Ilya N. Kurochkin

A laccase electrode for organic-phase enzymatic assays

The biocatalytic activity of catalase in non-aqueous environments is exploited for developing an organic-phase amperometric biosensor. The catalase is immobilized within the Eastman-AQ film on the glassy carbon surface. Various experimental variables influencing the response of the t-butyl hydroperoxide substrate are explored for optimum performance.

Direct electron transfer effect biosensors

A review of biosensors based on the direct electron transfer effect is carried out in this work. Different redox enzymes and proteins using the direct electron transport mechanism for electrocatalytical processes are described. The tunneling of electrons as a possible explanation of the direct electron transfer effect is also discussed.

Dual-stimuli-sensitive microgels as a tool for stimulated spongelike adsorption of biomaterials for biosensor applications.

This work examines the fabrication regime and the properties of microgel and microgel/enzyme thin films adsorbed onto conductive substrates (graphite or gold). The films were formed via two sequential steps: the adsorption of a temperature- and pH-sensitive microgel synthesized by precipitation copolymerization of N-isopropylacrylamide (NIPAM) and 3-(N,N-dimethylamino)propylmethacrylamide (DMAPMA) (poly(NIPAM-co-DMAPMA) at the pH-condition corresponding to its noncharged state (first step of adsorption), followed by the enzyme, tyrosinase, adsorption at the pH-condition when the microgel and the enzyme are oppositely charged (second step of adsorption). The stimuli-sensitive properties of poly(NIPAM-co-DMAPMA) microgel were characterized by potentiometric titration and dynamic light scattering (DLS) in solution as well as by atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) at solid interface. Enhanced deposition of poly(NIPAM-co-DMAPMA) microgel particles was shown at elevated temperatures exceeding the volume phase transition temperature (VPTT). The subsequent electrostatic interaction of the poly(NIPAM-co-DMAPMA) microgel matrix with tyrosinase was examined at different adsorption regimes. A considerable increase in the amount of the adsorbed enzyme was detected when the microgel film is first brought into a collapsed state but then was allowed to interact with the enzyme at T < VPTT. Spongelike approach to enzyme adsorption was applied for modification of screen-printed graphite electrodes by poly(NIPAM-co-DMAPMA)/tyrosinase films and the resultant biosensors for phenol were tested amperometrically. By temperature-induced stimulating both (i) poly(NIPAM-co-DMAPMA) microgel adsorption at T > VPTT and (ii) following spongelike tyrosinase loading at T < VPTT, we can achieve more than 3.5-fold increase in biosensor sensitivity for phenol assay. Thus, a very simple, novel, and fast strategy for physical entrapment of biomolecules by the polymeric matrix was proposed and tested. Being based on this unique stimuli-sensitive behavior of the microgel, this stimulated spongelike adsorption provides polymer films comprising concentrated biomaterial.

Potentiometric biosensors for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis.

A potentiometric method for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis has been developed. The method includes coimmobilization of three enzymes, butyrylcholinesterase, choline oxidase and peroxidase, on composite carbon electrodes. Catalytic hydrolysis of butyrylcholine and subsequent catalytic oxidation of choline result in the formation of hydrogen peroxide leads to a shift in the electrode potential. The detection limit for trichlorfon analysis is 2 x 10(-13) M. Electrodes remain stable for at least 4 weeks when stored at 277 K.

Study of cryostructuring of polymer systems: 27. Physicochemical properties of poly(vinyl alcohol) cryogels and specific features of their macroporous morphology

Based on aqueous poly(vinyl alcohol) (PVA) solutions with different content of polymer having different molecular masses and chain tacticity, macroporous viscoelastic gels (PVA cryogels) are prepared in various regimes of freezing-storage in a frozen state-thawing. Shear modulus and fusion temperature of corresponding samples are measured; the structure of thin sections is studied by optical microscopy and the images are processed and analyzed. It is shown that the rigidity and heat endurance of cryogels rise with an increase in the concentration of initial PVA solution and a decrease in the rate of thawing. The influence of the temperature of cryogenic treatment and the PVA molecular mass has an extreme character. At the same time, the effect of the main parameters of cryotropic gelation on the macroporous morphology of PVA cryogels is manifested in the form of more complex dependences because of its multiple-factor character. Therefore, distinct structure-property correlations are not observed in many cases. Cluster analysis of the morphometric characteristics of cryogels in comparison with data on their rigidity makes it possible to classify these systems.

Glucose potentiometric electrodes based on mediatorless bioelectrocatalysis. A new approach

A potentiometric method has been developed for analysis of glucose on the basis of glucose oxidase and peroxidase co-immobilized on the surface of an electrode made of composite carbonic material. Catalytic oxidation of glucose results in the formation of hydrogen peroxide. Mediatorless peroxidase catalysis of electroreduction of hydrogen peroxide leads to a shift in the electrode potential. The rate of the increase of the electrode potential is proportional to glucose concentration and calibration curve is linear at 0.025-2.0 mM. Electrodes permit at least 100 measurements without any loss in the activity. Electrodes remain stable for 90 days when stored at 277 K.

Luminescence lifetime-based sensor for relative air humidity

Abstract The development of new sensitive coatings for fibre-optic sensors that utilize the Langmuir-Blodgett technique is described. Monomolecular thin solid films containing phosphorescent water-soluble Pt- and Pd-porphyrins and amphiphilic surface-active polymer have been prepared by an original method and transferred onto solid supports. The solid-state coatings thus obtained show intense long-decay phosphorescence in ambient air, which is found to be sensitive to the water content in the environment, i.e., the relative humidity (RH) of air. The coatings have been studied by optical methods with emphasis on luminescence quenching and lifetime measurements. The results are applied to luminescence lifetime-based sensing of RH and a corresponding fibre-optic prototype device has been created.

Study of cryostructuring of polymer systems: 28. Physicochemical properties and morphology of poly(vinyl alcohol) cryogels formed by multiple freezing-thawing

Macroporous viscoelastic poly(vinyl alcohol) (PVA) cryogels are prepared from aqueous concentrated (80–120 g/l) PVA solutions subjected to 1–5 cycles of cryogenic treatment (freezing at −20°C for 19 h and subsequent thawing at a rate of 0.3°C/min). Shear moduli and fusion temperatures of corresponding samples are determined and the structure of thin sections is studied by optical microscopy with subsequent processing and analysis of images obtained. The previously described effect of a substantial increase in the rigidity and thermal stability of PVA cryogels resulted from the repeated freezing-thawing cycles is confirmed. The largest (jumpwise) changes in the physicochemical characteristics of such gels and their macroporous morphology take place after the second cycle of cryogenic treatment. Moreover, depending on the PVA concentration in the initial solution, the mean cross section of micropores increases by a factor of 2–3 and the total porosity of cryogel rises by a factor of 1.5–2; i.e., the imperfection of material increases. Nevertheless, this negative (from view-point of the integral properties of cryogel) effect is completely overpowered by processes of additional structuring, which result in the strengthening of polymer phase proceeding during the repeated freezing-thawing cycles.

Biosensor detection of neuropathy target esterase in whole blood as a biomarker of exposure to neuropathic organophosphorus compounds.

Abstract Neuropathy target esterase (NTE) is the target protein for neuropathic organophosphorus (OP) compounds that produce OP compound-induced delayed neurotoxicity (OPIDN). Inhibition/aging of brain NTE within hours of exposure predicts the potential for development of OPIDN in susceptible animal models. Lymphocyte NTE has also found limited use as a biomarker of human exposure to neuropathic OP compounds. Recently, a highly sensitive biosensor was developed for NTE activity using a tyrosinase carbon-paste electrode for amperometric detection of phenol produced by hydrolysis of the substrate, phenyl valerate. The I50 (20 min at 37°C) for N,N′-di-2-propylphosphorodiamid ofluoridate (mipafox) against hen lymphocyte NTE was 6.94 ± 0.28 μM amperometrically and 6.02 ± 0.71 μM colorimetrically. For O,O-di-1-propyl O-2,2-dichlorvinyl phosphate (PrDChVP), the I50 against hen brain NTE was 39 ±8 nM amperometrically and 42 ±2 nM colorimetrically. The biosensor enables NTE to be assayed in whole blood, whereas this cannot be done with the usual colorimetric method. Amperometrically, I50 values for PrDChVP against hen and human blood NTE were 66 ±3 and 70 ± 14nM, respectively. To study the possibility of using blood NTE inhibition as a biochemical marker of neuropathic OP compound exposure, NTE activities in brain and lymphocytes as well in brain and blood were measured 24 h after dosing hens with PrDChVP. Brain, lymphocyte, and blood NTE were inhibited in a dose-responsive manner, and NTE inhibition was highy correlated between brain and lymphocyte (r=.994) and between brain and blood (r=.997). The results suggest that the biosensor NTE assay for whole blood could serve as a biomarker of exposure to neuropathic OP compounds as well as a predictor of OPIDN and an adjunct to its early diagnosis.

Fibre-optic lifetime-based enzyme biosensor

Abstract A fibre-optic reagent-free glucose biosensor that utilizes both luminescent intensity and lifetime measurement principles is described. A solid-state luminescent oxygen-sensitive composition based on platinum-octaethylporphine (oxygen probe) and polystyrene (matrix) is used as a transducer and a support for immobilization of the oxidase enzyme. The glucose membrane and biosensor is capbale of determining and continuous monitoring of glucose within the range 0.03–1.2 mM. The performance of the new sensor is studied.