Novel sucrose three-enzyme conductometric biosensor

Transcription

1 Available online at Materials Science and Engineering C 28 (2008) Novel sucrose three-enzyme conductometric biosensor O.O. Soldatkin a,, V.M. Peshkova a,b, S.V. Dzyadevych a, A.P. Soldatkin a, N. Jaffrezic-Renault c, A.V. El'skaya a a Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, 150 Zabolotny Str., Kyiv, Ukraine b National Taras Shevchenko University of Kiev, 64 Volodymyrska Str., Kyiv, Ukraine c Laboratoire des Sciences Analytiques, UMR CNRS 5180, Universite Claude Bernard-Lyon 1, Villeurbanne Cedex, France Available online 13 October 2007 Abstract For the first time a conductometric biosensor for sucrose determination has been developed using a complex three-enzyme (invertase, mutarotase, and glucose oxidase) containing membrane as a sensitive element immobilized on the conductometric interdigitated planar electrodes. The time of measurement of sucrose concentration in the solution was about 1 2 min. The dynamic range of biosensor depends on buffer capacity, being 2 μm 5 mm of sucrose in 5 mm phosphate buffer. The conductometric biosensor developed demonstrates high selectivity, operational stability and reproducibility. The dependence of sensor response on ph and ionic strength of tested solution has been studied in this work. Storage conditions have also been under investigation. The sensor appeared to be eligible towards application in practice Elsevier B.V. All rights reserved. Keywords: Conductometric biosensor; Invertase; Mutarotase; Sucrose 1. Introduction Application of biosensors for substances monitoring in some technological and biotechnological practices, such as production of alcoholic and nonalcoholic drinks and sweets can be prospective for process optimization and control [1]. Sugar beet molasses are often used in some enzymatic processes as a nutrient medium, besides it is a natural resource in various products manufacture [2]. Sucrose, a key component of molasses, is used in food industry as liquid sugar; some special sugars are consumed in pharmaceutics and cosmetic industry [3,4]. Since sucrose is a component of foodstuff and beverages, precise information on the sucrose presence and concentration is very important for assessment of their quality. Current standard methods of sucrose accurate determination, liquid chromatography, chemical and optical methods, are disadvantageous in necessity of highly skilled personnel and Corresponding author. Tel.: ; fax: address: alex_sold@yahoo.com (O.O. Soldatkin). expensive complicated equipment [5,6] as well as in quite complex sample pretreatment. Other methods based on determination of density or refractory index, though simpler and faster, are less precise. Besides, they are sensitive to the presence of interfering components in the solutions [7]. Therefore, development of more convenient, precise, selective, fast and inexpensive method of determination of sugar content in various alcoholic and non-alcoholic beverages and foodstuff is an issue of the day. Enzyme biosensors for sucrose determination are potential in eliminating the disadvantages and meeting the requirements above mentioned. Currently, there is certain information on development of a number of sucrose biosensors [7 13]. The first biosensor [11] based on the enzymes invertase, mutarotase and glucose oxidase immobilized on oxygen electrode surface, was very sensitive to the oxygen level in a reactor and had poor selectivity. Phosphate ions instead of mutarotase were used in [7,12] to transform α-glucose into β-glucose. The yeast cells, as an invertase resource, were co-immobilized with glucose oxidase and used in [12], oxygen consumption being a measure in this case. Similar sucrose electrode was described for the determination of sucrose without interference with glucose or /$ - see front matter 2007 Elsevier B.V. All rights reserved. doi: /j.msec

2 960 O.O. Soldatkin et al. / Materials Science and Engineering C 28 (2008) fructose based on three-enzyme system of sucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase [13]. However, these biosensors being based on amperometric transducers have, along with their inherent advantages, some essential disadvantages. First, need in high measurement potential that results in errors associated with the presence of other electrooxidizing components, e.g. ascorbic acid, in the tested solutions. The interfering current can be decreased by usage of additional membranes [14] or polymer films [15], though this complicates manufacture of these systems and thus raises the total analysis price. Conductometric biosensor methods of sucrose analysis have not been hitherto used, though in our opinion this approach is rather promising, since these methods are sufficiently simple, convenient, accurate, and allow meeting important scientific and industrial challenges [16]. Conductometric transducers are considerably beneficial as compared with other electrochemical transducers [16, 17]. Their advantages are as follows: there is no need in technologically complex referent electrode; alternating voltage of low amplitude allows avoiding Faraday processes at electrodes; the transducers are not light-sensitive in contrast to ion-selective fieldeffect transistors; potential of miniaturization and high-level integration using inexpensive thin-film standard technology; low cost in industrial production. Considering the advantages mentioned, development of conductometric sucrose biosensor with reaction cascade of three-enzyme system seems very prospective. 2. Materials and methods Fig. 1. General view of a conductometric planar interdigitated electrode Materials The frozen-dried preparations of enzymes used in the experiments were as follows: glucose oxidase (GOD) from Penicillium vitale (ЕС ) with activity of 130 U/mg from Diagnosticum (L'viv, Ukraine); invertase (ЕС ) from baker's yeast with activity of 355 U/mg from Sigma-Aldrich Chemie; mutoratase(ес ) with activity of 100 U/mg from Biozyme Laborаtories Ltd., (UK). Bovine serum albumin (BSA) (V fraction) and 50% aqueous solution of glutaraldehyde (GA) were obtained from Sigma-Aldrich Chemie. Sucrose was used as a substrate and analyzed substance, potassium-phosphate solution (КН 2 Р0 4 NаОН), рн 7.2 from Меrck was used as a buffer. Other non-organic compounds were of analytical grade Sensor design The conductometric transducers produced according to our recommendations and outline in Lashkarev Institute of Semiconductor Physics of National Academy of Sciences of Ukraine (Kyiv, Ukraine) consisted of two identical pairs of gold interdigitated electrodes made by gold vacuum evaporation onto pyroceramic substrate (5 40 mm) (Fig. 1). The surface of sensitive area of each electrode pair was about mm. The width of each of interdigital spaces and digits was 20 μm. Fig. 2. Scheme of conductometric set-up.

3 O.O. Soldatkin et al. / Materials Science and Engineering C 28 (2008) Bioselective membrane production The solution consisting of invertase 4%, mutarotase 3%, glucose oxidase 3% in 20 mm phosphate buffer, ph 7.2, with 10% glycerol (further three-enzyme solution) was used to produce the working membrane while the same mixture with BSA instead of enzymes for the referent membrane. Prior deposition onto transducer surfaces, the solutions for both working and referent membranes were mixed with 2% glutaraldehyde aqueous solution in 1:1 ratio. The solutions obtained were immediately deposited onto the transducer working part by micropipette Eppendorf (of total volume μl) to cover completely the working surface of interdigitated electrode pair, the volume of each of the solutions less than 0.2 μl being enough per an electrode pair. The protein content was the same in both membranes. Before usage, the sensors dried during 20 min in the air at room temperature were then washed out in the working buffer solution Scheme of experimental measuring set-up Scheme of experimental measuring set-up is shown in Fig. 2. The alternating voltage with the frequency of 100 khz and amplitude 10 mv was applied from the low-frequency signal generator GZ-118 (Ukraine) to the differential pair of interdigitated electrodes placed in an experimental vessel with the solution tested. The signal obtained at the electrodes was transferred from the 1 kohm load resistance via differential amplifier Unipan onto selective nanovoltmeter Unipan- 233 (Poland) whereupon at the registering apparatus. The dependence of the output signal on the substrate concentration in the solution was measured The measurement procedure Fig. 4. Calibration curves of sucrose biosensor at different buffer concentrations. Measurements were done in 2.5 mm (1), 5 mm (2), 10 mm (3) and 20 mm (4) phosphate buffer, ph 7.2. adding given portions of the substrate of standard initial concentrations. The experiments were performed at least in three series. The effect of nonspecific variations of output signal owing to temperature and ph changes and electric interferences was avoided by differential mode of measurement. 3. Results and discussion The basic cascade of enzymatic reactions for sucrose detection by conductometric biosensor is as follows: invertase sucrose þ H 2 OYb D fructose þ a D glucose mutarotase ð1þ The measurements were carried out in an open cell at room temperature. The 5 mm phosphate buffer or universal buffers at different ph were used at intensive stirring. The necessary substrate concentration in the working buffer was achieved by a D glucoseyb D glucose glucose oxidase b D glucose þ O 2 YD gluconolactone þ H 2 O 2 x ð2þ ð3þ D gluconic acid þ Н 2 ОTacid residuum þ Н þ ð4þ Fig. 3. Dependence of biosensor responses on ionic strength of solution. Measurements were done in 5 mm phosphate buffer, ph 7.5. As a result, sucrose is gradually decomposed with invertase, mutarotase and glucose oxidase to hydrogen peroxide and D- gluconolactone. In its turn, the latter is spontaneously hydrolyzed to gluconic acid which dissociates to the acid residuum and a proton, the solution conductivity being changed which can be registered by the conductometric transducer [16]. The conductometric method is known to be based on measurement of the change in conductivity of the solution analyzed. This change can depend on both the enzyme reaction as such and characteristics of the solution in which this reaction takes place. Therefore, the influence of the solution parameters on the sensor response was studied in the first place.

4 962 O.O. Soldatkin et al. / Materials Science and Engineering C 28 (2008) Fig. 6. Reproducibility of biosensor response during one working day. Measurements were done in 5 mm phosphate buffer, ph 7.2. Ionic strength is one of the basic buffer characteristics which can have a negative effect on measurements by conductometric biosensor. To study this effect, the signal dependence on addition of different KCl concentration (1 100 mm) to the substrate solution of constant concentration (1 mm sucrose) was studied (Fig. 3). As can be seen, an increase in ionic strength results in exponential decrease of the response to the substrate concentration, a remarkable reduction at the beginning, 5% signal drop at KCl concentration of 50 mm, while a stable signal is observed at further KCl addition. One of key reasons of this effect is an increase in the solution background conductivity; therefore the ionic strength of the samples analyzed by conductometric biosensor is to be strictly controlled. Second parameter of analyzing sample that can have influence on the sensor response is buffer capacity. That is why the calibration curves of dependence of the biosensor responses on the sucrose concentration in buffer solution are plotted for various buffer capacities of the solution (Fig. 4). The biosensor responses and linear range of measurements are seen to vary to some extent when the concentration of buffer solution changes. The highest sucrose sensitivity of conductometric biosensor was in 2.5 mm phosphate buffer, ph 7.2, however the linear range in this case shifts towards the region of low concentrations, i.e mm (Fig. 4), while in 5-mM phosphate buffer the linear range was slightly wider ( mm). In 10- and 20-mM phosphate buffers the biosensor sensitivity towards sucrose slightly decreased. Therefore, the biosensor for measurement of sucrose concentration in given ranges with required sensitivity can be attained by varying buffer concentration, thus being adapted to the actual practical needs. Any enzyme is known to have an optimal ph, which can be shifted to either alkaline or acid region as a result of immobilization. Since a mixture of three enzymes with different ph optimums is immobilized on the sensor surface in our case, consequently the optimal buffer ph for work of the conductometric biosensor for sucrose determination should be optimized which was the next step of our study. As a capacity of onecomponent buffer changes at varying ph, the experiments were carried out to avoid a buffer capacity effect on the sensor response. Specifically, the dependence of sensor response on ph was studied for a universal multicomponent buffer characterized in similar buffer capacity within a wide range of ph values. It consists of a mixture of different buffer solutions phosphorous, acetic and boric acids of 0.04 M concentration, ph [18]. The curve of dependence of the biosensor signal to 1 mm sucrose introduction on ph was bell-like with maximum at ph 6 (Fig. 5). Operational stability and signal reproducibility, essential characteristics of biosensors, were tested. The responses to the samesucroseconcentration(0.5mm) weremeasuredevery30min forfourdays,thesensorduringintervalswaskeptinthecontinuously stirredbuffer.whilestayingidleatnightbiosensorswerestoreddryat room temperature. The chosen sucrose concentration was on the linearregionofsensorcalibrationcurve.ascanbeseeninfig.6,the measurement data were highly reproducible every day of the experiment,whilesufficientoperationalstabilitywasrevealedfora week(fig.7). Fig. 5. ph-dependence of biosensor response. Measurements were done in the universal buffer, sucrose concentration 1 mm. Fig. 7. Operational stability test for sucrose conductometric biosensor. Measurements were done in 5 mm phosphate buffer, ph 7.2.

5 O.O. Soldatkin et al. / Materials Science and Engineering C 28 (2008) Biosensor stability is an important characteristic of developed biosensor especially for its further commercialization. The research on biosensor stability at different storage conditions was performed (Fig. 8). The biosensors were stored in 5 mm phosphate buffer solution, ph 6.0, at temperatures of + 20 С and +4 С, as well as in dry conditions at +20 С, +4 С and 5 С. In 3 days after fabrication of sucrose biosensors, their responses to the injection of 1 mm sucrose into the model solution were obtained and these values taken as 100%. Next measurements were performed in 5 8-day intervals. The activity of biosensors stored dry at 5 С and +4 С was close to stable for a month; further storage during 4 months resulted in 10-fold reducing activity. Sucrose biosensors stored in phosphate buffer at + 4 С or dry at +20 С lost their activity during a month, while those kept at +20 С in phosphate buffer gave no response on the 20th day. The loss of activity of the developed three-enzyme biosensor can be a result of denaturation or deactivation of at least one enzyme of the membrane. Since immobilized glucose oxidase is known to be highly stable [8,19] the key factors of decreasing biosensor activity are deactivation of mutarotase or invertase. One of the reasons of mutarotase activity loss can be its oxidation by hydrogen peroxide generated at glucose hydrolysis by glucose oxidase [20]. The invertase stability was shown [21] to be a limiting factor of the biosensor stability. Anyway, being stored dry at low temperatures the developed sucrose biosensor had stable working characteristics longer than upon storage in buffer solution. Since sensor selectivity is of great importance in experiments with real samples, the influence of interfering components on the sensor response was studied. 10 mm phosphate buffer, ph 7.0, was used for measurements. The solution of 1 mm interfering substances was injected into a cell (Table 1). The sensor responses to these compounds were calculated in percentage terms, the response to 1 mm sucrose being taken as 100%. Basically, the sucrose biosensor turned out to be selective towards a number of interfering substances potentially present Table 1 Selectivity test of the sucrose biosensor 1 mm interfering substance Response of the sucrose biosensor (%) Sucrose 100 Maltose 10 Lactose 0 Glucose 165 Fructose 1 Sorbitol 0 Ramnose 0 Mannitol 0 Arabinose 0 Ascorbic acid 0 in juices, except for glucose. The response to 1 mm glucose was 165% relative to 1 mm sucrose. It is quite reasonable as glucose oxidase is a component of the enzyme membrane of sucrose biosensor. However, the response to glucose can differ depending on the enzymes proportion in membrane which is controllable. 4. Conclusion The conductometric biosensor for sucrose determination has been first developed. A complex three-enzyme membrane was immobilized onto conductometric transducer and used as a sensitive element of the biosensor. The analytical parameters have been optimized for the operation with real samples. The conductometric biosensor developed was characterized by high operational stability and signal reproducibility. This biosensor has better selectivity than amperometric analogues because it has no response to the presence of electrooxidizing components, e.g. ascorbic acid, in the tested solutions. The results of preliminary measurements performed in real juices can be considered as promising for usage of the biosensor for sucrose determination in real solutions, such as juices, wines, honey, etc. Acknowledgement Part of this work was financially supported by National Academy of Sciences of Ukraine in the frame of Scientific and Technical Program Sensors systems for medical ecological and industrial technological problems. References Fig. 8. Storage stability test for biosensors stored in 5 mm phosphate buffer, ph 6, at +4 С (1), +20 С (2) and in dry state at +20 С (3), +4 С (4) and 4 С (5). Measurements were done in 5 mm phosphate buffer, ph 6. [1] D.R. Schmid, F. Scheller, Biosensors. Application in medicine, environmental protection and process control, VCH, Weinheim, [2] J.C.Y. Tsao, Sugar Azúcar 59 (1964) 98. [3] H. Schiweck, Zuckerindustrie 119 (1994) 272. [4] K. Thielecke, Branntweinwirtschaft 127 (1987) 193. [5] B. Herbreteau, M. Lafosse, L. Morin-Allory, M. Dreux, J. High Resolut. Chromatogr. 13 (1990) 239. [6] S.V. Vercelotti, M.A. Clarke, Int. Sugar J. 96 (1994) 437. [7] J.L. Lima Filho, P.C. Pandey, H.H. Weet-all, Biosens. Bioelectron. 11 (1996) 719. [8] M. Filipiak, K. Fludra, E. Gosciminska, Biosens. Bioelectron. 11 (1996) 355. [9] M.D. Gouda, M.A. Kumar, M.S. Thakur, N.G. Karanth, Biosens. Bioelectron. 17 (2002) 503.

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