- Email: [email protected]
Yeast cells sucrose biosensor based on a potentiometric oxygen electrode
Analytica Chimica Acta 458 (2002) 215–222
Yeast cells sucrose biosensor based on a potentiometric oxygen electrode Lucian Rotariu∗ , Camelia Bala, Vasile Magearu Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, Sos. Panduri, No. 90, 76229-Bucharest 5, Romania Received 20 June 2001; received in revised form 14 September 2001; accepted 13 November 2001
Abstract A new microbial biosensor based on an immobilised microorganisms (Saccharomyces cerevisiae) and a potentiometric oxygen electrode is described. Determination is based on the respiratory activity of the microorganism in presence of different sugars (sucrose and glucose). A response time of ca. 4 min for the steady-state method and 2 min for the initial slope method was obtained. Potentiometric detection has the advantage of an extended calibration range and a low detection limit. The calibration curve for sucrose was linear in the range 1 × 10−5 to 3 × 10−2 M. This biosensor was used for selective monitoring of sucrose in the presence of glucose, using a second anti-interference enzymatic layer with glucose oxidase (GOD) and catalase (CAT). Interference of glucose in the determination of sucrose decreases from 15% for a microbial biosensor to a maximum 3.5% for the hybrid biosensor. The hybrid biosensor was used to determine sucrose in soft drinks. A good correlation between the results for the biosensor and a spectrophotometric method with dinitrosalicylic acid was achieved. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sucrose; Biosensor; Potentiometry; Yeast; Food analysis
1. Introduction Glucides are always present in biological, food products and biotechnological processes. The most important sugars are glucose and sucrose. It is easy to determinate glucose enzymatically without interferences from other glucides. Different enzyme electrodes for sucrose, based on invertase (EC 3.2.1.26), mutarotase (EC 5.1.3.3) and glucose oxidase (GOD) (EC 1.1.3.4), were reported using a oxygen electrode [1–7] or a platinum electrode [8,9]. This procedure is straight-forward for glucose-free media. Direct determination of sucrose in glucosecontaining samples was achieved using a multi-layer ∗ Corresponding author. Tel.: +40-1-315-92-49; fax: +40-1-315-92-49. E-mail address: [email protected] (L. Rotariu).
enzyme electrode [9,10]. An outer anti-interference layer containing GOD and catalase (CAT) that is separated from the indicator layer by a dialysis membrane eliminates the glucose interference. To prepare the enzyme layer, physical [10,11] and chemical [1,12,13] immobilisation procedures have been employed. The -d-glucose and oxygen are converted by GOD to the electro-inactive gluconic acid and the electrode-active hydrogen peroxide; the latter is converted in the catalytic reaction to no-interfering H2 O and O2 . The sucrose reaches the GOD/invertase layer, thus providing H2 O2 , which is measured. Food products were analysed successfully and up to 2 mM glucose could be tolerated [10]. Mediators [14] or modified carbon paste electrodes [15] were also used for sucrose biosensors. A potentiometric enzymatic biosensor [16] was designed using
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 5 2 9 - X
216
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
a three enzyme system and a pH electrode. The main problem for all enzymatic biosensors for sucrose is the multi-enzymatic system, sometimes requiring expensive or unstable enzymes. In order to reduce the cost of a biosensor some authors [17] proposed to replace the isolated and purified invertase with fragments of yeast cell wall, the main source for this enzyme. Bacteria and yeasts are recognised as organisms which metabolise very well all kind of sugars. Incubation with sucrose was used as a good method to improve the selectivity of a microbial biosensor for sucrose [18]. A hybrid biosensor based on Zymomonas mobilis and invertase [19] was successfully used for sucrose determination in fruit juices. A microbial amperometric biosensor for sucrose was previously presented [20] and consists in using yeast cells of S. cerevisiae immobilised on the surface of a Clark oxygen electrode. The selectivity of this microbial sensor was improved by using a second anti-interference enzymatic layer. The yeast cells do not assimilate gluconic acid obtained by enzymatic oxidation of glucose. The main disadvantage of this biosensor is the response range limited to the concentration between 6 × 10−3 to 0.1 M. This paper describes, a potentiometric microbial biosensor for the determination of sucrose. By changing the classical oxygen electrode with a potentiometric oxygen electrode [21] a more sensitive biosensor was obtained. To increase the biosensor selectivity the same enzymatic layer was used. 2. Experimental 2.1. Materials A yeast strain of Saccharomyces cerevisiae, purchased from the University of Galati (Romania), was used as a biocatalyst for the microbial sensor. GOD, CAT, glucose and sucrose were purchased from Fluka Chemie AG; all other reagents were of analytical grade. Standard solutions of glucose and sucrose were prepared in disodium phosphate–citric acid buffer, 0.5 M. 2.2. Preparation of microbial electrode and assay procedure Yeast cells were previously incubated in a medium containing 1.0 g l−1 KH2 PO4 , 3.5 g l−1 (NH4 )2 SO4 ,
0.3 g l−1 MgSO4 , 0.1 g l−1 CaCl2 and 2% sucrose as unique carbon source. Incubation of the cells in a medium based on the substrate for which the biosensor is intended, is a well-known method to improve the selectivity of the selected cells. The S. cerevisiae cells were maintained for 12 h at 30 ◦ C, under continuing oxygenation of the cells suspension. After centrifugation, the solid deposit was suspended in distilled water and centrifuged again. The cell mass obtained was suspended in 0.9% NaCl solution and successive dilutions were made (absorbance between 0.05 and 0.4 at 660 nm). The 0.5 ml of each suspension was filtered through a nitrocellulose membrane. After drying, each membrane was kept at 4 ◦ C before utilisation. The oxygen sensor consists in a platinum electrode as redox indicator electrode and an Ag/AgCl electrode as reference electrode, covered with an oxygen permeable PTFE membrane. The internal electrolyte is based on 0.1 M KCl and 5×10−4 M Mn(OH)2 . Potential measurements were made using a digital mV/pH— meter Cole Parmer model CP 059H3-H5. The immobilised microorganisms were placed on the oxygen membrane, covered with a dialysis membrane and fixed with a rubber ring. A schematic representation of the device is given in Fig. 1. All the determinations were achieved in a 10 ml measuring cell and all the solutions were previously saturated with oxygen. Before every determination, the biosensor was kept in oxygen-saturated phosphate buffer solution. After the output signal of the microbial sensor became stable, the sensor was removed to a buffered standard solution of sucrose (saturated with oxygen). The potential change indicates that sucrose passes through the membrane and is assimilated by the immobilised yeast cells. Oxygen consumption due to respiratory activity of the microorganism caused a decrease in dissolved oxygen concentration around the membrane and consequently brought about the decrease in output signal. The decrease of oxygen concentration was taken as the measure of sucrose concentration. The first step is considered to be the enzymatic hydrolysis of sucrose catalysed by invertase (E.C.3.2.1.26) localised in the cell wall of S. cerevisiae. Then glucose and fructose are assimilated in the presence of oxygen inside the cell. invertase
sucrose + H2 O → glucose + fructose
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
217
Fig. 1. Schematic representation of potentiometric microbial biosensor for sucrose.
2C6 H12 O6 (glucose) + 16O2
S. cerevisiae
→
CO2 + 24H2 O
Decrease of the oxygen concentration around the oxygen electrode is measured and correlated with sucrose concentration from the sample. In principle, there are two possibilities for measuring the biosensor response: (a) endpoint determination (steady-state method); (b) kinetic measurements (initial slope method). For selective determination of sucrose in the presence of glucose a second anti-interference layer containing GOD and CAT was added. Glucose is converted to gluconic acid that is not used by yeast cells as a carbon source in respiration activity.
3. Results and discussion 3.1. Optimisation of the microorganism concentration in the biocatalytic layer The membranes with immobilised yeast were tested on a 1 mM sucrose solution. Response curves were
registered and the difference between the steady-state signal and base signal in oxygen-saturated buffer is shown in Fig. 2. Low immobilised cell concentrations do not modify the baseline response of the biosensor in the presence of sucrose. Increasing the cell concentration on the surface of the membrane leads to a very low base signal for the microbial biosensor and the signal decreases rapidly to zero in the presence of sucrose in the sample solution. In extremes, for a very high concentration of yeast cells, the oxygen electrode could not detect dissolved oxygen around the biocatalytic membrane. Membranes made by depositing 0.5 ml of a cell suspension with absorbance of 0.08 showed the maximum response. These membranes have been used for further experiments. 3.2. Response curve of microbial biosensor When the microbial sensor was immersed in the oxygen saturated buffer solution the output current of the sensor became stable within 5 min. After the
218
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
Fig. 2. Optimisation of yeast cell concentration (temperature 25 ◦ C, pH = 7.00, sucrose 1 mM). Each point represents the average of three determinations.
steady current was obtained, the sensor was transferred to solutions containing different concentrations of sucrose in the range 10−5 to 10−2 M. The output current began to decrease and the minimum current was observed within 4 min. The amperometric sucrose biosensor previously reported [6] has fast response time of about 90 s. This relatively long response is due to the potentiometric oxygen electrode [7] and it is independent of the sucrose concentration. This behaviour shows that oxygen consumption by the yeast cells is faster than the response of the oxygen electrode. Unusually for a microbial biosensor, the response is determined by the detector. Kinetic measurements could be done in the first 2 min when the response curve is linear. 3.3. Effect of temperature The respiration activity of the yeast cells depends on the presence of a carbon source and on the temperature. Fig. 3 shows the effect of temperature upon the response of the microbial sensor. At 20 ◦ C, the response of the sensor was less than at higher temperatures. The optimal temperature for growth of S. cerevisiae was reported to be 30 ◦ C [22], in accord with our results. A temperature of 25 ◦ C was chosen as the
working temperature. A lower baseline for the oxygen electrode, worse reproducibility and limited response range for sucrose was observed at higher temperatures. 3.4. Effect of pH The effect of pH on the response of the microbial biosensor was investigated in the pH range 4–8. The response to a standard solution of 2 mM sucrose was checked and the optimum pH was 5. The optimum pH for invertase activity is 4.6 [23] for the isolated enzyme, and for a S. cerevisiae culture is ca. 5 [22]. 3.5. Calibration curve Fig. 4 shows the calibration graphs for glucose and sucrose using the steady-state method under conditions of pH = 5 and 25 ◦ C. Each point represents the average for five determinations. A linear relationship between the oxygen concentration decrease and concentration of sucrose and glucose was observed, up to 30 mM. Parameters for linear regression (y = ax + b) are presented in Table 1. A slope of 24 mV/decade shows the relatively low sensitivity of the biosensor. The main advantage of the potentiometric sucrose biosensor is
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
219
Fig. 3. Effect of temperature on biosensor response (pH = 7.00, sucrose 1 mM). Each point represents the average of three determinations.
Fig. 4. Biosensor calibration graph (steady-state, temperature 25 ◦ C, pH = 5.00). Table 1 Parameters for linear regression (y = ax + b)a Compound
Method
a
b
R
S.D.
N
Sucrose Sucrose Glucose
Steady-state Kinetic Steady-state
132.2 ± 1.9 80.5 ± 1.1 19.01 ± 0.42
24.08 ± 0.57 15.27 ± 0.34 3.28 ± 0.13
0.9983 0.9985 0.9957
1.8 1.1 0.35
8 8 7
a
R correlation coefficient, S.D. standard deviation, N no. of data points.
220
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
the extended response range for more than 3 decades of concentration between 0.01–30 mM. The detection limit for sucrose was 3.2 M. Glucose, the main interferent in the determination of sucrose, gives a linear response between 0.05–30 mM with a smaller slope than sucrose (see Table 1). This fact confirms the proposed model for the sucrose biosensor. Theoretically, glucose has to present a response that should be about 50% from the sucrose biosensor. Practically, a glucose response is not more than 15% from the sucrose response was observed. This leads to the conclusion that incubation of the yeast cells on a sucrose medium is a good method to improve biosensor selectivity. 3.6. Selective determination of sucrose in the presence of glucose Using a second layer consisting on GOD and CAT, the sucrose can be determined in the presence of glucose. The influence of glucose on the response of a hybrid sucrose sensor is shown in Table 2. The yeast cells do not assimilate gluconic acid, formed by oxidation of glucose in the presence of GOD. Higher concentrations of H2 O2 could have an inhibition effect on the enzyme (GOD) and on the yeast cells.
Table 2 Glucose interference in sucrose determination (0.1 mM) Sucrose
(1 mM) Sucrose Ea
Glucose concentration (mM)
(mV)
Glucose concentration (mM)
Ea (mV)
0 0.05 0.075 0.1
38 38 38 39
0 0.5 0.75 1
59 59 60 61
a
Each value represents the average of five determinations.
To prevent this effect CAT is used, which decomposes the hydrogen peroxide. In the layer containing yeast cells, only sucrose is assimilated and the oxygen consumption is related to the concentration of sucrose. Glucose can be tolerated up to 0.5 mM for a sucrose concentration of 0.5 mM. Interference of glucose in sucrose determination was no more than 3.5% for glucose concentrations >0.5 mM. 3.7. Stability Biosensor stability was tested on a 0.5 mM sucrose solution, pH = 5, 25 ◦ C, for 1 week with five determinations per day (Fig. 5). The detector, a potentiometric
Fig. 5. Biosensor stability (temperature 25 ◦ C, pH = 5.00, sucrose 0.5 mM). Each point represents the average of three determinations.
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222 Table 3 Determination of sucrose in soft drinks Sample
Biosensor: average valuea (mM)
Spectrophotometric method: average valuea (mM)
1 2 3 4 5
4.25 1.9 1.4 1.2 0.7
4.1 2 1.3 1 0.8
a
Five determinations for each sample.
oxygen electrode, affects the stability of the analytical signal. After 4 days, the signal decreases to 70% of its initial value and reaches 40% after 1 week. 3.8. Soft drinks sucrose determination The hybrid biosensor was used to determine sucrose in soft drinks. Different dilutions 50, 100, 150, 200 and 250 times, respectively were performed. The results were compared with those of spectrophotometric method with dinitrosalicylic acid (see Table 3). The correlation coefficient of the experimental data is 0.9951, showing a good correlation between biosensor and spectrophotometric methods. The coefficient of variation was no more than 4.5% for the biosensor and 2.8% for the spectrophotometric method, for determinations carried out on the same day.
4. Conclusions The main advantage of the potentiometric microbial biosensor is the extended response range of more than three concentration decades for sucrose determination. Previously reported microbial biosensor [20] presented a response for only one concentration decade and a high detection limit. Optimum working conditions were the same as for the amperometric sucrose biosensor—pH = 5 and a temperature of 25 ◦ C. The slower response of the potentiometric oxygen electrode affects the biosensor response. The interference of glucose in sucrose determination is decreased, firstly because of the selectivity of the yeast cells, achieved by previous yeast incubation on a sucrose medium and, secondly, because of the enzymatic anti-interference layer which further reduces
221
the interference of glucose. No interference from glucose was observed up to a concentration of 0.5 mM. Even in the absence of the enzymatic anti-interference layer, the selectivity of the microbial biosensors for sucrose is obviously better than the selectivity of any tri-enzyme biosensor. While the potentiometric microbial biosensor gives a glucose response which represents 15% from the sucrose response, for the enzymatic sucrose biosensors, the interference of glucose is 100% from the sucrose signal. The potentiometric biosensor for sucrose presented in this paper has also the advantage over the amperometric biosensor of a very simple construction, and use of a simple mV-meter for potential measurements. Logarithmic dependence between potential and concentration of the potentiometric detector gives nonuniform biosensor sensitivity for all response ranges. Comparable sensitivity and good correlation with the spectrophotometric method with dinitrosalicylic acid was observed. References [1] I. Satoh, I. Karube, S. Suzuki, Biotechnol. Bioeng. 18 (1976) 269. [2] Y. Xu, G.G. Guibault, Anal. Chem. 61 (1989) 782. [3] F. Mitzutani, M. Asai, Anal. Chim. Acta 236 (1990) 245. [4] E. Watanabe, T. Takagi, S. Takei, Biotechnol. Bioeng. 38 (1991) 99. [5] M.A.N. Rahni, G.J. Lubrano, G.G. Guibault, J. Agric. Food Chem. 35 (1987) 1001. [6] M. Filipiak, K. Fludra, E. Gosciminska, Biosens. Bioelectron. 11 (4) (1996) 355. [7] M.A. Krysteva, L.K. Yotova, J. Chem. Biotechnol. 54 (1992) 13. [8] M. Mason, J. Assoc. Off. Anal. Chem. 66 (1983) 981. [9] J.A. Hamid, G.J. Moody, J.D.R. Thomas, Analyst 113 (1998) 81. [10] F. Scheller, R. Renneberg, Anal. Chim. Acta 152 (1983) 265. [11] F. Scheller, Ch. Karsten, Anal. Chim. Acta 155 (1983) 29. [12] M. Cordonnier, F. Lawny, D. Chapot, D. Thomas, FEBS Lett. 59 (1975) 263. [13] C. Bertrand, P.R. Coulet, D.C. Gautheron, Anal. Chim. Acta 126 (1981) 23. [14] H. Gulce, S.S. Celebi, H. Ozyoruk, A. Yidiy, J. Electroanal. Chem. 397 (1995) 217. [15] J.L.L. Filho, P.C. Pandey, H.H. Weetall, Biosens. Bioelectron. 11 (8) (1996) 719. [16] J.K. Park, H.S. Ro, H.S. Kim, Biotechnol. Bioeng. 38 (1991) 217.
222
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
[17] A. Barlikova, J. Svorc, S. Miertus, Anal. Chim. Acta 247 (1991) 83. [18] H. Gulce, H. Ozyoruk, A. Yildiz, Electroanalysis 7 (1995) 178. [19] J.-K. Park, H.-S. Ro, H.-S. Kim, Biotechnol. Bioeng. 38 (3) (1991) 217. [20] L. Rotariu, C. Bala, V. Magearu, Rev. Roum. Chim. 45 (2000) 21.
[21] L. Rotariu, C. Bala, S. Fleschin, V. Magearu, Anal. Univ. Buc. Anul III (serie noua) (1994) 45. [22] G. Zarnea, Gh. Mencinicopschi, St. Bragarea, St., Bioingineria preparatelor enzimatice microbiene, Editura Tehnica, Bucuresti, 1980, p. 241. [23] G. Zarnea, Gh. Mencinicopschi, St. Bragarea, Bioingineria preparatelor enzimatice microbiene, Editura Tehnica, Bucuresti, 1980, p. 100.
Yeast cells sucrose biosensor based on a potentiometric oxygen electrode Lucian Rotariu∗ , Camelia Bala, Vasile Magearu Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, Sos. Panduri, No. 90, 76229-Bucharest 5, Romania Received 20 June 2001; received in revised form 14 September 2001; accepted 13 November 2001
Abstract A new microbial biosensor based on an immobilised microorganisms (Saccharomyces cerevisiae) and a potentiometric oxygen electrode is described. Determination is based on the respiratory activity of the microorganism in presence of different sugars (sucrose and glucose). A response time of ca. 4 min for the steady-state method and 2 min for the initial slope method was obtained. Potentiometric detection has the advantage of an extended calibration range and a low detection limit. The calibration curve for sucrose was linear in the range 1 × 10−5 to 3 × 10−2 M. This biosensor was used for selective monitoring of sucrose in the presence of glucose, using a second anti-interference enzymatic layer with glucose oxidase (GOD) and catalase (CAT). Interference of glucose in the determination of sucrose decreases from 15% for a microbial biosensor to a maximum 3.5% for the hybrid biosensor. The hybrid biosensor was used to determine sucrose in soft drinks. A good correlation between the results for the biosensor and a spectrophotometric method with dinitrosalicylic acid was achieved. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sucrose; Biosensor; Potentiometry; Yeast; Food analysis
1. Introduction Glucides are always present in biological, food products and biotechnological processes. The most important sugars are glucose and sucrose. It is easy to determinate glucose enzymatically without interferences from other glucides. Different enzyme electrodes for sucrose, based on invertase (EC 3.2.1.26), mutarotase (EC 5.1.3.3) and glucose oxidase (GOD) (EC 1.1.3.4), were reported using a oxygen electrode [1–7] or a platinum electrode [8,9]. This procedure is straight-forward for glucose-free media. Direct determination of sucrose in glucosecontaining samples was achieved using a multi-layer ∗ Corresponding author. Tel.: +40-1-315-92-49; fax: +40-1-315-92-49. E-mail address: [email protected] (L. Rotariu).
enzyme electrode [9,10]. An outer anti-interference layer containing GOD and catalase (CAT) that is separated from the indicator layer by a dialysis membrane eliminates the glucose interference. To prepare the enzyme layer, physical [10,11] and chemical [1,12,13] immobilisation procedures have been employed. The -d-glucose and oxygen are converted by GOD to the electro-inactive gluconic acid and the electrode-active hydrogen peroxide; the latter is converted in the catalytic reaction to no-interfering H2 O and O2 . The sucrose reaches the GOD/invertase layer, thus providing H2 O2 , which is measured. Food products were analysed successfully and up to 2 mM glucose could be tolerated [10]. Mediators [14] or modified carbon paste electrodes [15] were also used for sucrose biosensors. A potentiometric enzymatic biosensor [16] was designed using
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 5 2 9 - X
216
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
a three enzyme system and a pH electrode. The main problem for all enzymatic biosensors for sucrose is the multi-enzymatic system, sometimes requiring expensive or unstable enzymes. In order to reduce the cost of a biosensor some authors [17] proposed to replace the isolated and purified invertase with fragments of yeast cell wall, the main source for this enzyme. Bacteria and yeasts are recognised as organisms which metabolise very well all kind of sugars. Incubation with sucrose was used as a good method to improve the selectivity of a microbial biosensor for sucrose [18]. A hybrid biosensor based on Zymomonas mobilis and invertase [19] was successfully used for sucrose determination in fruit juices. A microbial amperometric biosensor for sucrose was previously presented [20] and consists in using yeast cells of S. cerevisiae immobilised on the surface of a Clark oxygen electrode. The selectivity of this microbial sensor was improved by using a second anti-interference enzymatic layer. The yeast cells do not assimilate gluconic acid obtained by enzymatic oxidation of glucose. The main disadvantage of this biosensor is the response range limited to the concentration between 6 × 10−3 to 0.1 M. This paper describes, a potentiometric microbial biosensor for the determination of sucrose. By changing the classical oxygen electrode with a potentiometric oxygen electrode [21] a more sensitive biosensor was obtained. To increase the biosensor selectivity the same enzymatic layer was used. 2. Experimental 2.1. Materials A yeast strain of Saccharomyces cerevisiae, purchased from the University of Galati (Romania), was used as a biocatalyst for the microbial sensor. GOD, CAT, glucose and sucrose were purchased from Fluka Chemie AG; all other reagents were of analytical grade. Standard solutions of glucose and sucrose were prepared in disodium phosphate–citric acid buffer, 0.5 M. 2.2. Preparation of microbial electrode and assay procedure Yeast cells were previously incubated in a medium containing 1.0 g l−1 KH2 PO4 , 3.5 g l−1 (NH4 )2 SO4 ,
0.3 g l−1 MgSO4 , 0.1 g l−1 CaCl2 and 2% sucrose as unique carbon source. Incubation of the cells in a medium based on the substrate for which the biosensor is intended, is a well-known method to improve the selectivity of the selected cells. The S. cerevisiae cells were maintained for 12 h at 30 ◦ C, under continuing oxygenation of the cells suspension. After centrifugation, the solid deposit was suspended in distilled water and centrifuged again. The cell mass obtained was suspended in 0.9% NaCl solution and successive dilutions were made (absorbance between 0.05 and 0.4 at 660 nm). The 0.5 ml of each suspension was filtered through a nitrocellulose membrane. After drying, each membrane was kept at 4 ◦ C before utilisation. The oxygen sensor consists in a platinum electrode as redox indicator electrode and an Ag/AgCl electrode as reference electrode, covered with an oxygen permeable PTFE membrane. The internal electrolyte is based on 0.1 M KCl and 5×10−4 M Mn(OH)2 . Potential measurements were made using a digital mV/pH— meter Cole Parmer model CP 059H3-H5. The immobilised microorganisms were placed on the oxygen membrane, covered with a dialysis membrane and fixed with a rubber ring. A schematic representation of the device is given in Fig. 1. All the determinations were achieved in a 10 ml measuring cell and all the solutions were previously saturated with oxygen. Before every determination, the biosensor was kept in oxygen-saturated phosphate buffer solution. After the output signal of the microbial sensor became stable, the sensor was removed to a buffered standard solution of sucrose (saturated with oxygen). The potential change indicates that sucrose passes through the membrane and is assimilated by the immobilised yeast cells. Oxygen consumption due to respiratory activity of the microorganism caused a decrease in dissolved oxygen concentration around the membrane and consequently brought about the decrease in output signal. The decrease of oxygen concentration was taken as the measure of sucrose concentration. The first step is considered to be the enzymatic hydrolysis of sucrose catalysed by invertase (E.C.3.2.1.26) localised in the cell wall of S. cerevisiae. Then glucose and fructose are assimilated in the presence of oxygen inside the cell. invertase
sucrose + H2 O → glucose + fructose
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
217
Fig. 1. Schematic representation of potentiometric microbial biosensor for sucrose.
2C6 H12 O6 (glucose) + 16O2
S. cerevisiae
→
CO2 + 24H2 O
Decrease of the oxygen concentration around the oxygen electrode is measured and correlated with sucrose concentration from the sample. In principle, there are two possibilities for measuring the biosensor response: (a) endpoint determination (steady-state method); (b) kinetic measurements (initial slope method). For selective determination of sucrose in the presence of glucose a second anti-interference layer containing GOD and CAT was added. Glucose is converted to gluconic acid that is not used by yeast cells as a carbon source in respiration activity.
3. Results and discussion 3.1. Optimisation of the microorganism concentration in the biocatalytic layer The membranes with immobilised yeast were tested on a 1 mM sucrose solution. Response curves were
registered and the difference between the steady-state signal and base signal in oxygen-saturated buffer is shown in Fig. 2. Low immobilised cell concentrations do not modify the baseline response of the biosensor in the presence of sucrose. Increasing the cell concentration on the surface of the membrane leads to a very low base signal for the microbial biosensor and the signal decreases rapidly to zero in the presence of sucrose in the sample solution. In extremes, for a very high concentration of yeast cells, the oxygen electrode could not detect dissolved oxygen around the biocatalytic membrane. Membranes made by depositing 0.5 ml of a cell suspension with absorbance of 0.08 showed the maximum response. These membranes have been used for further experiments. 3.2. Response curve of microbial biosensor When the microbial sensor was immersed in the oxygen saturated buffer solution the output current of the sensor became stable within 5 min. After the
218
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
Fig. 2. Optimisation of yeast cell concentration (temperature 25 ◦ C, pH = 7.00, sucrose 1 mM). Each point represents the average of three determinations.
steady current was obtained, the sensor was transferred to solutions containing different concentrations of sucrose in the range 10−5 to 10−2 M. The output current began to decrease and the minimum current was observed within 4 min. The amperometric sucrose biosensor previously reported [6] has fast response time of about 90 s. This relatively long response is due to the potentiometric oxygen electrode [7] and it is independent of the sucrose concentration. This behaviour shows that oxygen consumption by the yeast cells is faster than the response of the oxygen electrode. Unusually for a microbial biosensor, the response is determined by the detector. Kinetic measurements could be done in the first 2 min when the response curve is linear. 3.3. Effect of temperature The respiration activity of the yeast cells depends on the presence of a carbon source and on the temperature. Fig. 3 shows the effect of temperature upon the response of the microbial sensor. At 20 ◦ C, the response of the sensor was less than at higher temperatures. The optimal temperature for growth of S. cerevisiae was reported to be 30 ◦ C [22], in accord with our results. A temperature of 25 ◦ C was chosen as the
working temperature. A lower baseline for the oxygen electrode, worse reproducibility and limited response range for sucrose was observed at higher temperatures. 3.4. Effect of pH The effect of pH on the response of the microbial biosensor was investigated in the pH range 4–8. The response to a standard solution of 2 mM sucrose was checked and the optimum pH was 5. The optimum pH for invertase activity is 4.6 [23] for the isolated enzyme, and for a S. cerevisiae culture is ca. 5 [22]. 3.5. Calibration curve Fig. 4 shows the calibration graphs for glucose and sucrose using the steady-state method under conditions of pH = 5 and 25 ◦ C. Each point represents the average for five determinations. A linear relationship between the oxygen concentration decrease and concentration of sucrose and glucose was observed, up to 30 mM. Parameters for linear regression (y = ax + b) are presented in Table 1. A slope of 24 mV/decade shows the relatively low sensitivity of the biosensor. The main advantage of the potentiometric sucrose biosensor is
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
219
Fig. 3. Effect of temperature on biosensor response (pH = 7.00, sucrose 1 mM). Each point represents the average of three determinations.
Fig. 4. Biosensor calibration graph (steady-state, temperature 25 ◦ C, pH = 5.00). Table 1 Parameters for linear regression (y = ax + b)a Compound
Method
a
b
R
S.D.
N
Sucrose Sucrose Glucose
Steady-state Kinetic Steady-state
132.2 ± 1.9 80.5 ± 1.1 19.01 ± 0.42
24.08 ± 0.57 15.27 ± 0.34 3.28 ± 0.13
0.9983 0.9985 0.9957
1.8 1.1 0.35
8 8 7
a
R correlation coefficient, S.D. standard deviation, N no. of data points.
220
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
the extended response range for more than 3 decades of concentration between 0.01–30 mM. The detection limit for sucrose was 3.2 M. Glucose, the main interferent in the determination of sucrose, gives a linear response between 0.05–30 mM with a smaller slope than sucrose (see Table 1). This fact confirms the proposed model for the sucrose biosensor. Theoretically, glucose has to present a response that should be about 50% from the sucrose biosensor. Practically, a glucose response is not more than 15% from the sucrose response was observed. This leads to the conclusion that incubation of the yeast cells on a sucrose medium is a good method to improve biosensor selectivity. 3.6. Selective determination of sucrose in the presence of glucose Using a second layer consisting on GOD and CAT, the sucrose can be determined in the presence of glucose. The influence of glucose on the response of a hybrid sucrose sensor is shown in Table 2. The yeast cells do not assimilate gluconic acid, formed by oxidation of glucose in the presence of GOD. Higher concentrations of H2 O2 could have an inhibition effect on the enzyme (GOD) and on the yeast cells.
Table 2 Glucose interference in sucrose determination (0.1 mM) Sucrose
(1 mM) Sucrose Ea
Glucose concentration (mM)
(mV)
Glucose concentration (mM)
Ea (mV)
0 0.05 0.075 0.1
38 38 38 39
0 0.5 0.75 1
59 59 60 61
a
Each value represents the average of five determinations.
To prevent this effect CAT is used, which decomposes the hydrogen peroxide. In the layer containing yeast cells, only sucrose is assimilated and the oxygen consumption is related to the concentration of sucrose. Glucose can be tolerated up to 0.5 mM for a sucrose concentration of 0.5 mM. Interference of glucose in sucrose determination was no more than 3.5% for glucose concentrations >0.5 mM. 3.7. Stability Biosensor stability was tested on a 0.5 mM sucrose solution, pH = 5, 25 ◦ C, for 1 week with five determinations per day (Fig. 5). The detector, a potentiometric
Fig. 5. Biosensor stability (temperature 25 ◦ C, pH = 5.00, sucrose 0.5 mM). Each point represents the average of three determinations.
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222 Table 3 Determination of sucrose in soft drinks Sample
Biosensor: average valuea (mM)
Spectrophotometric method: average valuea (mM)
1 2 3 4 5
4.25 1.9 1.4 1.2 0.7
4.1 2 1.3 1 0.8
a
Five determinations for each sample.
oxygen electrode, affects the stability of the analytical signal. After 4 days, the signal decreases to 70% of its initial value and reaches 40% after 1 week. 3.8. Soft drinks sucrose determination The hybrid biosensor was used to determine sucrose in soft drinks. Different dilutions 50, 100, 150, 200 and 250 times, respectively were performed. The results were compared with those of spectrophotometric method with dinitrosalicylic acid (see Table 3). The correlation coefficient of the experimental data is 0.9951, showing a good correlation between biosensor and spectrophotometric methods. The coefficient of variation was no more than 4.5% for the biosensor and 2.8% for the spectrophotometric method, for determinations carried out on the same day.
4. Conclusions The main advantage of the potentiometric microbial biosensor is the extended response range of more than three concentration decades for sucrose determination. Previously reported microbial biosensor [20] presented a response for only one concentration decade and a high detection limit. Optimum working conditions were the same as for the amperometric sucrose biosensor—pH = 5 and a temperature of 25 ◦ C. The slower response of the potentiometric oxygen electrode affects the biosensor response. The interference of glucose in sucrose determination is decreased, firstly because of the selectivity of the yeast cells, achieved by previous yeast incubation on a sucrose medium and, secondly, because of the enzymatic anti-interference layer which further reduces
221
the interference of glucose. No interference from glucose was observed up to a concentration of 0.5 mM. Even in the absence of the enzymatic anti-interference layer, the selectivity of the microbial biosensors for sucrose is obviously better than the selectivity of any tri-enzyme biosensor. While the potentiometric microbial biosensor gives a glucose response which represents 15% from the sucrose response, for the enzymatic sucrose biosensors, the interference of glucose is 100% from the sucrose signal. The potentiometric biosensor for sucrose presented in this paper has also the advantage over the amperometric biosensor of a very simple construction, and use of a simple mV-meter for potential measurements. Logarithmic dependence between potential and concentration of the potentiometric detector gives nonuniform biosensor sensitivity for all response ranges. Comparable sensitivity and good correlation with the spectrophotometric method with dinitrosalicylic acid was observed. References [1] I. Satoh, I. Karube, S. Suzuki, Biotechnol. Bioeng. 18 (1976) 269. [2] Y. Xu, G.G. Guibault, Anal. Chem. 61 (1989) 782. [3] F. Mitzutani, M. Asai, Anal. Chim. Acta 236 (1990) 245. [4] E. Watanabe, T. Takagi, S. Takei, Biotechnol. Bioeng. 38 (1991) 99. [5] M.A.N. Rahni, G.J. Lubrano, G.G. Guibault, J. Agric. Food Chem. 35 (1987) 1001. [6] M. Filipiak, K. Fludra, E. Gosciminska, Biosens. Bioelectron. 11 (4) (1996) 355. [7] M.A. Krysteva, L.K. Yotova, J. Chem. Biotechnol. 54 (1992) 13. [8] M. Mason, J. Assoc. Off. Anal. Chem. 66 (1983) 981. [9] J.A. Hamid, G.J. Moody, J.D.R. Thomas, Analyst 113 (1998) 81. [10] F. Scheller, R. Renneberg, Anal. Chim. Acta 152 (1983) 265. [11] F. Scheller, Ch. Karsten, Anal. Chim. Acta 155 (1983) 29. [12] M. Cordonnier, F. Lawny, D. Chapot, D. Thomas, FEBS Lett. 59 (1975) 263. [13] C. Bertrand, P.R. Coulet, D.C. Gautheron, Anal. Chim. Acta 126 (1981) 23. [14] H. Gulce, S.S. Celebi, H. Ozyoruk, A. Yidiy, J. Electroanal. Chem. 397 (1995) 217. [15] J.L.L. Filho, P.C. Pandey, H.H. Weetall, Biosens. Bioelectron. 11 (8) (1996) 719. [16] J.K. Park, H.S. Ro, H.S. Kim, Biotechnol. Bioeng. 38 (1991) 217.
222
L. Rotariu et al. / Analytica Chimica Acta 458 (2002) 215–222
[17] A. Barlikova, J. Svorc, S. Miertus, Anal. Chim. Acta 247 (1991) 83. [18] H. Gulce, H. Ozyoruk, A. Yildiz, Electroanalysis 7 (1995) 178. [19] J.-K. Park, H.-S. Ro, H.-S. Kim, Biotechnol. Bioeng. 38 (3) (1991) 217. [20] L. Rotariu, C. Bala, V. Magearu, Rev. Roum. Chim. 45 (2000) 21.
[21] L. Rotariu, C. Bala, S. Fleschin, V. Magearu, Anal. Univ. Buc. Anul III (serie noua) (1994) 45. [22] G. Zarnea, Gh. Mencinicopschi, St. Bragarea, St., Bioingineria preparatelor enzimatice microbiene, Editura Tehnica, Bucuresti, 1980, p. 241. [23] G. Zarnea, Gh. Mencinicopschi, St. Bragarea, Bioingineria preparatelor enzimatice microbiene, Editura Tehnica, Bucuresti, 1980, p. 100.