Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation

Aim. Optimization of a new method of enzyme immobilization for amperometric biosensor creation. Methods. The amperometric biosensor with glucose oxidase immobilized on zeolites as bioselective elements and platinum disk electrode as transducers of biochemical signal into the electric one was used in...

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Опубліковано в: :	Вiopolymers and Cell
Дата:	2014
Автори:	Soldatkin, O.O., Ozansoy Kasap, B., Akata Kurc, B., Soldatkin, A.P., Dzyadevych, S.V., El’skaya, A.V.
Формат:	Стаття
Мова:	English
Опубліковано:	Інститут молекулярної біології і генетики НАН України 2014
Теми:	Molecular and Cell Biotechnologies
Онлайн доступ:	https://nasplib.isofts.kiev.ua/handle/123456789/154426
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Цитувати:	Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation / O.O. Soldatkin, B. Ozansoy Kasap, B. Akata Kurc, A.P. Soldatkin, S.V. Dzyadevych, A.V. El’skaya // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 291-298. — Бібліогр.: 24 назв. — англ.

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spelling	Soldatkin, O.O. Ozansoy Kasap, B. Akata Kurc, B. Soldatkin, A.P. Dzyadevych, S.V. El’skaya, A.V. 2019-06-15T15:26:42Z 2019-06-15T15:26:42Z 2014 Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation / O.O. Soldatkin, B. Ozansoy Kasap, B. Akata Kurc, A.P. Soldatkin, S.V. Dzyadevych, A.V. El’skaya // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 291-298. — Бібліогр.: 24 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008A3 https://nasplib.isofts.kiev.ua/handle/123456789/154426 577.15.08:543.9 Aim. Optimization of a new method of enzyme immobilization for amperometric biosensor creation. Methods. The amperometric biosensor with glucose oxidase immobilized on zeolites as bioselective elements and platinum disk electrode as transducers of biochemical signal into the electric one was used in the work. Results. The biosensors based on glucose oxidase adsorbed on zeolites were characterized by a higher sensitivity to glucose and a better inter-reproducibility. The best analytical characteristics were obtained for the biosensors based on nano beta zeolite. It has been found that an increase in the amount of zeolite on the surface of amperometric transducer may change such biosensor parameters as sensitivity to the substrate and duration of the analysis. Conclusions. The proposed method of enzyme immobilization by adsorption on zeolites is shown to be quite promising in the development of amperometric biosensors and therefore should be further investigated. Мета. Оптимізація нового методу іммобілізації ферментів для розробки амперометричних біосенсорів. Методи. Використано іммобілізовану глюкозооксидазу на цеоліті як біоселективний елемент біосенсора та платиновий дисковий електрод – як амперометричний перетворювач біохімічного сигналу в електричний. Результати. Біосенсор на основі глюкозооксидази, адсорбованої на цеолітах, вирізняється високою чутливістю до глюкози та покращеною інтер-відновлюваністю виготовлення біосенсорів. Найкращі аналітичні характеристики притаманні біосенсору на основі нано-бета цеоліту. Встановлено, що при зміні кількості цеолітів на поверхні амперметричного перетворювача можна варіювати параметри біосенсора, такі як чутливість до субстрату та час аналізу. Висновки. Показано, що запропонований метод іммобілізації, а саме – адсорбція ферментів на цеолітах є дуже перспективним при розробці амперометричних біосенсорів. Цель. Оптимизация нового метода иммобилизации ферментов для разработки амперометрических биосенсоров. Методы. Использовали иммобилизованную глюкозооксидазу на цеолитах как биоселективный элемент биосенсора и платиновый дисковый электрод – как амперометрический преобразователь биохимического сигнала в электрический. Результаты. Биосенсор на основе глюкозооксидазы, адсорбированной на цеолитах, отличается высокой чувствительностью к глюкозе и улучшенной интер-воспроизводимостью приготовления биосенсоров. Наилучшими аналитическими характеристиками обладает биосенсор на основе нано-бета цеолита. Установлено, что при изменении количества цеолитов на поверхности амперометрического преобразователя можно менять параметры биосенсора, такие как чувствительность к субстрату и время анализа. Выводы. Показано, что предложенный метод иммобилизации, а именно – адсорбция ферментов на цеолитах является перспективным при разработке амперометрических биосенсоров. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Molecular and Cell Biotechnologies Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation Розробка нового методу на основі адсорбції ферменту на силікаліті та нано-бета цеоліті для створення амперометричних біосенсорів Розработка нового метода на основе адсорбции фермента на силикалите и нано-бета цеолите для создания амперометрических биосенсоров. Article published earlier
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
title	Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation
spellingShingle	Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation Soldatkin, O.O. Ozansoy Kasap, B. Akata Kurc, B. Soldatkin, A.P. Dzyadevych, S.V. El’skaya, A.V. Molecular and Cell Biotechnologies
title_short	Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation
title_full	Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation
title_fullStr	Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation
title_full_unstemmed	Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation
title_sort	elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation
author	Soldatkin, O.O. Ozansoy Kasap, B. Akata Kurc, B. Soldatkin, A.P. Dzyadevych, S.V. El’skaya, A.V.
author_facet	Soldatkin, O.O. Ozansoy Kasap, B. Akata Kurc, B. Soldatkin, A.P. Dzyadevych, S.V. El’skaya, A.V.
topic	Molecular and Cell Biotechnologies
topic_facet	Molecular and Cell Biotechnologies
publishDate	2014
language	English
container_title	Вiopolymers and Cell
publisher	Інститут молекулярної біології і генетики НАН України
format	Article
title_alt	Розробка нового методу на основі адсорбції ферменту на силікаліті та нано-бета цеоліті для створення амперометричних біосенсорів Розработка нового метода на основе адсорбции фермента на силикалите и нано-бета цеолите для создания амперометрических биосенсоров.
description	Aim. Optimization of a new method of enzyme immobilization for amperometric biosensor creation. Methods. The amperometric biosensor with glucose oxidase immobilized on zeolites as bioselective elements and platinum disk electrode as transducers of biochemical signal into the electric one was used in the work. Results. The biosensors based on glucose oxidase adsorbed on zeolites were characterized by a higher sensitivity to glucose and a better inter-reproducibility. The best analytical characteristics were obtained for the biosensors based on nano beta zeolite. It has been found that an increase in the amount of zeolite on the surface of amperometric transducer may change such biosensor parameters as sensitivity to the substrate and duration of the analysis. Conclusions. The proposed method of enzyme immobilization by adsorption on zeolites is shown to be quite promising in the development of amperometric biosensors and therefore should be further investigated. Мета. Оптимізація нового методу іммобілізації ферментів для розробки амперометричних біосенсорів. Методи. Використано іммобілізовану глюкозооксидазу на цеоліті як біоселективний елемент біосенсора та платиновий дисковий електрод – як амперометричний перетворювач біохімічного сигналу в електричний. Результати. Біосенсор на основі глюкозооксидази, адсорбованої на цеолітах, вирізняється високою чутливістю до глюкози та покращеною інтер-відновлюваністю виготовлення біосенсорів. Найкращі аналітичні характеристики притаманні біосенсору на основі нано-бета цеоліту. Встановлено, що при зміні кількості цеолітів на поверхні амперметричного перетворювача можна варіювати параметри біосенсора, такі як чутливість до субстрату та час аналізу. Висновки. Показано, що запропонований метод іммобілізації, а саме – адсорбція ферментів на цеолітах є дуже перспективним при розробці амперометричних біосенсорів. Цель. Оптимизация нового метода иммобилизации ферментов для разработки амперометрических биосенсоров. Методы. Использовали иммобилизованную глюкозооксидазу на цеолитах как биоселективный элемент биосенсора и платиновый дисковый электрод – как амперометрический преобразователь биохимического сигнала в электрический. Результаты. Биосенсор на основе глюкозооксидазы, адсорбированной на цеолитах, отличается высокой чувствительностью к глюкозе и улучшенной интер-воспроизводимостью приготовления биосенсоров. Наилучшими аналитическими характеристиками обладает биосенсор на основе нано-бета цеолита. Установлено, что при изменении количества цеолитов на поверхности амперометрического преобразователя можно менять параметры биосенсора, такие как чувствительность к субстрату и время анализа. Выводы. Показано, что предложенный метод иммобилизации, а именно – адсорбция ферментов на цеолитах является перспективным при разработке амперометрических биосенсоров.
issn	0233-7657
url	https://nasplib.isofts.kiev.ua/handle/123456789/154426
citation_txt	Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation / O.O. Soldatkin, B. Ozansoy Kasap, B. Akata Kurc, A.P. Soldatkin, S.V. Dzyadevych, A.V. El’skaya // Вiopolymers and Cell. — 2014. — Т. 30, № 4. — С. 291-298. — Бібліогр.: 24 назв. — англ.
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fulltext	UDC 577.15.08:543.9 Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation O. O. Soldatkin1, 2, B. Ozansoy Kasap3, B. Akata Kurc3, 4, A. P. Soldatkin1, 2, S. V. Dzyadevych1, 2, A. V. El’skaya1 1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 2Institute of High Technologies, Taras Shevchenko National University of Kyiv 64, Volodymyrska Str., Kyiv, Ukraine, 01601 3Central Laboratory, Middle East Technical University Ankara, Turkey, 06531 4Micro and Nanotechnology Department, Middle East Technical University Ankara, Turkey, 06531 alex_sold@yahoo.com Aim. Optimization of a new method of enzyme immobilization for amperometric biosensor creation. Methods. The amperometric biosensor with glucose oxidase immobilized on zeolites as bioselective elements and platinum disk electrode as transducers of biochemical signal into the electric one was used in the work. Results. The bio- sensors based on glucose oxidase adsorbed on zeolites were characterized by a higher sensitivity to glucose and a better inter-reproducibility. The best analytical characteristics were obtained for the biosensors based on na- no beta zeolite. It has been found that an increase in the amount of zeolite on the surface of amperometric transdu- cer may change such biosensor parameters as sensitivity to the substrate and duration of the analysis. Con- clusions. The proposed method of enzyme immobilization by adsorption on zeolites is shown to be quite promi- sing in the development of amperometric biosensors and therefore should be further investigated. Keywords: Biosensor, amperometric transducer, enzyme adsorption, silicalite, nano beta zeolite, glucose oxidase. Introduction. It is well known that enzyme immobili- zation plays a key role in the development of biosen- sors. In recent years, the study and optimization of the methods of immobilization have attracted considerable interest of researchers. Special additives injected in the sensitive membrane upon immobilization can improve the sensitivity and stability of the immobilized enzyme [1]. Recently a great deal of attention has been paid to the immobilization of proteins on nanoparticles, in par- ticular zeolites, which are able to retain the biological activity of proteins [2]. Zeolites are an important group of minerals for in- dustrial and other purposes. They combine rarity, comp- lexity and unique crystal habits [3]. Formed in cracks or cavities of volcanic rocks, zeolites are a result of a very slow progressive metamorphism. Some of them, which are formed due to barely perceptible heat and pressure, can be called metamorphic only conditionally whereas others are found in obviously metamorphic regimes. Zeolites are of great interest due to their large surface areas, rigid and well defined pore structures, thermal stability, and tailorable surface charges with respect to other types of nanomaterials [4]. Particularly, the nano- sized pores of zeolites can be adjusted to precisely deter- mined uniform openings allowing the molecules smal- ler than the pores diameter to be adsorbed. Various size of pores in synthetic zeolites opens up a wide range of possibilities in terms of sieving molecules of different 291 ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 4. P. 291–298 doi: http://dx.doi.org/10.7124/bc.0008A3 � Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014 size or shape from gases and liquids [2, 5]. Finally, zeo- lites are known to be stable under both wet and dry con- ditions and well-tolerated by microorganisms, which provides an enhanced compatibility with biochemical analyses [2]. All these properties make zeolites unique nanomaterials and promising candidates for the immo- bilization of biological molecules and for the advanced analytical tasks. At present, some variants of biosensors containing zeolite crystals are known. Zeolites can be embedded in- to bioselective elements to improve analytical characte- ristics of biosensors, i. e. their sensitivity to the substra- te, linear and dynamic ranges, signal inter- and intra- reproducibility. In [6–8] the use of zeolite in the biosen- sor structure has been shown to increase the sensitivity and improve the selectivity. As demonstrated in [4], zeolites of various kinds can be effectively applied for the glucose oxidase immobilization while developing glucose amperometric biosensors to optimize their sen- sitivity, selectivity and stability. It was also shown that zeolites can be a basis for the creation of a new type of amperometric biosensors without mediators since zeoli- tes can serve as charge carriers [7, 9]. As reported in [10, 11], zeolites are used in conductometric biosensors as alternative carriers for the enzyme immobilization. Di- verse variants of co-immobilisation of urease and BEA- zeolites onto the surface of conductometric transducers were analyzed in respect to the improvement of analyti- cal characteristics of biosensors for urea determination [11]. A similar increase in sensitivity of bioselective ele- ments was obtained when using BEA-zeolites in biosen- sors based on pH-sensitive field effect transistors [12– 14]. These studies have shown that the greatest effect of improving the sensitivity of potentiometric biosensors is observed with the biomembranes of complex archi- tecture [13]. The promising results were obtained when clinoptilolite was used at the development of enzyme biosensors based on pH-sensitive field-effect transis- tors [15–18]. The basic idea was to attain high sensiti- vity of the potentiometric transducer to NH4 + by depo- sition of a clinoptilolite layer on the transducer surface [15]. The described effect was also observed in the bio- sensors based on urease [17] and urease co-immobili- zed with arginase [18]. Another option of the use of na- noscale materials in the design of biosensors has been proposed in [19], namely silicalite as a carrier for the en- zyme sorption. The biosensors obtained were charac- terized by a significantly higher signal reproducibility. The goal of this study was to check a possibility of using silicalite and nano beta zeolite for creation of am- perometric glucose biosensor with enhancement analy- tical characteristics. Materials and methods. Materials. Glucose oxida- se (GOD, EC 1.1.3.4) from Aspergillus niger with acti- vity 272 U/mg («Genzyme», UK) was used in biore- cognition elements of biosensors. Bovine serum albu- min (BSA, fraction V), glucose, glycerol, ascorbic acid, HEPES, and 50 % aqueous solution of glutaraldehyde (GA) have been received from «Sigma-Aldrich Chemie» (Germany). All other chemicals were of p. a. grade. Synthesis of zeolite crystals. S i l i c a l i t e. To synthe- size the silicalite solution we used 1TPA-OH : 4 TEOS : 350�H2O. When hydrolysing tetraethoxysilane (TEOS) with tetrapropylammonium hydroxide (TPA-OH) we obtained a homogeneous solution by constant stirring for 6 h at room temperature. The crystallization took place at 125 °C during one day. The material, which did not react, was removed from the solution by centrifuga- tion. The size of silicalite particles was approximately 400 nm. N a n o b e t a z e o l i t e. The molar composition of the nano beta zeolite is 0.25 Al2O3 : 25 SiO2 : 490 H2O : : 9 TEAOH. Silica source was TEOS (98 %, «Al- drich»). Aluminum isopropoxide (98 %, «Aldrich»), tetraethylammonium hydroxide (TEA-OH) (20 wt.% in water, «Aldrich») and doubly distilled water were used as the other reactants. Aging was continued under static conditions for 4 h with clear solution. The crystal- lization was completed within 17 days under static con- ditions at 100 °C in teflon lined autoclaves. The pro- duct was purified using centrifugation [20]. Approxi- mate size of the nano beta zeolite particles is 60 nm. Characterization of zeolites. The resulting samples were characterized by powder X-ray diffraction (XRD) using Ni filtered Cu-K� radiation in a Philips PW 1729. Scanning electron microscopy (SEM) analysis were performed after AuPb coating in a 400 Quanta FEI. The energy dispersive X-ray spectroscopy (EDX) analyses of the all samples were carried out utilizing a Phoenix EDAX X-ray analyzer equipped with Sapphi- re super ultrathin window detector attached to the Hita- chi S-4700 FE-SEM. The nitrogen adsorption/desorp- 292 SOLDATKIN O. O. ET AL. tion experiments were carried out at by NOVA 3000 se- ries («Quantachrome Instruments», USA) instrument. Surface area of the samples were obtained by Multi- point BET, while the pore size and pore volumes were obtained by Saito-Foley (SF) and t-plot methods. A sample preparation method includes outgassing samp- les under vacuum at 300 K for 4 h before analysis. The morphologies of the produced silicalite and nano beta zeolite can be seen in Fig. 1. According to the X-ray diffraction data presented in Fig. 2, all samples exhibited the characteristic diffrac- tion lines of their structures. In Table, Si/Al ratios, particle sizes, pore sizes, external and total surface area, micro- and mesopore volume are given. Design of amperometric transducers. Most of exis- ting amperometric systems use wire electrodes, which are structurally rather inconvenient for biosensors [21, 22]. It is much more promising to utilize planar thin- film electrode systems placed on a single glass or a cera- mic substrate. This is especially true for the multisensor systems. For the development of a planar amperometric multisensor, the design was chosen, in which the electro- chemical cell consisted of four working electrodes, re- ference and auxiliary electrodes. Enzyme immobilization in glutaraldehyde vapour. GA is a polyfunctional agent which forms covalent bonds between biocatalytic particles or proteins. Therefore, the enzyme immobilization with glutaraldehyde is often used for the development of enzyme biosensors [23]. This im- mobilization method produces a three-dimensional mat- rix, in which the enzyme is closely trapped with the electrode material, thus improving both retention of the biomolecule on the electrode surface and electrical com- munication. To produce a working bioselective membrane, a mixture of 5 % glucose oxidase (GOX), 5 % BSA, 10 % 293 ELABORATION OF NEW METHOD OF ENZYME ADSORPTION ON SILICALITE AND ZEOLITE Fig. 1. Scanning electron micro- scope image of silicalite (A) and nano beta zeolite (B) In te n si ty , cp s In te n si ty , cp s 2 theta, deg2 theta, deg B 0 5 10 15 20 25 30 35 40 45 50 55 0 200 400 600 800 1000 1200 1400 0 5 10 15 20 25 30 35 40 45 50 55 -200 0 200 400 600 800 1000 1200 1400 1600 1800A Fig. 2. XRD spectrum of synthesized zeolites: A – nano beta zeolite; B – silicalite glycerol was prepared in 20 mM phosphate buffer, pH 7.2. This mixture was deposited on the transducer sur- face using Eppendorf microsampler (total volume 0.1– 2.5 �l) until the working surface is covered completely. The volume of each membrane was about 0.05 µl. All membrane mixtures contained the same total amount of protein. Afterwards, the transducers with membrane mixtu- res were placed in saturated vapor of GA for 15–25 min, dried in the air at room temperature for 15 min, and prior to usage washed with the buffer solution from un- bound GA. Dip-coating zeolite onto transducers. A zeolite layer was formed on the transducer surface by dip-coating. 10 % zeolite solution in 5 mM PBS, pH 6.5, was used. 0.4 �l of the solution was deposited onto all active zo- nes of multitransducer, then it was heated during 3 min to 150 °C. This temperature had no effect on silicalite and did not influence the transducer working parameters. Modification procedure for zeolite monolayers. First- ly, we tried to attach zeolite to the electrodes surface but failed in our attempts. Then, we used a layer of poly(ethy- leneimine) (PEI) between zeolite and electrode surface. In this case, the zeolite attachment was attained but there appeared a problem of homogeneity. To solve it, we used mucasol (1/6 v/v) in distilled water, which gave good results due to changing the surface hydrophility. The electrode surfaces were dip-coated with muca- sol for 15 min, rinsed with copious amount of distilled water and dried under air. For the formation of homoge- neous layers of PEI, both dip-coating and spin-coating techniques had been tried. Since spin-coating gave mo- re homogeneous layers, it was used further on. The ef- fects of PEI solvent type (hot water and ethanol), PEI concentration (0.5, 1, 3, 5 %), spin-coating time (3000 rpm 15 s, 7 s), calcination temperature (100, 90, 50 °C) were investigated. The obtained monolayers were che- cked with microscope. The suitable conditions for zeo- lite monolayer production were chosen as follows: spin- coating with 0.5 % PEI in ethanol at 3000 rpm during 15 s, and calcination at temperature 90 °C for 30 min. The synthesized zeolites were directly attached to the obtained electrode surfaces simply by rubbing zeo- lites with a finger, a technique called direct attachment. These electrodes were used in further studies. Enzyme adsorption on zeolites. 0.1 �l of 5 % GOD solution in 20 mM phosphate buffer, pH 6.5 was depo- sited onto the active zones of multitransducer previous- ly coated with silicalite, then the transducer was expo- sed to complete air-drying (for 20 min). Neither GA nor any other auxiliary compounds were used. Next, the transducers were submerged into the working buffer for 20–30 min to wash off the unbound enzyme. After experiments, the transducer surface was cleaned from enzyme with ethanol-wetted cotton. Experimental setup for amperometric measure- ments. A three-electrode scheme of amperometric ana- lysis was used. The working amperometric transducers were developed, which were connected to the Palm Sens potentiostat (Netherlands) along with the auxilia- ry nickel electrode (with a much larger area of the ni- ckel surface compared to the working electrode) and the Ag/AgCl reference electrode. Each electrode has its own function in the ampero- metric analysis. When positive potential is applied to the working electrode, all the molecules in solution on the electrode surface are oxidized and an electron tran- sition from the solution to the electrode takes place. If there was no additional electrode, a large potential diffe- rence would be generated due to the stoichiometric im- balance. The function of the auxiliary electrode is to form the external circuit providing the electrons a pathway back to the solution. Obviously, this results in the reduc- tion process on the auxiliary electrode, equivalent to the oxidation process on the working electrode. This flow of electrons generates a current in the amperometric sen- sor. The third electrode is a reference electrode, which should contain a known chemical compound, which in- 294 SOLDATKIN O. O. ET AL. Sample name Si/Ala Part. size (nm)b Pore size (nm)c Sext (m2/g)d Stotal (m2/g)e Pore volume (cc/g)c Nano beta zeolite 26.54 ~ 60 0.43 190 472 0.20 Silicalite No Al ~ 400 0.45 52 185 0.08 N o t e. Measured by: a EDX; b SEM; c Saito-Foley (SF) method; d t-plot method; e BET. Characteristics of zeolites cludes both forms of the redox pair. Usually it is either Hg/HgCl2 (saturated calomel electrode) or Ag/AgCl (chloro-silver electrode). Since the applied potential is fixed, the reference electrode has a stable point, which can be also fixed on the working electrode for measure- ment. That is, the applied potential is controlled between the working and reference electrodes, whereas the cur- rent is measured between the working and auxiliary electrodes [24]. All measurements were carried out in an open mea- suring cell with permanent stirring at a constant poten- tial of +1 V vs Ag/AgCl reference electrode. Measurement procedure. Measurements were car- ried out in 20 mM HEPES, pH 7.4, in a voltampero- metric mode at a constant potential of +1 V vs Ag/AgCl reference electrode in an open cell with vigorous stir- ring. The substrate concentration in the measuring cell was specified by the introduction of aliquots of the sub- strate standard stock solution to the working buffer. All experiments were performed in at least three series. Results and discussion. The main goal of this re- search was to develop a new amperometric biosensor with enhanced analytical characteristics by using a no- vel method of immobilization of the enzyme based on its adsorption on zeolites. This method was first propo- sed for conductometric biosensors [19]. In this work we presented our attempt to use the mentioned method and to upgrade it for the amperometric transducers. The en- zyme GOD was chosen as one of the most stable and studied enzymes. The operation of amperometric biosensor for gluco- se determination is based on the enzymatic reaction with consequent hydrogen peroxide oxidation on the working electrode, which occurs when applying neces- sary potential and can be directly registered by the am- perometric transducer. In the presense of glucose, the reactions take place on the electrode surface as follows: GOD D-glucose + O2 � D-gluconic acid + H2O2; (1) + 1000 mV H2O2 � 2H+ + 2e– . (2) In this case, the biosensor response is proportional to the glucose concentration. At first, we fabricated a number of biosensors based on GOD immobilized by the method of enzyme adsorption on silicalite proposed in [19]. In details the immobilization technique is des- cribed in «Materials and Methods». The biosensors sen- sitivity to glucose was tested. The results obtained did not justify our expectations – it appeared that, along with good sensitivity, the biosensor responses were very slow and so biosensors lost one of their main advantages – fast analysis. For example, the response to 1 mM gluco- se amounted 40 min. We believe it is an effect of too thick layer of silicalite formed on the transducer surfa- ce by dip-coating. However, we considered that the va- lue of response of the biosensors based on GOD adsor- bed on silicalite was 3 times more than that of the bio- sensors based on the traditional methods of GOD immo- bilization in GA vapor. Therefore, we decided to modi- fy the method of zeolite application on the surface of amperometric transducer in order to obtain a mono- layer of zeolite. In details the technique proposed is des- cribed in «Materials and Methods». Using this tech- nique we created two types of biosensors based on sili- calite and nano beta zeolite. The characteristics of these biosensors were compared with those of the biosensors based on the traditional immobilization in GA vapor. Since the sensitivity and linear range of measure- ment are the most important working characteristics of any biosensor, we investigated an influence of different types of immobilization on these parameters. The cali- bration curves were obtained for each biosensor. The li- near ranges of measurement for all biosensors were iden- tical, whereas sensitivities differed. As shown in Fig. 3, 295 ELABORATION OF NEW METHOD OF ENZYME ADSORPTION ON SILICALITE AND ZEOLITE 0,0 0,5 1,0 1,5 2,0 2,5 3,0 0 50 100 150 200 250 R es p o n se , n A Glucose, mM 1 2 3 Fig. 3. Calibration curves for glucose detection by biosensors based on GOD adsorbed on nano beta (1) and silicalite (2), and GOD immobili- zed in GA vapor (3). Measurements were carried out in 5 mM HEPES buffer, pH 7.2 the biosensors based on using both zeolites were charac- terized by a higher sensitivity than the biosensors based on the GOD immobilized in GA vapor. It is known, that one of the most important biosen- sor characteristics is the reproducibility of the biosensor signal during operation. Therefore, it was necessary to explore this option for the biosensors with the GOD ad- sorbed on monolayers of zeolites and compare it with that for the biosensors based on the GOD immobilized in GA vapor. At the next stage, we studied reproducibility of res- ponses of all three biosensors during several hours of continuous operation. One measurement of glucose took 3–5 min. The interval between measurements was about 20 min, during this time we washed the biosensors from substrates by working buffer. We did not reveal any considerable decrease in responses of all three sensors after 10 subsequent measurements. As seen (Fig. 4), all biosensors demonstrated high signal reproducibility. The relative standard deviation of responses to glucose was similar for these biosensors (about 8 %). Since the reproducibility of biosensor manufac- turing is a well-known challenge in biosensorics, we should prove that this characteristic for the proposed by us procedure of enzyme immobilization, i. e. the enzy- me adsorption on zeolite monolayer, is not worse than for the immobilization in GA vapor. Therefore, we che- cked reproducibility of manufacturing of all three bio- sensors (with and without zeolite) and compared the re- sults obtained (Fig. 5). As can be seen, the biosensors based on the GOD adsorption on nano beta zeolite had better reproducibility as compared to the biosensors with the GOD adsorbed on silicalite, whereas the variant of enzyme immobilization in GA vapor was the worst. Summing up all the experiments conducted, we re- vealed that the biosensors based on the GOD adsorbed on nano beta zeolite had the best parameters. This type of immobilization was the most stable and reproducible, and the biosensors were characterized with the highest and fastest responses. However, though the promising results were obtained, we made further endeavour for improving the biosensors sensitivity by an increase in the number of nano beta zeolite particles on the elect- rode surface. It is known that, increasing the polymer concentration in course of applying zeolite monolay- ers, it is possible to increase an amount of zeolite on the surface of amperometric transducer. Nevertheless, as it was shown at the beginning of the work, the zeolite layer formed on the transducer surface by dip-coating proce- dure is too thick, which results, along with good sensiti- vity, in essential increase in the analysis time. Therefo- re, a compromise was required, i. e. it was necessary to find the immobilization conditions, which would ensu- re the greatest sensitivity to the substrate and short ana- lysis time. The results of this study are shown in Fig. 6. As can be seen at a higher polymer concentration used in the procedure of zeolite application, the value of bio- sensor response increases, but the response time increa- 296 SOLDATKIN O. O. ET AL. R es p o n se , n A Time, min 50 150 250 350 450 550 0 50 100 150 200 250 1 2 3 Fig. 4. Reproducibility of responses of biosensors based on GOD adsor- bed on nano beta (1) and silicalite (2), and GOD immobilized in GA va- por (3). Glucose concentration – 1 mM. Measurements were carried out in 20 mM HEPES buffer, pH 7.2 GOD immobilized in GA vapor GOD adsorbed on silicalite R es p o n se , n A 0 40 80 120 160 200 240 RSD = 38.9 % RSD = 12.0 % RSD = 12.6 % GOD adsorbed on Nano Beta Fig. 5. Reproducibility of preparation of biosensors based on GOD ad- sorbed on nano beta (1) and silicalite (2), and GOD immobilized in GA vapor (3). Glucose concentration – 1 mM. Measurements were carried out in 20 mM HEPES buffer, pH 7.2 ses as well. That is why we choose 0.5 % PEI for zeolite monolayer creation. In this case, we obtained enough high response and short response time. Conclusions. The enzyme adsorption on zeolites has been verified as a method of immobilization of bio- logical membranes for the development of amperomet- ric biosensors with improved analytical characteristics. For comparison the most common traditional method of immobilization in glutaraldehyde vapor has been chosen. The best results were obtained for the biosen- sor based on immobilization with nano beta zeolite par- ticles, the worst – for the biosensors based on the immo- bilization in glutaraldehyde. Besides sensitivity, stabi- lity and other analytical characteristics, it has been shown that the proposed biosensor based on nano beta zeolite particles is characterized by good repeatability of responses (error did not exceed 8 %) and reproduci- bility of biosensor manufacturing (error did not exceed 13 %). It has been shown that the proposed method of the enzyme immobilization by adsorption on zeolites is qui- te promising for further application in the development of amperometric biosensors. Funding. The authors gratefully acknowledge the fi- nancial support of this study by Project European IRSES- 318524-NANODEV. Furthermore, this study was partly supported by NASU in the frame of Scientific and Tech- nical Program «Sensor systems for medico-ecological and industrial-technological requirement: metrological support and experimental operation». Ðîçðîáêà íîâîãî ìåòîäó íà îñíîâ³ àäñîðáö³¿ ôåðìåíòó íà ñèë³êàë³ò³ òà íàíî-áåòà öåîë³ò³ äëÿ ñòâîðåííÿ àìïåðîìåòðè÷íèõ á³îñåíñîð³â O. O. Ñîëäàòê³í, Á. Îçàíñîé Êàñàï, Á. Àêàòà Êóðê, Î. Ï. Ñîëäàòê³í, Ñ. Â. Äçÿäåâè÷, Ã. Â. ªëüñüêà Ðåçþìå Ìåòà. Îïòèì³çàö³ÿ íîâîãî ìåòîäó ³ììîá³ë³çàö³¿ ôåðìåíò³â äëÿ ðîçðîáêè àìïåðîìåòðè÷íèõ á³îñåíñîð³â. Ìåòîäè. Âèêîðèñòàíî ³ììîá³ë³çîâàíó ãëþêîçîîêñèäàçó íà öåîë³ò³ ÿê á³îñåëåêòèâíèé åëå- ìåíò á³îñåíñîðà òà ïëàòèíîâèé äèñêîâèé åëåêòðîä – ÿê àìïåðî- ìåòðè÷íèé ïåðåòâîðþâà÷ á³îõ³ì³÷íîãî ñèãíàëó â åëåêòðè÷íèé. Ðå- çóëüòàòè. Á³îñåíñîð íà îñíîâ³ ãëþêîçîîêñèäàçè, àäñîðáîâàíî¿ íà öåîë³òàõ, âèð³çíÿºòüñÿ âèñîêîþ ÷óòëèâ³ñòþ äî ãëþêîçè òà ïîêðà- ùåíîþ ³íòåð-â³äíîâëþâàí³ñòþ âèãîòîâëåííÿ á³îñåíñîð³â. Íàéêðà- ù³ àíàë³òè÷í³ õàðàêòåðèñòèêè ïðèòàìàíí³ á³îñåíñîðó íà îñíîâ³ íàíî-áåòà öåîë³òó. Âñòàíîâëåíî, ùî ïðè çì³í³ ê³ëüêîñò³ öåîë³ò³â íà ïîâåðõí³ àìïåðìåòðè÷íîãî ïåðåòâîðþâà÷à ìîæíà âàð³þâàòè ïàðàìåòðè á³îñåíñîðà, òàê³ ÿê ÷óòëèâ³ñòü äî ñóáñòðàòó òà ÷àñ àíàë³çó. Âèñíîâêè. Ïîêàçàíî, ùî çàïðîïîíîâàíèé ìåòîä ³ììîá³ë³- çàö³¿, à ñàìå – àäñîðáö³ÿ ôåðìåíò³â íà öåîë³òàõ º äóæå ïåðñïåê- òèâíèì ïðè ðîçðîáö³ àìïåðîìåòðè÷íèõ á³îñåíñîð³â. Êëþ÷îâ³ ñëîâà: á³îñåíñîð, àìïåðîìåòðè÷íèé ïåðåòâîðþâà÷, àä- ñîðáö³ÿ ôåðìåíò³â, ñèë³êàë³ò, íàíî-áåòà öåîë³ò, ãëþêîçîîêñèäàçà. Ðîçðàáîòêà íîâîãî ìåòîäà íà îñíîâå àäñîðáöèè ôåðìåíòà íà ñèëèêàëèòå è íàíî-áåòà öåîëèòå äëÿ ñîçäàíèÿ àìïåðîìåòðè÷åñêèõ áèîñåíñîðîâ. À. À. Ñîëäàòêèí, Á. Îçàíñîé Êàñàï, Á. Àêàòà Êóðê, À. Ï. Ñîëäàòêèí, Ñ. Â. Äçÿäåâè÷, À. Â. Åëüñêàÿ Ðåçþìå Öåëü. Îïòèìèçàöèÿ íîâîãî ìåòîäà èììîáèëèçàöèè ôåðìåíòîâ äëÿ ðàçðàáîòêè àìïåðîìåòðè÷åñêèõ áèîñåíñîðîâ. Ìåòîäû. Èñ- ïîëüçîâàëè èììîáèëèçîâàííóþ ãëþêîçîîêñèäàçó íà öåîëèòàõ êàê áèîñåëåêòèâíûé ýëåìåíò áèîñåíñîðà è ïëàòèíîâûé äèñêîâûé ýëå- êòðîä – êàê àìïåðîìåòðè÷åñêèé ïðåîáðàçîâàòåëü áèîõèìè÷åñêî- ãî ñèãíàëà â ýëåêòðè÷åñêèé. Ðåçóëüòàòû. Áèîñåíñîð íà îñíîâå ãëþêîçîîêñèäàçû, àäñîðáèðîâàííîé íà öåîëèòàõ, îòëè÷àåòñÿ âû- ñîêîé ÷óâñòâèòåëüíîñòüþ ê ãëþêîçå è óëó÷øåííîé èíòåð-âîñïðî- èçâîäèìîñòüþ ïðèãîòîâëåíèÿ áèîñåíñîðîâ. Íàèëó÷øèìè àíàëè- òè÷åñêèìè õàðàêòåðèñòèêàìè îáëàäàåò áèîñåíñîð íà îñíîâå íà- íî-áåòà öåîëèòà. Óñòàíîâëåíî, ÷òî ïðè èçìåíåíèè êîëè÷åñòâà öåîëèòîâ íà ïîâåðõíîñòè àìïåðîìåòðè÷åñêîãî ïðåîáðàçîâàòåëÿ ìîæíî ìåíÿòü ïàðàìåòðû áèîñåíñîðà, òàêèå êàê ÷óâñòâèòåëü- íîñòü ê ñóáñòðàòó è âðåìÿ àíàëèçà. Âûâîäû. Ïîêàçàíî, ÷òî ïðåä- ëîæåííûé ìåòîä èììîáèëèçàöèè, à èìåííî – àäñîðáöèÿ ôåðìåí- òîâ íà öåîëèòàõ ÿâëÿåòñÿ ïåðñïåêòèâíûì ïðè ðàçðàáîòêå àìïå- ðîìåòðè÷åñêèõ áèîñåíñîðîâ. Êëþ÷åâûå ñëîâà: áèîñåíñîð, àìïåðîìåòðè÷åñêèé ïðåîáðàçî- âàòåëü, àäñîðáöèÿ ôåðìåíòîâ, ñèëèêàëèò, íàíî-áåòà öåîëèò, ãëþêîçîîêñèäàçà. REFERENCES 1. Vidinha P, Augusto V, Nunes J, Lima JC, Cabral JM, Barreiros S. Probing the microenvironment of sol-gel entrapped cutinase: the role of added zeolite NaY. 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Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation

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