I. S. Kucherenko

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.

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.

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.

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.

Effect of different modifications of BEA-zeolites on operational characteristics of conductometric biosensor

Effect of different modifications of zeolite Na(+)-BEA on working characteristics of urease-based conductometric biosensor was studied. As the biosensor sensitive elements were used bioselective membranes based on urease and various zeolites immobilised with bovine serum albumin on the surface of conductometric transducers. Influence of zeolites on sensitivity of urea biosensor was investigated as well as reproducibility of biosensor signal and reproducibility of activity of the bioselective element after different variants of urease immobilisation on the surface of conductometric transducer. The biosensors based on zeolites (NH4(+)-BEA 30 and H(+)-BEA 30) were shown to be the most sensitive. Concentration of these zeolites in the bioselective membrane was optimized. Use of zeolites modified with methyl viologen and silver was ascertained to be of no prospect for urea conductometric biosensors. It was demonstrated that characteristics of urea biosensors can be regulated, varying zeolites modifications and their concentrations in bioselective membranes.

Development of electrochemical biosensors with various types of zeolites

In the work, different types of zeolites were used for the development of enzyme-based electrochemical biosensors. Zeolites were added to the biorecognition elements of the biosensors and served as additional components of the biomembranes or adsorbents for enzymes. Three types of biosensors (conductometric, amperometric and potentiometric) were studied. The developed biosensors were compared with the similar biosensors without zeolites. The biosensors contained the following enzymes: urease, glucose oxidase, glutamate oxidase, and acetylcholinesterase and were intended for the detection of urea, glucose, glutamate, and acetylcholine, respectively. Construction of the biosensors using the adsorption of enzymes on zeolites has several advantages: simplicity, good reproducibility, quickness, absence of toxic compounds. These benefits are particularly important for the standardization and further mass production of the biosensors. Furthermore, a biosensor for the sucrose determination contained a three-enzyme system (invertase/mutatorase/glucose oxidase), immobilized by a combination of adsorption on silicalite and cross-linking via glutaraldehyde; such combined immobilization demonstrated better results as compared with adsorption or cross-linking separately. The analysis of urea and sucrose concentrations in the real samples was carried out. The results, obtained with biosensors, had high correlation with the results of traditional analytical methods, thus the developed biosensors are promising for practical applications.

Development of a New Biosensor by Adsorption of Creatinine Deiminase on Monolayers of Micro- and Nanoscale Zeolites

This work is dedicated to the development of creatinine-sensitive biosensor consisting of pH-sensitive field-effect transistor (pH-FET) and creatinine deiminase (CD) immobilized with various types of zeolites, in particular, silicalite, zeolite beta (BEA) and nanobeta, and BEA zeolites, modified with gold nanoparticles and ions. For comparison, the traditional method of CD immobilization in saturated glutaraldehyde (GA) vapor was used. To modify pH-FET with zeolites, a monolayer method of deposition was applied. All basic analytical characteristics of the developed biosensors were compared: linear range of creatinine determination, time of response and regeneration, minimum limit of detection, and response reproducibility within a single biosensor; the calibration curves were plotted. It is shown that the use of zeolites of different types as adsorbents in the development of creatinine-sensitive biosensors resulted in a decrease of time of response and regeneration, an increase in sensitivity of the bioselective element to creatinine, and improvement in reproducibility of preparation of various biosensors, as compared with the method of covalent cross-linking in GA vapor.

Gold Nanoparticle/Polymer/Enzyme Nanocomposite for the Development of Adenosine Triphosphate Biosensor

Development of the enzyme-containing nanocomposites provides an excellent opportunity for the development of the sensitive and effective analytical devices – biosensors. In the present work, we used the nanocomposites that contained two enzymes (glucose oxidase and hexokinase), polymers, and gold nanoparticles (GNPs). Such nanostructure was expected to increase electron transfer in the bioselective element of biosensor and to improve the enzyme stability during the immobilization process. An amperometric biosensor based on the bienzyme system has been developed. The biosensor is sensitive to adenosine-5′-triphosphate (ATP) and glucose. The enzymes were immobilized onto the surface of a platinum disk electrode that was used as amperometric transducer. Three different methods of immobilization were investigated: cross-linking of the enzymes in the presence of bovine serum albumin, entrapment in a photo-cross-linkable modified polyvinyl alcohol (PVA-SbQ) matrix, and entrapment of the enzymes in a PVA/polyethylenimine matrix. The best results during the ATP determination were obtained with PVA-SbQ, and this immobilization method was modified by addition of 18-nm GNPs to the reacting mixture. The ATP detection could be achieved in all cases except for PVA/polyethylenimine, the best sensitivity and linear range being achieved by co-immobilizing glucose oxidase/hexokinase into the photo-cross-linked PVA-SbQ/GNP polymer matrix.

ОПТИМІЗАЦІЯ АМПЕРОМЕТРИЧНОГО БІОСЕНСОРА ДЛЯ ОЦІНКИ ШВИДКОСТІ НАКОПИЧЕННЯ ГЛУТАМАТУ ІЗОЛЬОВАНИМИ НЕРВОВИМИ ТЕРМІНАЛЯМИ МОЗКУ

Розроблено глутаматний біосенсор та адаптовано його для аналізу швидкості на- копичення глутамату ізольованими нервовими терміналями головного мозку щурів. Для створення біомембрани біосенсора використовували глутаматоксидазу, коіммобілізовану з бичачим сироватковим альбуміном за допомогою глутарового альдегіду на поверхню амперометричного дискового платинового електроду. Для покращення селективності біосенсора на поверхню електроду наносилась додаткова напівпроникна мембрана на основі поліфенілендіаміну. Проведено оптимізацію умов іммобілізації ферменту на поверхню робочого електроду (перевірено залежність роботи біосенсора від концентрації ферменту, глутарового альдегіду та часу іммобілізації). Досліджено вплив параметрів розчину (іонна сила, буферна ємність, рН ) на роботу біосенсора. Лінійний діапазон роботи біосенсора був 2-600 мкМ глутамату, мінімальна межа визначення становила 0,5-2 мкМ, чутливість - 250-300 нА/мМ. Біосенсор характеризувався доброю відтворюваністю відгуків та операційною стабільністю. З використанням біосенсора виявлено наявність ендогенного глутамату у препаратах ізольо- ваних нервових терміналей головного мозку. Показано, що початкова швидкість Na-залежного накопичення глутамату та його акумуляція нервовими терміналями суттєво не відрізнялись від результатів, отриманих з використанням радіоактивно-міченого L-[14C]глутамату. Проведена робота дозволить широко використовувати розроблений біосенсор у медицині, а також у біохімічних та біотехнологічних дослідженнях