Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis

Aim. To determine an influence of serum components on the L-arginine biosensor sensitivity and to formulate practical recommendations for its reliable analysis. Methods. The L-arginine biosensor comprised arginase and urease co-immobilized by cross-linking. Results. The biosensor specificity was i...

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Datum:	2012
Hauptverfasser:	Saiapina, O.Y., Dzyadevych, S.V., Jaffrezic-Renault, N.
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Schriftenreihe:	Вiopolymers and Cell
Schlagworte:	Molecular and Cell Biotechnologies
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spelling	irk-123456789-1567562019-06-19T01:30:08Z Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis Saiapina, O.Y. Dzyadevych, S.V. Jaffrezic-Renault, N. Molecular and Cell Biotechnologies Aim. To determine an influence of serum components on the L-arginine biosensor sensitivity and to formulate practical recommendations for its reliable analysis. Methods. The L-arginine biosensor comprised arginase and urease co-immobilized by cross-linking. Results. The biosensor specificity was investigated based on a series of representative studies (namely, through urea determination in the serum; inhibitory effect studies of mercury ions; high temperature treatment of sensors; studying the biosensor sensitivity to the serum treated by enzymes, and selectivity studies). It was found that the response of the biosensor to the serum injections was determined by high sensitivity of the L-arginine biosensor toward not only to L-arginine but also toward two other basic amino acids (L-lysine and L-histidine). Conclusions. A detailed procedure of optimization of the conductometric biosensor for L-arginine determination in blood serum has been proposed. Keywords: L-arginine, conductometric biosensors, serum, optimization procedure. Мета. Визначити вплив компонентів сироватки крові на чутливість біосенсора при виявленні L-аргініну та сформулювати практичні рекомендації для забезпечення її надійного аналізу. Методи. Біосенсор для визначення L-аргініну містить аргіназу і уреазу, коіммобілізовані методом поперечного зшивання. Результати. Специфічність біосенсора вивчали на основі низки показників – вмісту сечовини у сироватці; інгібувального впливу іонів ртуті; високотемпературної обробки біосенсорів; чутливості біосенсора до сироватки крові, обробленої ліофілізованими препаратами ферментів, та селективності біосенсора. Встановлено, що відгук біосенсора на внесення сироватки зумовлений високою чутливістю біосенсора ще до двох, крім L-аргініну, основних амінокислот (L-лізину та L-гістидину). Висновки. Запропоновано детальну процедуру оптимізації кондуктометричного біосенсора для визначення L-аргініну у сироватці крові. Ключові слова: L-аргінін, кондуктометричні біосенсори, сироватка крові, процедура оптимізації. Цель. Определить влияние компонентов сыворотки крови на чувствительность биосенсора для выявления L-аргинина и сформулировать практические рекомендации для обеспечения ее надежного анализа. Методы. Биосенсор для определения L-аргинина содержит аргиназу и уреазу, ко-иммобилизованные методом поперечной сшивки. Результаты. Специфичность биосенсора изучали на основе серии показателей – содержания мочевины в сыворотке; ингибирующего эффекта ионов ртути; высокотемпературной обработки биосенсоров; чувствительности биосенсора к сыворотке крови, обработанной лиофилизованными препаратами ферментов, и селективности биосенсора. Установлено, что отклик биосенсора на внесение сыворотки обусловлен высокой чувствительностью биосенсора еще к двум, кроме L-аргинина, основным аминокислотам (L-лизину и L-гистидину). Выводы. Предложена детальная процедура оптимизации кондуктометрического биосенсора для определения L-аргинина в сыворотке крови. Ключевые слова: L-аргинин, кондуктометрические биосенсоры, сыворотка крови, процедура оптимизации. 2012 Article Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis / O.Y. Saiapina, S.V. Dzyadevych, N. Jaffrezic-Renault // Вiopolymers and Cell. — 2012. — Т. 28, № 6. — С. 441-448. — Бібліогр.: 27 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000134 http://dspace.nbuv.gov.ua/handle/123456789/156756 543.555 + 551.508.91 + 577.112.385 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies
spellingShingle	Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies Saiapina, O.Y. Dzyadevych, S.V. Jaffrezic-Renault, N. Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis Вiopolymers and Cell
description	Aim. To determine an influence of serum components on the L-arginine biosensor sensitivity and to formulate practical recommendations for its reliable analysis. Methods. The L-arginine biosensor comprised arginase and urease co-immobilized by cross-linking. Results. The biosensor specificity was investigated based on a series of representative studies (namely, through urea determination in the serum; inhibitory effect studies of mercury ions; high temperature treatment of sensors; studying the biosensor sensitivity to the serum treated by enzymes, and selectivity studies). It was found that the response of the biosensor to the serum injections was determined by high sensitivity of the L-arginine biosensor toward not only to L-arginine but also toward two other basic amino acids (L-lysine and L-histidine). Conclusions. A detailed procedure of optimization of the conductometric biosensor for L-arginine determination in blood serum has been proposed. Keywords: L-arginine, conductometric biosensors, serum, optimization procedure.
format	Article
author	Saiapina, O.Y. Dzyadevych, S.V. Jaffrezic-Renault, N.
author_facet	Saiapina, O.Y. Dzyadevych, S.V. Jaffrezic-Renault, N.
author_sort	Saiapina, O.Y.
title	Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis
title_short	Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis
title_full	Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis
title_fullStr	Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis
title_full_unstemmed	Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis
title_sort	potentiality of application of the conductometric l-arginine biosensors for the real sample analysis
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2012
topic_facet	Molecular and Cell Biotechnologies
url	http://dspace.nbuv.gov.ua/handle/123456789/156756
citation_txt	Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis / O.Y. Saiapina, S.V. Dzyadevych, N. Jaffrezic-Renault // Вiopolymers and Cell. — 2012. — Т. 28, № 6. — С. 441-448. — Бібліогр.: 27 назв. — англ.
series	Вiopolymers and Cell
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fulltext	MOLECULAR AND CELL BIOTECHNOLOGIES UDC 543.555 + 551.508.91 + 577.112.385 Potentiality of application of the conductometric L-arginine biosensors for the real sample analysis O. Y. Saiapina1, 2, S. V. Dzyadevych1, 3, N. Jaffrezic-Renault2 1Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Zabolotnoho Str., Kyiv, Ukraine, 03680 2Laboratory of Analytical Sciences, University Claude Bernard, Lyon 1 43, Boulevard du 11 Novembre 1918, Villeurbanne Cedex, France, 69622 3Institute of High Technologies, Taras Shevchenko Kyiv National University 64, Volodymyrska Str., Kyiv, Ukraine, 01003 osayapina4@gmail.com Aim. To determine an influence of serum components on the L-arginine biosensor sensitivity and to formulate practical recommendations for its reliable analysis. Methods. The L-arginine biosensor comprised arginase and urease co-immobilized by cross-linking. Results. The biosensor specificity was investigated based on a series of representative studies (namely, through urea determination in the serum; inhibitory effect studies of mercury ions; high temperature treatment of sensors; studying the biosensor sensitivity to the serum treated by enzymes, and selectivity studies). It was found that the response of the biosensor to the serum injections was determined by high sensitivity of the L-arginine biosensor toward not only to L-arginine but also toward two other basic amino acids (L-lysine and L-histidine). Conclusions. A detailed procedure of optimization of the conductometric bio- sensor for L-arginine determination in blood serum has been proposed. Keywords: L-arginine, conductometric biosensors, serum, optimization procedure. Introduction. For the last decade, the statistics of in- born errors of the amino acid metabolism is alarming, and screening of the related diseases in newborns is al- ways advisable [1–3]. In biotechnology and microbio- logy, the monitoring of amino acids in culture medium is also important since elevated consumption of certain amino acids serves as an indicator of microbial contami- nation [4]. Regarding the necessity of amino acid moni- toring in farming, the continuous evaluation of the nutri- tion efficiency can lead to sustainable improvements in productivity, allowing the development of feeding stra- tegies based on suitable local feedstuffs [5, 6]. The levels of L-arginine are commonly measured through its direct reaction with ninhydrin, Sakaguchi re- agent [7] or by biacetyl reaction [8, 9]. However, colori- metric methods have low specificity to L-arginine and at the same time the analysis may be performed only if large amount of probe is available. The arginine deter- mination based on arginase coupled with urease with the following spectrophotometric detection is also used [10, 11], however, the method is applicable only for the protein hydrolizates free of urea. If quantification of L- arginine in a complex mixture is required, high-preci- sion results can be obtained by liquid or ion-exchange chromatography [12, 13]. In clinical practices, arginine may also be detected using capillary electrophoresis [14], capillary electro- phoresis-time of flight-mass spectrometry or capillary electrophoresis-electrospray mass spectrometry [15, 16]. However, these methods are time consuming, ex- pensive and demand skilful personnel. Thus, there is an increasing challenge for inexpen- sive and reliable techniques which, along with the use in central or satellite laboratories, would be much more accessible in health care area, at farms, etc. Electro- chemical biosensors have been offered as a response to 441 ISSN 0233–7657. Biopolymers and Cell. 2012. Vol. 28. N 6. P. 441–448 doi 10.7124/bc.000134  Institute of Molecular Biology and Genetics, NAS of Ukraine, 2012 this challenge in virtue of their analytical capabilities, portability, simplicity, ease of mass manufacture. Our previous work on L-arginine biosensor [17] has demonstrated the feasibility of fabricating a highly sensitive conductometric biosensor for the model samp- le analysis. The biosensor reported was designed on the basis of arginase and urease immobilized as a single bio- selective membrane. This paper describes the possibili- ties of application of the biosensor developed for L-argi- nine determination in serum using the optimization pro- cedure, essential for the conductometric measurements. The aim of the work was to determine an influence of se- rum components on the biosensor sensitivity and to for- mulate practical recommendations and precautions for reliable real sample analysis. This study was the first stage in the elaboration of a reliable biosensor system for amino acid assay in complex media of different origin. Materials and method. Reagents. Arginase (E. C. 3.5.3.1, 136 U/mg solid) from bovine liver and urease (E. C. 3.5.1.5, 100 U/mg solid) from jack beans were purchased from «Sigma-Aldrich» (France). The solu- tion of bovine serum («Calf serum, iron supplemented from formula-fed calves, cell culture tested, sterile-fil- tered, for RD use only», «Sigma» C8056-100 ml, 018 K8406) was supplied by «Sigma-Aldrich» (Germany) and used without further purification. Bovine serum albumin (BSA), glutaraldehyde (GA, 25 % aq. solu- tion), urea (60.06 g/mol), L-amino acids and their derivatives were provided by «Sigma-Aldrich» (Fran- ce). The phosphate solution used was prepared with KH2PO4 and Na2HPO4 («Acros Organics», Belgium) unless otherwise stated. While the biosensor operating in a differential measuring mode, a phosphate solution (5 mM KH2PO4-Na2HPO4, pH 6.0) was useful to main- tain the necessary ionic strength of the measuring sys- tem and permitted to perform the efficient registration of newly generated ions. Glycerol was purchased from «Macrokhim» (Ukraine). The amino acid solutions and phosphate solution were made from the chemicals of at least analytical grade using ultra-pure (UP) water. UP water used was obtained from a Millipore («Milli Q purification system», France). Transducers. Each transducer chip consisted of two pairs of interdigitated thin film electrodes (150 nm thick) of identical configuration. The electrodes were fabricated by vapor deposition of gold onto a non-con- ducting pyroceramic substrate (5 × 30 mm). A 50 nm thick intermediate chromium layer was used to impro- ve the gold adhesion to the substrate. Both the digit width and interdigital distance were 10 µm, and their length was ~ 1.5 mm. Thus, the sensitive area of each pair of electrodes was ~ 2.9 mm2. The first pair of elect- rodes, covered with non-reactive BSA membrane, con- stituted a reference sensor. The second pair of electro- des, covered with the enzyme membrane, represented a working sensor. Preparation of selective elements of conductometric biosensor for L-arginine determination. The enzyme membranes cross-linked with glutaraldehyde were for- med using the immobilization technique originated from the previous experience [17]. Briefly, arginase (1.8 mg), urease (4.4 mg) and BSA (2 mg) were thoroughly dis- solved in 40 µl of 40 mM phosphate buffer (pH 7.4), containing glycerol (15 %). Afterwards, 0.15 µl of the prepared solution and 0.15 µl of the GA aqueous so- lution (2 %, v/v) were vigorously homogenized and de- posited onto the sensitive surface of one pair of elect- rodes. The reference sensor was prepared by the same procedure, except that arginase and urease were repla- ced by BSA. Time of the biomembrane immobilization was about 25 min. Before the measurements, the bio- sensor was carefully washed for 10–15 min in 5 mM phosphate solution (pH 6.0). Electrochemical measuring system. The conducto- metric biosensors were studied using the portable bio- sensor analyzer reported in the work [18]. The analyzer sensor assembly contained a stand with fixed block of holders; each holder was connected to the contact of an appropriate conductometric biosensor. The applied sinusoidal potential was of 30 kHz fre- quency and 10 mV amplitude which allowed avoiding faradaic processes, double-layer charging and polariza- tion of the microelectrodes. Illumination and tempera- ture variations had practically no influence on the bio- sensor characteristics. The measurements were carried out in a glass cell filled with phosphate solution (volu- me 3 ml), under vigorous magnetic stirring. An output potential of each conductometric transducer was propor- tional to an impedance difference between working and reference sensors [19]. The conductometric detection of L-arginine in the biosensor is based on the following processes and reac- 442 SAIAPINA O. Y., DZYADEVYCH S. V., JAFFREZIC-RENAULT N. tions. Species of the phosphate solution (H2PO4 – and HPO4 2–) which have high value of relative conductivity, significantly contribute to the overall conductivity of the membrane. While the measurements, these species as well as water hydroxyl ions serve as ammonium car- riers (NH4 + generated in the reactions (1) and (2)) from the arginase-urease membrane to the bulk solution. In particular, after the formation of ammonium in the en- zymatic reactions, its translocation from the membrane to the bulk solution is provided by the temporary asso- ciation of NH4 + with the species of the dissociated com- ponents of the phosphate background solution and water molecules. At the same time, species of the phosphate solution from the bulk penetrate the enzymatic membra- ne and maintain its conductivity. Arginase L-arginine → L-ornithine + urea; (1) Urease (NH2)2CO + 2H2O + H+ → 2NH4 + + HCO3 –. (2) Accordingly, the analytical signal of the biosensor has the following nature. Before the injection of the substrate to the measuring cell, a conductivity of a boun- dary layer (a thin layer of the solution adjacent to the electrode surface where the protein-based membrane is located) is registered as an initial signal of the biosen- sor and has a form of the continuous baseline. After- wards, when L-arginine is added, its enzymatic decom- position results in the generation of new ions (ammoni- um) contributing to the change of overall conductivity at the boundary layer of the biosensor. When the equi- librium between the rate of the ammonium production inside the arginase-urease membrane and the speed of protons influx into the membrane (from the bulk solu- tion) is established, it is reflected in the steady-state bio- sensor response, corresponding to the end of the enzy- matic transformations. Thus, the initial biochemical changes, occurring within the bioselective element of the biosensor, are registered finally as a physical para- meter (conductivity changes). Operating in the differen- tial measuring mode, the output signal of the biosensor is a difference between the newly established conducti- vities at the boundary layer of the working and referen- ce electrodes. Results and discussion. Optimization of ionic strength of working solutions for real sample analysis. Blood serum is a complex, high ionic strength medium, which composition is similar to that of plasma, except that the latter contains fibrinogen and prothrombin required for blood clotting. Accurate measurements of L-arginine in serum can be performed if the working so- lution and the serum sample have equal initial conducti- vities. It was important to be sure that serum aliquot injection to the measuring cell will change conductivity only in connection with L-arginine concentration in the sample. For that reason, we compared the initial con- ductivities of all solutions used, namely bovine serum, 5 mM phosphate solution, pH 6.0 (the composition of the phosphate solution was previously determined as optimal for the reliable performance of the L-arginine biosensor [17]), stock solutions of L-arginine, and urea. The measurements were performed for both solutions and after their appropriate dilution. The measured conductivity of pure serum and that of the working solution (5 mM phosphate solution, pH 6.0) was 10.43 ± 0.03 mS and 0.52 ± 0.03 mS, res- pectively. Such considerable difference in the conducti- vities could be significantly expressed in the biosensor response, even if serum comprised neither L-arginine nor urea. However, it is noteworthy that when injecting certain volumes of serum to the measuring cell, the sam- ple conductivity is likely to reduce because of its dilu- tion in the working solution. The serum sample volume was 30 µl, thus its dilution in the measuring cell was 100. The measured conductivity of such sample was 0.7 ± 0.03 mS. Since the conductivities of the diluted serum and the proper phosphate solution still differed (0.7 ± 0.03 mS and 0.52 ± 0.03 mS, respectively), for higher precision we regulated the working solution con- ductivity by adjusting potassium chloride to its final concentration in the phosphate solution of 1 mM. The conductivities of the stock solutions of L-arginine and urea were 0.75 ± 0.03 mS and 0.42 ± 0.03 mS, res- pectively. These values were quite comparable with the conductivity of the phosphate solution used. Determination of urea in serum. Since the develo- ped bi-enzyme biosensor comprised urease, it was ne- cessary to find out whether or not the tested sample con- tained urea. For that purpose, the urea biosensor was fabricated. Bioselective membranes for urea sensor we- 443 POTENTIALITY OF APPLICATION OF THE CONDUCTOMETRIC L-ARGININE BIOSENSORS re prepared according to the procedure described in our work [20]. Bovine serum, taken for L-arginine analysis, was initially examined towards the presence of urea. The sensitivity of the urea biosensor in 5 mM phosphate so- lution (pH 6.0) comprising 1 mM KCl was found to be quite satisfied (no less than 5 µS/mM). However, the functionally active urea biosensor did not respond to serum. The results of measurements carried out at least in 3 series were the same. Therefore, it was concluded that the tested sample was urea-free. Nevertheless, urea determination in the real sample is necessary in each particular case. Preliminary evaluation of amino acid content in the serum. Since at the beginning it was not known what contributed to the biosensor response exactly, a condi- tional term «L-arginine» was used to substitute all fac- tors having impact on the biosensor signal while the se- rum adjustment. After the serum sample being tested for urea, L-ar- ginine biosensor was calibrated for the model solution of L-arginine (the biosensor calibration curve was ob- tained in 5 mM phosphate solution, pH 6.0, with 1 mM KCl added). The calibration parameters were the fol- lowing: dynamic range 0.025–13.3 mM; linear range 0.025–5 mM (y = 0.06894 + 6.9367x, with the correla- tion coefficient R = 0.99928). Afterwards, the biosen- sor response to the serum aliquot (30 µl) was studied at least in 3 series and L-arginine content in the serum was evaluated using the calibration curve. The determined concentration of L-arginine was 1.372 ± 0.049 mM. Ta- king into account 100-fold dilution of the aliquot sample, the determined concentration ranged between 132.3 and 142.1 mM. Compared to the literature data, this value was sig- nificantly higher than the normal level of L-arginine in the blood (depending of the age and gender, the normal level of L-arginine ranges between 72.4 ± 6.7 µmol/l and 113.7 ± 19.8 µmol/l [21]). Therefore, the determined concentration of L-argi- nine was likely a sum of the biosensor responses to cer- tain compounds. Meanwhile, it was significant that the serum analysis, using the urea biosensor, did not reveal the traces of urea in the sample. Thus, it was important to find a suitable explanation for such behavior of the biosensor. Inhibitory effect of mercury ions on the enzyme ac- tivity of the biosensor for L-arginine determination. The biosensor specificity to L-arginine was verified based on several approaches. One of them was an inhibi- tion of the enzyme activity of the biosensor by mercury ions. For this purpose, the biosensor was immersed into the solution of mercury ions (its concentration was about 100 µM [22]), for 20 min. The inhibition efficiency was evaluated in the further measurements of the bio- sensor response to the model samples of L-arginine and urea. After the incubation of L-arginine biosensor in the mercury ion solution, it was observed no response to the elevated volumes of serum aliquots as well as to increased concentrations of L-arginine. The studies of the enzyme activity of the biosensor after its incubation with mercury allowed concluding that initial biosensor response to serum was due to the presence of certain compounds to which the biosensor was sensitive. High temperature treatment of L-arginine biosen- sor. The aim of further investigations was to find out whether the conductivity changes, recorded by the bio- sensor while serum adjustment, were related to the dif- ference in the ionic strength of the solutions used, or to the level of L-arginine in serum. The responses of L-ar- ginine biosensor to serum (30 µl), L-arginine (1 mM) and urea (1 mM) were measured before and after tempe- rature treatment of the biosensor. High temperature treatment of the immobilized enzymes was carried out as follows. The functionally active biosensor was pla- ced in the boiling UP water for 1, 4, 10, 20, and 40 min. After each boiling, the amplitudes of the biosensor res- ponses to serum, L-arginine and urea were carefully do- cumented. According to the observations, after the biosensor was subjected to high temperatures for 1 min, its respon- ses to serum and L-arginine slightly reduced (compa- red to the initial response, the signal decrement was about 20 %). Thereafter, each following boiling caused a slow decline in the biosensor response to serum and L-ar- ginine. Eventually, after the fourth boiling, the biosen- sor did not respond to both serum and L-arginine at all. Enzyme activity of the biosensor toward the enzyma- tically treated serum. The third approach to prove the biosensor specificity to L-arginine was testing the bio- sensor response to the serum sample, treated with free 444 SAIAPINA O. Y., DZYADEVYCH S. V., JAFFREZIC-RENAULT N. enzymes. The experiments were carried out in the fol- lowing way. The initial responses of the functionally ac- tive biosensor to serum (30 µl), L-arginine (1 mM), and urea (1 mM) were obtained and their amplitudes were documented. Afterwards, 2 mg of the lyophilized argi- nase (E. C. 3.5.3.1, 136 U/mg solid) and 2 mg of lyophi- lized urease (E. C. 3.5.1.5, 100 U/mg solid) were added to the individual microtube containing the pure serum sample (volume 1.8 ml). The enzymes were carefully solubilized in serum and the response of L-arginine bio- sensor to the aliquot of obtained suspension was measu- red each 20 min after adjustment of free enzymes, the last measurement was carried out in three hours. An analysis of the biosensor responses obtained af- ter pure serum treatment revealed quite interesting facts. Before the free enzymes were adjusted to the 30 µl se- rum sample, the biosensor response was 10.59 µS. Af- ter 20 min incubation the biosensor signal amplitude de- creased (9.048 µS). During further serum incubation, the biosensor response remained relatively constant (the coefficient of variation of the response intensity was about 0.5 %). In three hours after the moment when pu- re serum was subjected to arginase and urease, the bio- sensor response was about 9 µS. Thus, a conclusion was drawn about the impact of some compounds, other than L-arginine, on the biosensor signal. These observations implied to the biosensor selectivity. Selectivity of the bi-enzyme conductometric biosen- sor for L-arginine determination. The biosensor selecti- vity studies [17] demonstrated that the L-arginine bio- sensor had remarkable sensitivity to two other basic amino acids (i. e., L-lysine, L-histidine), was less sensi- tive to γ-aminobutyric acid and almost insensitive to others. Interestingly, in the literature L-lysine and L-histi- dine are widely considered as competitive inhibitors of arginase [23–25]. Xie et al. speculated [24] that L-ly- sine inhibited the arginase activity and switched off the EPR signal of the binuclear center by removing a brid- ging ligand or by increasing the inter-manganese se- paration. Analysis and interpretation of the results. Summa- rizing the results of all optimization procedures it was drawn the following conclusion. While testing the en- zyme activity of the biosensor toward the enzymati- cally treated serum, it was revealed that after a moment when free arginase and urease hydrolyzed the serum L-arginine, the biosensor yet responded to serum. To explain that observation we assume that the serum injection provoked the interactions between arginase and small amounts of L-histidine (His) and L-lysine (Lys), present in serum, to such extent that the observed response was initially interpreted as that to L-arginine (according to [24, 26], the inhibitory influence of these amino acids on the arginase activity is observed at high concentrations). Eventually, diminution of the biosen- sor response to serum from 10.59 µS to about 9 µS sug- gested that pure serum (without free enzymes added) did contain Arg, and the difference in the biosensor sig- nal (1.59 µS) could be exactly attributed to the response to Arg. The biosensor response monitored after first series of the high temperature treatment of the immobilized membranes, was also explained by the presence of Lys and His in the sample. To interpret the nature of the biosensor response toward Lys and His, it was made the following assumption. It is known that both enzy- mes (arginase and urease) as well as BSA are histidine- rich compounds. As it was stated in [27], His and Lys tend to the formation of the salt-bridges between their residual chains in the aqueous solutions (the most pro- nounced ability to the ion-pairing was observed for His-residues). Consequently, ion-pairing between His, belonging to the active centers of enzymes, and exoge- nous His and Lys affects the arginase ability to bind L- arginine properly but, at the same time, causes the con- ductivity changes at the boundary layer, and, thus, the biosensor response to these amino acids. Evidently that the biosensor response after the first series of the high temperature treatment originated from the interactions between exogenous His and Lys (from the serum samp- le) and His and Lys, present within the denaturated pro- tein molecules. According to the test on the biosensor specificity ba- sed on the inhibition with mercury ions, it was confir- med that the initial sensitivity of the biosensor was rela- ted to the interactions of L-arginine and two other basic amino acids with enzymes. After incubation of the immo- bilized enzymes in the mercury-based solution, the bio- sensor did not respond to serum (it was useful observa- tion, since it allowed to ensure that the conductivity of the electrolytes, present in serum, was fitted properly 445 POTENTIALITY OF APPLICATION OF THE CONDUCTOMETRIC L-ARGININE BIOSENSORS and, therefore, it was not a part of the initial respon- siveness of the biosensor to the serum injections). Summarizing all the observations, to eliminate the distortion of the biosensor response to serum by Lys and His, and aiming for the accurate determination of L-ar- ginine, we propose the following strategy. After the pro- cedure of fitting the conductivity of the working solu- tion in accordance to the conductivity of that of serum, and calibration the L-arginine biosensor in model solu- tion of L-arginine, the necessary steps are the follow- ing: 1) to analyze the serum sample using the urea bio- sensor (if the result is negative, to follow the further steps. If the result is positive, to obtain the calibration curve of the L-arginine biosensor in model solution of urea and then follow the further steps); 2) to document the initial response of the L-arginine biosensor to the se- rum aliquot; 3) to treat the serum sample with histidine ammonia-lyase (E. C. 4.3.1.3) (in order to free sample from L-histidine); 4) to document periodically the bio- sensor responses to the serum aliquot until reaching the invariable response of the biosensor; 5) to treat the se- rum sample with lysine-2,3 aminomutase (E. C. 5.4.3.2) (in order to free sample from L-lysine); 6) to document periodically the biosensor response to the serum aliquot until reaching the invariable response of the biosensor. Afterwards, the measure, at which the invariable res- ponse of the biosensor is reached, may be used for the determination of the L-arginine concentration in serum using a calibration curve of the L-arginine biosensor. Besides the L-arginine measurement in serum, the developed conductometric biosensor was applied to the analysis of several pharmaceutical items, namely the commercially available drinkable solution «Arginine- Veyron» (Laboratoires Pierre Fabre Medicament, Fran- ce) and the tablets «Arginotri-B» (Bouchara-Recordati, Italy). The solution «Arginine-Veyron» had the following composition: 5 ml of the solution (1 ampoule) contai- ned L (+) arginine hydrochloride 1 g (corresponds to the quantity of L (+) arginine: 0.8266 g), excipients: ca- ramel flavor, methyl parahydroxybenzoate (E218), sac- charin, saccharose solution at 67 % (m/m), purified water. The result of the arginine determination in the so- lution «Arginine-Veyron», obtained by the method of calibration curve, is presented in Table. The standard deviation between five repeatable mea- surements (n = 5) was found as 20.84 mM, with stan- dard error of 9.32 mM (Table). Taking the reliability assessment as γ = 0.95, the arginine measurement may be given within the following confidence interval: (946.13 ≤ a ≤ 982.67) mM. The coefficient of variation of the obtained measures was 2.16 %. Comparing the stated value (922.4 mM) and that one, obtained experi- mentally, we suppose that some variance between these values could be caused by the interference of the back- ground (i. e., presence of the additives contained in the examined solution). At measuring the arginine in the tablets «Arginotri- B», it was observed a pronounced inhibitory effect of vitamins B1 and B6, present in the tablets, on the enzy- me activity of the biosensor. However, the studies on biosensor responses to B1 and B6 alone showed that the inhibition was reversible. Conclusions. A detailed procedure of optimization of the conductometric biosensor for L-arginine deter- mination in bovine serum has been proposed. The non- specific conductivity changes in the course of analysis were eliminated by the regulation of ionic strength of the phosphate solution used. It was confirmed that con- siderable amplitude of the biosensor signal in response to the serum sample injection can be attributed to the presence of two basic amino acids, L-lysine and L-his- tidine. For the accurate determination of L-arginine in real samples it was suggested the treatment of the serum samples with the free enzymes specific to L-histidine and L-lysine (histidine ammonia-lyase and lysine-2,3 aminomutase, respectively). To date, the adaptation and 446 SAIAPINA O. Y., DZYADEVYCH S. V., JAFFREZIC-RENAULT N. CC, mM CDD, mM SD/SE, mM SC, mM 0.927 954 20.84/9.32 922.4 0.955 983 – – 0.955 983 – – 0.940 968 – – 0.908 934 – – Indications: CC – individual Concentration obtained from the Calibra- tion curve for Arg-HCl; CDD – obtained concentration including Deg- ree of Dilution (total DD = 1029.25 times); SD – Standard Deviation; SE – Standard Error of the mean; SC – Concentration Stated by the producer. Determination of arginine in the ampoules «Arginine-Veyron» application of the L-arginine biosensor for the analysis of biological fluids may be considered as an attractive diagnostic tool in the modern medicine. Acknowledgements. The authors would like to thank National Academy of Sciences of Ukraine (complex sci- entific-technical program «Sensor systems for medi- cal-ecological and industrial purposes»), the European Commission for their funding of the Project IRSES- NANODEV and Rhone-Alpes Region for MIRA project. О. Я. Саяпіна, С. В. Дзя де вич, Н. Жаф фре зик-Рено По тенційна мож ливість за сто су ван ня кон дук то мет рич них біосен сорів для виз на чен ня L-аргініну при аналізі ре аль них зразків Ре зю ме Мета. Виз на чи ти вплив ком по нентів си ро ват ки крові на чут ли- вість біосен со ра при ви яв ленні L-аргініну та сфор му лю ва ти прак- тичні ре ко мен дації для за без пе чен ня її надійно го аналізу. Ме то - ди. Біосен сор для виз на чен ня L-аргініну містить аргіназу і уре а зу, ко-іммобілізо вані ме то дом по пе реч но го зши ван ня. Ре зуль та ти. Спе цифічність біосен со ра вив ча ли на основі низ ки по каз ників – вмісту се чо ви ни у си ро ватці; інгібу валь но го впли ву іонів ртуті; ви со ко тем пе ра тур ної об роб ки біосен сорів; чут ли вості біосен со - ра до си ро ват ки крові, об роб ле ної ліофілізо ва ни ми пре па ра та ми фер ментів, та се лек тив ності біосен со ра. Вста нов ле но, що відгук біосен со ра на вне сен ня си ро ват ки зу мов ле ний ви со кою чут ливі- стю біосен со ра ще до двох, крім L-аргініну, основ них аміно кис - лот (L-лізину та L-гісти ди ну). Вис нов ки. Зап ро по но ва но де таль - ну про це ду ру оптимізації кон дук то мет рич но го біосенсо ра для ви- зна чен ня L-аргініну у си ро ватці крові. Клю чові сло ва: L-аргінін, кон дук то мет ричні біосен со ри, си ро - ват ка крові, про це ду ра оптимізації. О. Я. Са я пи на, С. В. Дзя де вич, Н. Жаф фре зик-Рено По тен ци аль ная воз мож ность при ме не ния кон дук то мет ри чес ких би о сен со ров для опре де ле ния L-ар ги ни на при ана ли зе ре аль ных об раз цов Ре зю ме Цель. Опре де лить вли я ние ком по нен тов сы во рот ки кро ви на чув- стви тель ность би о сен со ра для вы яв ле ния L-ар ги ни на и сфор му - ли ро вать прак ти чес кие ре ко мен да ции для об ес пе че ния ее над еж - но го ана ли за. Ме то ды. Би о сен сор для опре де ле ния L-ар ги ни на со- дер жит ар ги на зу и уре а зу, ко-им мо би ли зо ван ные ме то дом по пе - реч ной сшив ки. Ре зуль та ты. Спе ци фич ность би о сен со ра из уча - ли на осно ве се рии по ка за те лей – со дер жа ния мо че ви ны в сыво- рот ке; ин ги би ру ю ще го эф фек та ио нов рту ти; вы со ко тем пе ра - тур ной об ра бот ки би о сен со ров; чу встви тель нос ти би о сен со ра к сы во рот ке кро ви, об ра бо тан ной ли о фи ли зо ван ны ми пре па ра та - ми фер мен тов, и се лек тив нос ти би о сен со ра. 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