have great economical effect because determination by biosensors is less expensive, labor-intensive, and lengthy than traditional methods of analysis.