- Email: [email protected]
Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation
Biosensors and Bioelectronics 18 (2003) 1125 /1134 www.elsevier.com/locate/bios
Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation Jan Tkac a,*,1, Igor Vostiar b, Lo Gorton c, Peter Gemeiner a, Ernest Sturdik b a
b
Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, SK-842 38 Bratislava, Slovak Republic Department of Biochemical Technology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, SK-812 37 Bratislava, Slovak Republic c Department of Analytical Chemistry, Lund University, PO Box 124, SE-22 100 Lund, Sweden Received 24 June 2002; received in revised form 3 October 2002; accepted 24 October 2002
Abstract A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 mM (S/N /3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l1. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cellulose acetate; Ferricyanide; Enhanced selectivity; Ethanol detection; Microbial biosensor; Gluconobacter oxydans
1. Introduction Microbial cells are very promising for biosensor construction because of several advantages: the enzyme does not need to be isolated, enzymes are usually more stable in their natural environment in the cell, coenzymes and activators are already present in the system (D’Souza, 2001; Racek, 1994). Cell-based biosensors are frequently used for determination of BOD, toxic agents and assimilable sugars (Riedel, 1998). Microbial biosensors can be also used for selective detection of a single analyte, but the low selectivity has to be overcome. To enhance the selectivity of microbial biosensors several approaches were used. The possible methods for
* Corresponding author. Present address: Division of Biotechnology, Department of Biochemical Technology, IFM, Linko¨pings Universitet, SE-581 83, Linko¨ping, Sweden. E-mail address: [email protected] (J. Tkac). 1 Tel.: / 421-2-5941-0318; fax: / 421-2-5941-0222; e-mail: [email protected].
enhancement of the selectivity of microbial biosensors were well reviewed (Riedel, 1991; Racek, 1994), including induction of transport/metabolic systems, inhibition of undesired transport/metabolic pathways, the coupling of enzymes with immobilized cells, the transfer of desired plasmid-controlled pathways, the choice of a proper transducer and even pH for measurement. Permeabilization of cells using digitonin, which releases cofactors from the cells, (Gonchar et al., 1998) and application of a gas-permeable Teflon membrane (Hikuma et al., 1979) effectively increased selectivity of microbial biosensors. Another possibility is the use of genetically engineered cells lacking specific receptors (Aravanis et al., 2001), fusing of reporter gene for green fluorescent protein with genes induced by the analyte (Shrestha et al., 2001), by overexpression of natural enzymes in the cells (Svorc et al., 1990) or by improving of analyte affinity (Sode, 2000). Other approaches used to enhance the selectivity of whole-cell systems using genetic engineering are well reviewed elsewhere (Daunert et al., 2000). Different chemometric tools were
0956-5663/03/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 2 4 4 - 0
1126
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
recently successfully used to enhance the selectivity of microbial biosensor assays including application of the artificial neural networks (Lobanov et al., 2001) and multivariate calibration techniques (Reshetilov et al., 1998; Plegge et al., 2000). The genera Gluconobacter have been frequently used for preparation of microbial biosensors (Kitagawa et al., 1987b; Lobanov et al., 2001; Reshetilov et al., 2001; Svitel et al., 1998; Tkac et al., 2000a,b) because of high efficiency and speed of substrate oxidation due to incomplete oxidation of substrates (Gupta et al., 2001; Matsushita et al., 2002). Moreover, pyrroloquinoline quinone (PQQ) containing enzymes of this genera involved in substrate oxidation are bound to the periplasmic membrane, providing fast response of microbial biosensors. Several enzymes were purified from Gluconobacter and one of them, fructose dehydrogenase, is commercially available, but low stability and activity hamper wider use of these enzymes for biosensors preparation. Thus, it is better to use whole cells with improved selectivity for preparation of biosensors. The cellulose acetate (CA) membrane is often used in order to enhance the selectivity of detection of hydrogen peroxide (Wilson and The´venot, 1990) and now the CA membrane is widely used for covering of electrodes, microelectrodes and modified screen-printed electrodes. To our knowledge the CA membrane has not been used to enhance the selectivity of any microbial biosensor. A simple and generally applicable concept for enhancement of the selectivity of microbial biosensors is here proposed. For this purpose a size exclusion membrane cast from CA solution on the electrode modified with Gluconobacter oxydans was successfully used to discriminate between ethanol and glucose providing fast and sensitive ethanol detection. This approach is cheap when compared to the use of the expensive, unstable and methanol oxidizing alcohol oxidase; or alcohol dehydrogenase with unfavorable reaction equilibrium (Vijayakumar et al., 1996), which depends on the soluble cofactor (NAD). Moreover, oxidation of ethanol by PQQ-dependent alcohol dehydrogenase is irreversible and the enzyme is unable to oxidize methanol (Jongejan et al., 2000).
biomass production of G. oxydans . These carbon sources were purchased either from Merck (Darmstadt, Germany) or from Sigma (St. Louis, USA). All other reagents were of analytical grade and were supplied by Lachema (Brno, Czech Republic). McIlvaine buffers were prepared from a 0.1 M solution of citric acid and a 0.2 M solution of Na2HPO4 with addition of 0.1 M KCl. Phosphate buffers were prepared from 0.1 M solutions of KH2PO4 and Na2HPO4 containing 0.1 M of KCl. 2.2. Cell cultivation of Gluconobacter oxydans The strain of G. oxydans CCM 1783 (/ATCC 621) was maintained on the agar containing (g l 1): Dglucose, 100; yeast extract (Oxoid Ltd., Basingstoke, UK), 10; calcium carbonate, 20; agar (Oxoid Ltd., Basingstoke, UK), 20; and transferred monthly (Tkac et al., 2000a). The cell biomass was prepared by aerobic cultivation at 28 8C on a rotary shaker in 500 ml flasks filled with 100 ml of media. The growth medium contained (g l 1): carbon source, 5; yeast extract, 5. The culture, inoculated from the slant agar, was incubated until reaching the late exponential phase (preferentially 12 h), when the cells contained the most active PQQ-dependent alcohol dehydrogenase in the highest yield (Shinagawa et al., 1989). Then the cells from one cultivation flask were collected by centrifugation (10 min, 3500 /g ), resuspended in 2 ml of cold and sterile 0.9% NaCl solution containing 2 mM CaCl2 and this procedure was repeated three times to assure a cell suspension free from fermentation broth. The biomass concentration was expressed as the dry weight matter of cells determined by drying to a constant weight at 105 8C. 2.3. Apparatus
2. Material and methods
Biosensor measurements were carried out on an Amperometric Detector ADLC2 (Laboratorni pristroje, Prague, Czech Republic) using a glassy carbon electrode as the working (diameter 6 mm) and a saturated calomel electrode (SCE) as the reference electrode. Cyclic voltammetry experiments were performed using the GCE as the working electrode, a platinum wire as the auxiliary electrode and a SCE as the reference. In both cases data were collected using a PC.
2.1. Reagents
2.4. Biosensor preparation
CA (approx. 40% of acetyl groups). Dialysis membrane (cut off 12 000), sodium alginate from Macrocystis perifea were purchased from Sigma (St. Louis, USA). As sugar source D-glucose, glycerol, D-mannitol, Dsorbitol, galactitol (dulcitol), D-ribose, D-gluconate, Larabinitol, L-sorbose and D-fructose were used for
On the surface of the GCE, 10 ml of a 1 gDW l 1 of G. oxydans suspension in McIlvaine buffer pH 6.0 containing 0.1 M KCl and 2 mM CaCl2 (if not stated otherwise) were spread together with 10 ml of a 1 mM ferricyanide solution in distilled water and then the water was allowed to evaporate at 25 8C using a fan (15 min).
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Then 25 ml of a 1% CA solution (if not stated otherwise) was spread and the solvents were allowed to evaporate (40 min, 25 8C). The CA solution was prepared by dissolving a desired amount of CA powder, preferentially 1 g, in a mixture of 55 ml of acetone and 45 ml of cyclohexanone (Tkac et al., 2001). The resulting thickness of the CA membrane on the surface of cell modified glassy carbon electrode was estimated to be about 12 mm (Yamamoto et al., 2000). Before measurement, the sensor was equilibrated for 30 min in McIlvaine buffer pH 6.0 containing 0.1 M KCl, 2 mM CaCl2 and 10 mM ferricyanide at 289/0.2 8C. All experiments were done in a 20 ml thermostated vessel at 289/0.2 8C containing 10 ml of measurement buffer using magnetic stirring (300 rpm) at a working potential of /300 mV, if not mentioned otherwise. 2.5. Fermentation experiments The distillery strain of Saccharomyces cerevisiae GY2 (from the Collection of Microorganisms of the Department of Biochemical Technology, Faculty of Chemical Technology, Bratislava, Slovak Republic) was propagated in 100 ml of liquid medium containing glucose, 30 g l1; yeast extract, 5 g l 1 and peptone (Difco, Detroit, USA), 10 g l 1 on a rotary shaker at 28 8C. The cells were collected by centrifugation (10 min, 3500 /g) after reaching the late exponential phase. The suspension containing the biomass from five cultivation flasks in 300 ml of sterile NaCl (9 g l 1) solution was used to prepare a 2% sodium alginate solution. This solution was dropped with an injection needle into 2% CaCl2. The diameter of the gel beads was about 1.5 mm. The beads were placed in 700 ml of liquid medium containing glucose, 100 g l1; yeast extract, 5 g l 1; and peptone 10 g l 1. After 12 h of aerobic cultivation at 309/0.5 8C, the beads were transferred into 700 ml of fresh medium containing glucose, 200 g l 1; yeast extract, 5 g l1; peptone, 10 g l 1; (NH4)2SO4, 10 g l 1; KH2PO4, 4 g l 1; MgSO4 ×/ 7H2O, 10 g l 1; and CaCl2, 0.2 g l 1. Batch ethanol fermentation was carried out at 309/0.5 8C using S. cerevisiae cells immobilized in the alginate beads under anaerobic conditions. The ethanol concentration was monitored during 10 h by the biosensor and by a reference analytical method (HPLC). 2.6. HPLC measurements Ethanol was determined by HPLC (Waters, Milford, USA) using a column (TESSEK Steel 8 /250 mm) packed with Ostion LGKS 0800 ion exchange particles in H form (TESSEK, Prague, Czech Republic) with differential refractometric detection at 25 8C (Waters R403, Milford, USA). The column was used at 50 8C with a flow rate of 0.5 ml min1 of 1.25 mM H2SO4 (in
1127
deionised water) used as the mobile phase. For detection a sample volume of 5 ml was injected.
3. Results and discussion 3.1. Influence of the physiological state of G. oxydans on the substrate oxidation 3.1.1. Effect of carbon source It was found that the carbon source used in the cultivation medium for biomass production influenced very strongly the sensitivity of the G. oxydans sensor toward glucose and ethanol. Among the ten different carbon sources used for cultivation (see Section 2.1), only two had a positive effect on the ethanol/glucose (EtOH/Glc) sensitivity ratio. Although the EtOH/Glc sensitivity ratio for cells cultivated on sorbitol was similar to that of glycerol, for further work a cultivation medium containing glycerol as carbon source was used because of the shorter time needed for biomass production of G. oxydans . 3.1.2. Effect of cultivation time For evaluation of the influence of the physiological state on the glucose and ethanol oxidation rates, a biosensor with G. oxydans cells immobilized on the GCE behind a dialysis membrane was used. It was found that with an increase of the cultivation time from 10 to 16 h, the EtOH/Glc sensitivity ratio measured by the biosensor decreased from 14.5 to 5.0 and the ethanol/glycerol (EtOH/Gly) sensitivity ratio also decreased from 326.7 to 44.0 (Fig. 1). On the other hand, the linear range of the biosensor was wider, when the cells were collected after 12 h of cultivation. Thus, a cultivation time of 12 h for bacterial biomass production was used for further work. 3.2. Influence of physico/chemical parameters on the biosensor response 3.2.1. Effect of pH and buffer When comparing the effect of pH on the response of the whole cell biosensor with G. oxydans immobilized behind a dialysis membrane for glucose and ethanol it was found that the response for glucose was highly sensitive having a sharp pH optimum at pH 5.5, whereas, that for ethanol was not affected significantly by pH (5.0 /7.0) with an optimum at 6.0. This should reflect the difference in pH optima between aldose and alcohol dehydrogenase, the enzymes responsible for glucose and ethanol oxidation inside intact cells of G. oxydans . When a CA membrane was used for enhancement of the biosensor selectivity, the sensor sensitivity toward ethanol was not affected by pH in the studied range (5.0 /7.0).
1128
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
its inactive apo-form, while phosphate buffer caused hardly any apoenzyme formation (Shinagawa et al., 1989). The maximum holoenzyme occurred at pH 6.0 in the presence of Ca2 (Shinagawa et al., 1989). Thus, for further work a McIlvaine buffer was chosen containing 2 mM CaCl2 and with the pH set to 6.0, resulting in high operational stability (see Section 3.4.4). 3.2.2. Effect of the working potential The effect of the working potential on the sensor performance was studied in the range between 0 and 340 mV. The biosensor sensitivity increased with increasing the working potential up to 220 and then slightly decreased (Fig. 2). At a working potential of 250 mV (cyclic voltammetry study), an anodic current raised after ethanol addition, indicating efficient ethanol oxidation by the biosensor (Fig. 3). For further work the working potential was set to/300 mV. 3.3. Influence of other parameters on the biosensor performance
Fig. 1. The effect of cultivation time on the oxidation of ethanol, glucose and glycerol by G. oxydans cells. First, the sensitivity on additions of ethanol, glucose or glycerol was expressed in nA mM 1 and then the sensitivity to EtOH was expressed as a ratio to the glucose sensitivity (EtOH/Glc) or to glycerol sensitivity (EtOH/Gly) of the biosensor.
It is well known that Ca2 ions are essential for proper function of PQQ-dependent alcohol dehydrogenases (Jongejan et al., 2000). Since phosphate buffer may chelate Ca2 from the active site of the enzyme causing an inactivation, a sodium acetate buffer has been used for the preparation of the ethanol biosensor based on the PQQ-dependent dehydrogenase isolated from G. oxydans (Ramanavicius et al., 1999). On the other hand, acetate is a product of ethanol oxidation by the cells (oxidation by PQQ-dependent alcohol and aldehyde dehydrogenases); therefore, an inhibition of ethanol oxidation by G. oxydans in the presence of sodium acetate buffer is expected. Moreover, incubation of ADH with acetate buffer converted 90% of ADH into
3.3.1. Effect of the amount of CA For investigation of the effect of the amount of CA on the biosensor performance, four membranes were tested prepared by casting 25 ml of CA solution (0.5, 1, 2 and 4% of CA) on the modified GCE electrode. With increasing the thickness of the CA membrane, the sensitivity of the sensor for ethanol decreased rapidly from 5660 to 22 nA mM 1 and the response time increased linearly from 13 to 51 s (Table 1). For further work a CA membrane prepared from 1% solution was used, because spreading of 0.5% solution of CA produced an unstable sensor with high background noise. During 8.5 h of measurement the response time of the sensor on standard ethanol addition was practically the same, indicating stable diffusional parameters of the membrane in course of measurements (see Section 3.4.4). 3.3.2. Effect of the amount of biomass The biosensor sensitivity increased with increasing the amount of G. oxydans cells on the electrode in the range tested from 1 to 30 mgDW of G. oxydans per electrode. The response time decreased from 40 to 10 s, when the cell amount increased from 1 to 3 mgDW of G. oxydans per electrode. This is probably caused by kinetic limitations of ethanol oxidation, when 1 mgDW of G. oxydans was applied on the GCE resulting in a longer response time. The response time was the same for the biosensor containing either 3 or 10 mgDW of G. oxydans per electrode and then increased (Table 2). This is probably caused by a diffusion controlled process, because of higher cell loading. Spreading of 10 mgDW of G. oxydans per electrode was used for further work as
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
1129
Fig. 2. The influence of the working potential on the sensor performance (A). A typical sensor response for successive addition of 10 mM ethanol measured at a working potential of /300 mV at 289/0.2 8C (B). A glassy carbon electrode covered with 10 mgDW of G. oxydans and 10 nmol of ferricyanide. The cell-modified electrode was covered with 25 ml of 1% CA solution. Measurements were performed in stirred (250 rpm) McIlvaine buffer pH 6.0 containing 0.1 M KCl and 10 mM ferricyanide at 289/0.2 8C.
Table 2 The effect of the amount of G. oxydans cells spread on the GCE on the sensitivity and response time of the ethanol sensor (10 nmol of ferricyanide and 20 ml of 1% CA solution on the GCE) Amount of G. oxydans (mg)
Sensitivity (nA mM 1)
Response time (s)
1 3 10 30
120 520 1140 2100
40 10 10 25
a compromise between the sensitivity and the response time.
Fig. 3. Cyclic voltammetry of the whole cell ethanol biosensor before and after ethanol addition (1 mM). Sweep rate 50 mV s 1, for other conditions used see Fig. 2.
Table 1 The effect of the amount of CA spread on the GCE on the sensitivity and response time of the ethanol sensor (10 nmol of ferricyanide and 10 mgDW of G. oxydans on the GCE) Concentration of CA (%)
Sensitivity (nA mM 1)
Response time (s)
0.5 1 2 4
5660 1140 350 22
13 16 31 51
3.3.3. Effect of the amount of ferricyanide It was observed that even ferricyanide penetrates slightly through the CA membrane. Thus, a decrease of the sensor sensitivity to 53% of the initial value within 1 h occurred. When 1 ml of 100 mM ferricyanide solution was added to the measurement solution, restoration of the initial sensitivity was observed and the signal was stable. Hence, the measurement buffer used further contained 10 mM ferricyanide. 3.4. Characterization of the ethanol biosensor 3.4.1. Discrimination of a cellulose acetate membrane between ethanol and glucose It was confirmed that even a CA membrane spread on a G. oxydans layer hampers the diffusion of glucose
1130
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Fig. 4. The microbial biosensor response for ethanol (10 mM) addition or mixture of ethanol (10 mM) and glucose (100 mM) addition using a dialysis and a CA membrane (A). The response time of the biosensor for glucose or ethanol additions using a dialysis or a CA membrane coverage (B). For other conditions used see Fig. 2.
through the membrane, whereas, ethanol diffusion was possible. The response time for ethanol addition was the same using a dialysis membrane or a CA membrane (Fig. 4). The response time for glucose of the sensor covered by a dialysis membrane was 26 s, twice longer than that for ethanol detection. The response time of 430 s for glucose, when the CA membrane was applied on the modified GCE, indicating limited diffusion of glucose (Fig. 4). In order to prove a discrimination effect of the CA membrane between ethanol and glucose, standard solutions of ethanol (10 mM) or a mixture of glucose (100 mM) and ethanol (10 mM) were analyzed by biosensors with surface coverage either by a dialysis membrane or by the size-exclusion CA membrane. Detection of ethanol by the CA modified biosensor was not influenced by the presence of glucose, which is in contrast to the biosensor covered by a dialysis membrane (Fig. 4). The EtOH/Glc sensitivity ratio increased from 9.3 (a dialysis membrane application) to 541.1, when a CA was used. This membrane also prevents diffusion of glycerol, as the EtOH/Gly sensitivity ratio increased from 183.4 to 1366.6, under the same conditions. 3.4.2. Selectivity of the sensor toward alcohols Alcohol oxidase or alcohol dehydrogenases are able to oxidize several alcohols including ethanol. Moreover, alcohol oxidase is also able to oxidize methanol even more effectively than ethanol. The higher methanol response is attributed to the reaction product, formaldehyde, which is a substrate for alcohol oxidase, whereas, aldehydes formed from higher alcohols are not (Vijayakumar et al., 1996). The effect of several alcohols on the sensor sensitivity and response time was carefully investigated and the sensitivity of our ethanol
biosensor toward different alcohols is compared with that of previously reported biosensors based on alcohol oxidase and alcohol dehydrogenases (Table 3). The CA membrane is not an important diffusion barrier for ethanol because the same response time was achieved using a CA and a dialysis membrane (Table 3). The CA membrane, due to the hydrophobic nature is more permeable for higher alcohols than for ethanol. The response time for the CA modified electrode is shorter for propanol (7 s) and also shorter for butanol (11 s) than that for ethanol (13 s). When a dialysis membrane for G. oxydans retention was used, the response time for ethanol, propanol and butanol were practically the same (around 13 /15 s). Isopropanol does not diffuse well through the CA membrane, when compared with the dialysis membrane, with doubled response time (62 s respectively 30 s). This behavior can be explained by restricted diffusion of isopropanol through the CA membrane. Alcohol oxidases from various sources oxidize methanol, but their ability to oxidize higher alcohols decreases with increasing molecular weight. Moreover, these enzymes slightly oxidize branched alcohols. In the case AOD from Candida boindii was covalently immobilized on graphite particles mixed into carbon paste, isopropanol was oxidized with the same efficiency as propanol (Johansson et al., 1993). PQQ-dependent alcohol dehydrogenase (Acetobacter aceti or G. oxydans) oxidizes propanol and butanol almost as effectively as ethanol, but not methanol and only slightly branched alcohols. The affinity of PQQ-dependent alcohol dehydrogenase purified from Comamonas testosteroni for aliphatic and secondary alcohols increases with the chain length of the substrate. NAD-dependent alcohol dehydrogenase does not catalyze the oxidation of methanol, but higher and
C. boindii (Patel et al., 2001; Vijayakumar et al., 1996); P. pastoris (Lubrano et al., 1991; Patel et al., 2001; Vijayakumar et al., 1996); H. polymorpha (Karyakin et al., 1996; Patel et al., 2001); NADdependent ADH (Chi and Dong, 1994; Lobo Castanon et al., 1997; Sprules et al., 1996); PQQ-dependent ADH (acetic acid bacteria-G. oxydans or A. aceti ) (Kitagawa et al., 1987a,b, 1989); PQQdependent ADH (C. testosteroni ) (Somers et al., 1998; Stigter et al., 1996, 1997); RT, response time (90% of steady state response); Sensit., sensitivity expressed in nA mM 1, then the sensitivity to addition of ethanol was set to 100%; an/5; b; n /25; ca response time in the case of direct electron transfer of PQQ-dependent ADH (Ikeda et al., 1993); / not determined or not known.
/ / 20 /60 0.8 /2.2 / 0.06 / 0.005 /0.006 / 22 / / / 0 /1 / 15 /45 100 3c, 60 /300 / 39 /90 / / 41 /76 / / 16 /78 / / 0 /35 / Methanol Ethanol Propanol Butanol 2-propanol 2-butanol
0a 0a 13.09/0.8b 100.09/1.7b 7.39/0.6a 151.79/3.4a 11.29/0.6a 85.69/1.6a a 0.49/0.2a 62.39/3.1 / /
0a B/0.1a / 13.29/0.5b 100.09/1.5b 26 15.39/0.6a 108.19/1.7a / 13.19/0.7a 99.19/1.3a / a 30.49/1.9 5.09/0.5a / / / /
113 /128 / 117 /160 / 100 10 /19 100 12 4 /19 / 5 /29 / 3 /16 / 4 /16 / 0 /2 / 0 /8 / 0 /2 / 0 /2 /
120 /690 100 5 /18 0 /4 0 /1 0 /1
0 100 69 /130 73 /160 0 0
Kapp m [mM] RT [s] Sensit. [%] Sensit. RT [%] [s] RT [s] Sensit. [%] RT [s] RT [s] RT [s]
Sensit. [%]
RT [s]
Sensit. [%]
RT Sensit. [s] [%]
Sensit. [%]
PQQ (acetic acid bacteria) NAD Pichia pastoris CA Membrane
Dialysis membrane
C. boindii
Hansenula polymorpha
Alcohol dehydrogenase Alcohol oxidase This work
Table 3 Substrate specificity and response time of ethanol biosensors based on alcohol oxidase; NAD- and PQQ-dependent alcohol dehydrogenase; and G. oxydans cells
PQQ (C. testosteroni )
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
1131
branched alcohols are oxidized (Table 3). From this point of view, PQQ-dependent alcohol dehydrogenase isolated from acetic bacteria or kept within intact G. oxydans cells can be considered as the most selective. 3.4.3. Basic biosensor parameters When applying all the optimal conditions, the basic characteristics of the ethanol biosensor were evaluated. The sensitivity of the sensor was very high (3.5 mA mM 1) resulting in a low detection limit (0.85 mM, S/ N /3). The linear response range was wide (2 /270 mM) and the response fast (13.0 s, n /30, 90% of steady state, Fig. 2B). A comparison of response time of presented ethanol biosensor with those already published is shown in Table 3. The relative standard deviation for measurement of 10 mM ethanol solution was within 1.79% (n/ 30). Moreover, the biosensor is very cheap; it is possible to construct approximately 2000 sensors from one cultivation flask of G. oxydans suspension. Immobilizing G. oxydans cells on mass-produced electrodes prepared by ink- or screen-printing techniques, the price of one sensor will be negligible. 3.4.4. Operational stability During examination of the operational stability of the ethanol biosensor it was found that the sensor sensitivity decreased to 75% of its initial sensitivity within 4.5 h, when a cell suspension was prepared in phosphate buffer not containing Ca2. When the cells were resuspended in McIlvaine buffer containing 2 mM CaCl2, the sensitivity remained unchanged for 8.5 h of continuous use (100.49/1.8%, initial sensitivity /2.9 mA mM 1, n /16, Fig. 5). The operational stability of the biosensor during measurement of real samples was very good indicating effective anti-fouling effect of the membrane to compounds present in yeast extract and peptone, which are part of the fermentation medium. 3.4.5. Storage stability The sensor sensitivity examined during biosensor storage at 4 8C in McIlvaine buffer containing ferricyanide decreased to 7% of its initial value within 6 days. The low stability can be ascribed either to the uncoupling of the respiratory chain by ferricyanide or by cell starvation during storage. If no artificial redox compound was used for G. oxydans sensor preparation, the storage stability of G. oxydans sensor was good (50% of initial sensitivity after 8 days storage at 20 8C) (Reshetilov et al., 2001). 3.5. Analysis of real samples Ethanol fermentation has been realized under anaerobic conditions with S. cerevisiae cells immobilized in alginate gel prepared by ionotropic gelation in 2% calcium chloride. Ionotropic gelation allows immobili-
1132
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Fig. 5. Operational stability of the whole cell ethanol biosensor measured in k 0.1 M phosphate buffer pH 6.0 containing 0.1 M KCl and 10 mM of ferricyanide; in I McIlvaine buffer pH 6.0 containing 0.1 M KCl, 2 mM CaCl2 and 10 mM ferricyanide m in McIlvaine buffer pH 6.0 containing 0.1 M KCl, 2 mM CaCl2 and 10 mM ferricyanide during measurement of real samples. For other conditions used see Fig. 2.
zation of cells under mild condition with high cell loading. This method of immobilization preserves a high viability of the immobilized cells (Gemeiner et al., 1996). Samples taken from the S. cerevisiae cultivation were analyzed by the biosensor in the order; standardsample-standard. The sensitivity of the sensor on standard ethanol addition before and after sample measurement was averaged and applied for the determination of ethanol in the sample. Each sample was analyzed by the biosensor at least three times and the R.S.D. varied in the range 0.50 /4.19% with an average R.S.D. of 2.63%. During the measurement of real samples the sensitivity for the ethanol standard remained stable for 8.5 h of continuous operation, thus, revealing an excellent operational stability. During this time 150 samples or ethanol standards were analyzed, including the time needed for washing and equilibration of the sensor signal. The microbial biosensor with enhanced selectivity was successfully applied in an offline monitoring of S. cerevisiae batch fermentation, even in the presence of 200 g l 1 glucose in the initial stage. Only a mild influence of the highly excessive amount of glucose on ethanol detection with the microbial biosensor was observed (Fig. 6). The results obtained with the biosensor were in very good agreement with the HPLC measurements (Fig. 6), cHPLC /0.996*cBiosensor/0.143 (R2 /0.998, n /20).
4. Conclusion The presented microbial biosensor represents sensitive and selective alternative for ethanol detection even in the presence of high excess of glucose. The biosensor has a low detection limit combined with a fast response time. The high operational stability of the sensor makes it very promising for on-line monitoring, but the storage stability should be improved. The selectivity of the ethanol biosensor toward different alcohols was better compared to published ethanol biosensors based on alcohol oxidase, PQQ- and NAD-dependent alcohol dehydrogenases. Reliability of biosensor assay was confirmed by comparison with reference analytical method, performed on real samples. Moreover, the application of a permselective CA membrane might serve as a model for developing other redox sensor systems either with immobilized microbial cells or enzymes.
Acknowledgements The authors thank L. Kristofikova for the HPLC measurements. This work was supported by following the VEGA grants: 1/6252/99, 1/7347/20 and 2/1047/21 and by the Swedish Research Council (VR).
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Fig. 6. Correlation between biosensor and HPLC assays of ethanol during batch fermentation with immobilized S. cerevisiae cells. Bars at each point represent R.S.D. of sample measurement by both methods of analysis (A). Residuals between ethanol concentrations obtained by both methods of analysis (B).
References Aravanis, A.M., DeBusschere, B.D., Chruscinski, A.J., Gilchrist, K.H., Kobilka, B.K., Kovacs, G.T.A., 2001. A genetically engineered cell-based biosensor for functional classification of agents. Biosens. Bioelectron. 16, 571 /577. Chi, Q., Dong, S., 1994. Electrocatalytic oxidation of reduced nicotinamide coenzymes at Methylene Green-modified electrodes and fabrication of amperometric alcohol biosensors. Anal. Chim. Acta 285, 125 /133. Daunert, S., Barrett, G., Feliciano, J.S., Shetty, R.S., Shrestha, S., Smith-Spencer, W., 2000. Genetically engineered whole-cell sensing systems: Coupling biological recognition with reporter genes. Chem. Rev. 100, 2705 /2738. D’Souza, S.F., 2001. Microbial biosensors. Biosens. Bioelectron. 16, 337 /353. Gemeiner, P., Rexova-Benkova, L., Svec, F., Norrlow, O., 1996. Immobilized Biosystems. Theory and Practical Applications. Blackie Academic & Professional with Chapman & Hall, Glasgow. Gonchar, M.V., Maidan, M.M., Moroz, O.M., Woodward, J.R., Sibirny, A.A., 1998. Microbial O2 and H2O2-electrode sensors for alcohol assays based on the use of permeabilized mutant yeast cells as the sensitive bioelements. Biosens. Bioelectron. 13, 945 / 952.
1133
Gupta, A., Singh, V.K., Qazi, G.N., Kumar, A., 2001. Gluconobacter oxydans : its biotechnological applications. J. Mol. Microb. Biotechnol. 3, 445 /456. Hikuma, M., Kubo, T., Yasuda, T., Karube, I., Suzuki, S., 1979. Microbial electrode sensor for alcohols. Biotechnol. Bioeng. 21, 1845 /1853. Johansson, K., Jo¨nsson-Pettersson, G., Gorton, L., Marko-Varga, G., Cso¨regi, E., 1993. A reagentless amperometric biosensor for alcohol detection in column liquid-chromatography based on coimmobilized peroxidase and alcohol oxidase in carbon-paste. J. Biotechnol. 31, 301 /316. Jongejan, A., Machado, S.S., Jongejan, J.A., 2000. The enantioselectivity of quinohaemoprotein alcohol dehydrogenases: mechanistic and structural aspects. J. Mol. Catal. B: Enzyme 8, 121 /163. Karyakin, A.A., Karyakina, E.E., Gorton, L., 1996. Prussian-Bluebased amperometric biosensors in flow-injection analysis. Talanta 43, 1597 /1606. Kitagawa, Y., Ameyama, M., Nakashima, K., Tamiya, E., Karube, I., 1987a. Amperometric alcohol sensor based on an immobilized bacteria cell-membrane. Analyst 112, 1747 /1749. Kitagawa, Y., Tamiya, E., Karube, I., 1987b. Microbial-FET alcohol sensor. Anal. Lett. 20, 81 /96. Kitagawa, Y., Kitabatake, K., Kubo, I., Tamiya, E., Karube, I., 1989. Alcohol sensor based on membrane-bound alcohol dehydrogenase. Anal. Chim. Acta 218, 61 /68. Lobanov, A.V., Borisov, I.A., Gordon, S.H., Greene, R.V., Leathers, T.D., Reshetilov, A.N., 2001. Analysis of ethanol glucose mixtures by two microbial sensors: Application of chemometrics and artificial neural networks for data processing. Biosens. Bioelectron. 16, 1001 /1007. Lobo Castanon, M.J., Miranda Ordieres, A.J., Tunon Blanco, P., 1997. Amperometric detection of ethanol with poly-(o-phenylenediamine)-modified enzyme electrodes. Biosens. Bioelectron. 12, 511 /520. Lubrano, G.J., Faridnia, M.H., Palleschi, G., Guilbault, G.G., 1991. Amperometric alcohol electrode with extended linearity and reduced interferences. Anal. Biochem. 198, 97 /103. Matsushita, K., Toyama, H., Yamada, M., Adachi, O., 2002. Quinoproteins: structure, functions and biotechnological applications. Appl. Microb. Biotechnol. 58, 13 /22. Patel, N.G., Meier, S., Cammann, K., Chemnitius, G.-C., 2001. Screen-printed biosensors using different alcohol oxidases. Sens. Actuat. B. 75, 101 /110. Plegge, V., Slama, M., Suselbeck, B., Wienke, D., Spener, F., Knoll, M., Zaborosch, C.H., 2000. Analysis of ternary mixtures with a single dynamic microbial sensor and chemometrics using a nonlinear multivariate calibration. Anal. Chem. 72, 2937 /2942. Racek, J., 1994. Cell-Based Biosensors. Technomic Publishing Co, Lancaster. Ramanavicius, A., Habermu¨ller, K., Cso¨regi, E., Laurinavicius, V., Schuhmann, W., 1999. Polypyrrole entrapped quinohemoprotein alcohol dehydrogenase. Evidence for direct electron transfer via conducting-polymer chains. Anal. Chem. 71, 3581 /3586. Reshetilov, A.N., Lobanov, A.V., Morozova, N.O., Gordon, S.H., Greene, R.V., Leathers, T.D., 1998. Detection of ethanol in a twocomponent glucose/ethanol mixture using a nonselective microbial sensor and a glucose enzyme electrode. Biosens. Bioelectron. 13, 787 /793. Reshetilov, A.N., Trotsenko, J.A., Morozova, N.O., Iliasov, P.V., Ashin, V.V., 2001. Characteristics of Gluconobacter oxydans B1280 and Pichia methanolica MN4 cell based biosensors for detection of ethanol. Proc. Biochem. 36, 1015 /1020. Riedel, K., 1991. Biochemical fundamentals and improvement of the selectivity of microbial sensors */a minireview. Bioelectrochem. Bioenerg. 25, 19 /30. Riedel, K., 1998. Enzyme and Microbial Biosensors: Techniques and Protocols. Humana Press, Totowa.
1134
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Shinagawa, E., Matsushita, K., Adachi, O., Ameyama, M., 1989. Formation of the apo-form of quinoprotein alcohol-dehydrogenase from Gluconobacter-suboxydans . Agric. Biol. Chem. 53, 1823 / 1828. Shrestha, S., Shetty, R.S., Ramanathan, S., Daunert, S., 2001. Simultaneous detection of analytes based on genetically engineered whole cell sensing systems. Anal. Chim. Acta 444, 251 /260. Sode, K., 2000. Water-soluble Acinetobacter pyrroloquinoline quinone glucose dehydrogenase mutants with improved glucose affinity for use in glucose assay and sensor. PCT Int. Appl., 40. Somers, W.A.C., Stigter, E.C.A., van Hartingsveldt, W., van der Lugt, J.P., 1998. Enantioselective oxidation of secondary alcohols at a quinohaemoprotein alcohol dehydrogenase electrode. Appl. Biochem. Biotechnol. 75, 151 /162. Sprules, S.D., Hartley, I.C., Wedge, R., Hart, J.P., Pittson, R., 1996. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages. Anal. Chim. Acta 329, 215 /221. Stigter, E.C.A., de Jong, G.A.H., Jongejan, J.A., Duine, J.A., van der Lugt, J.P., Somers, W.A.C., 1996. Electron transfer between a quinohemoprotein alcohol dehydrogenase and an electrode via a redox polymer network. Enzyme Microb. Tech. 18, 489 /494. Stigter, E.C.A., de Jong, G.A.H., Jongejan, J.A., Duine, H.A., van der Lugt, J.P., Somers, W.A.C., 1997. Electron transfer and stability of a quinohaemoprotein alcohol dehydrogenase electrode. J. Chem. Tech. Biotechnol. 68, 110 /116.
Svitel, J., Curilla, O., Tkac, J., 1998. Microbial cell-based biosensor for sensing glucose, sucrose or lactose. Biotechnol. Appl. Biochem. 27, 153 /158. Svorc, J., Miertus, S., Barlikova, A., 1990. Hybrid biosensor for the determination of lactose. Anal. Chem. 62, 1628 /1631. Tkac, J., Gemeiner, P., Svitel, J., Benikovsky, T., Sturdik, E., Vala, V., Petrus, L., Hrabarova, E., 2000a. Determination of total sugars in lignocellulose hydrolysate by a mediated Gluconobacter oxydans biosensor. Anal. Chim. Acta 420, 1 /7. Tkac, J., Svitel, J., Novak, R., Sturdik, E., 2000b. Triglyceride assay by amperometric microbial biosensor: Sample hydrolysis and kinetic approach. Anal. Lett. 33, 2441 /2452. Tkac, J., Vostiar, I., Sturdik, E., Gemeiner, P., Mastihuba, V., Annus, J., 2001. Fructose biosensor based on D-fructose dehydrogenase immobilized on a ferrocene-embedded cellulose acetate membrane. Anal. Chim. Acta 439, 39 /46. Vijayakumar, A.R., Cso¨regi, E., Heller, A., Gorton, L., 1996. Alcohol biosensors based on coupled oxidase-peroxidase systems. Anal. Chim. Acta 327, 223 /234. Wilson, G.S., The´venot, D.R., 1990. Biosensors. A practical approach. Oxford University Press, Oxford. Yamamoto, K., Ohgaru, T., Torimura, M., Kinoshita, H., Kano, K., Ikeda, T., 2000. Highly-sensitive flow injection determination of hydrogen peroxide with a peroxidase-immobilized electrode and its application to clinical chemistry. Anal. Chim. Acta 406, 201 /207.
Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation Jan Tkac a,*,1, Igor Vostiar b, Lo Gorton c, Peter Gemeiner a, Ernest Sturdik b a
b
Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, SK-842 38 Bratislava, Slovak Republic Department of Biochemical Technology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, SK-812 37 Bratislava, Slovak Republic c Department of Analytical Chemistry, Lund University, PO Box 124, SE-22 100 Lund, Sweden Received 24 June 2002; received in revised form 3 October 2002; accepted 24 October 2002
Abstract A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 mM (S/N /3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l1. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cellulose acetate; Ferricyanide; Enhanced selectivity; Ethanol detection; Microbial biosensor; Gluconobacter oxydans
1. Introduction Microbial cells are very promising for biosensor construction because of several advantages: the enzyme does not need to be isolated, enzymes are usually more stable in their natural environment in the cell, coenzymes and activators are already present in the system (D’Souza, 2001; Racek, 1994). Cell-based biosensors are frequently used for determination of BOD, toxic agents and assimilable sugars (Riedel, 1998). Microbial biosensors can be also used for selective detection of a single analyte, but the low selectivity has to be overcome. To enhance the selectivity of microbial biosensors several approaches were used. The possible methods for
* Corresponding author. Present address: Division of Biotechnology, Department of Biochemical Technology, IFM, Linko¨pings Universitet, SE-581 83, Linko¨ping, Sweden. E-mail address: [email protected] (J. Tkac). 1 Tel.: / 421-2-5941-0318; fax: / 421-2-5941-0222; e-mail: [email protected].
enhancement of the selectivity of microbial biosensors were well reviewed (Riedel, 1991; Racek, 1994), including induction of transport/metabolic systems, inhibition of undesired transport/metabolic pathways, the coupling of enzymes with immobilized cells, the transfer of desired plasmid-controlled pathways, the choice of a proper transducer and even pH for measurement. Permeabilization of cells using digitonin, which releases cofactors from the cells, (Gonchar et al., 1998) and application of a gas-permeable Teflon membrane (Hikuma et al., 1979) effectively increased selectivity of microbial biosensors. Another possibility is the use of genetically engineered cells lacking specific receptors (Aravanis et al., 2001), fusing of reporter gene for green fluorescent protein with genes induced by the analyte (Shrestha et al., 2001), by overexpression of natural enzymes in the cells (Svorc et al., 1990) or by improving of analyte affinity (Sode, 2000). Other approaches used to enhance the selectivity of whole-cell systems using genetic engineering are well reviewed elsewhere (Daunert et al., 2000). Different chemometric tools were
0956-5663/03/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 2 4 4 - 0
1126
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
recently successfully used to enhance the selectivity of microbial biosensor assays including application of the artificial neural networks (Lobanov et al., 2001) and multivariate calibration techniques (Reshetilov et al., 1998; Plegge et al., 2000). The genera Gluconobacter have been frequently used for preparation of microbial biosensors (Kitagawa et al., 1987b; Lobanov et al., 2001; Reshetilov et al., 2001; Svitel et al., 1998; Tkac et al., 2000a,b) because of high efficiency and speed of substrate oxidation due to incomplete oxidation of substrates (Gupta et al., 2001; Matsushita et al., 2002). Moreover, pyrroloquinoline quinone (PQQ) containing enzymes of this genera involved in substrate oxidation are bound to the periplasmic membrane, providing fast response of microbial biosensors. Several enzymes were purified from Gluconobacter and one of them, fructose dehydrogenase, is commercially available, but low stability and activity hamper wider use of these enzymes for biosensors preparation. Thus, it is better to use whole cells with improved selectivity for preparation of biosensors. The cellulose acetate (CA) membrane is often used in order to enhance the selectivity of detection of hydrogen peroxide (Wilson and The´venot, 1990) and now the CA membrane is widely used for covering of electrodes, microelectrodes and modified screen-printed electrodes. To our knowledge the CA membrane has not been used to enhance the selectivity of any microbial biosensor. A simple and generally applicable concept for enhancement of the selectivity of microbial biosensors is here proposed. For this purpose a size exclusion membrane cast from CA solution on the electrode modified with Gluconobacter oxydans was successfully used to discriminate between ethanol and glucose providing fast and sensitive ethanol detection. This approach is cheap when compared to the use of the expensive, unstable and methanol oxidizing alcohol oxidase; or alcohol dehydrogenase with unfavorable reaction equilibrium (Vijayakumar et al., 1996), which depends on the soluble cofactor (NAD). Moreover, oxidation of ethanol by PQQ-dependent alcohol dehydrogenase is irreversible and the enzyme is unable to oxidize methanol (Jongejan et al., 2000).
biomass production of G. oxydans . These carbon sources were purchased either from Merck (Darmstadt, Germany) or from Sigma (St. Louis, USA). All other reagents were of analytical grade and were supplied by Lachema (Brno, Czech Republic). McIlvaine buffers were prepared from a 0.1 M solution of citric acid and a 0.2 M solution of Na2HPO4 with addition of 0.1 M KCl. Phosphate buffers were prepared from 0.1 M solutions of KH2PO4 and Na2HPO4 containing 0.1 M of KCl. 2.2. Cell cultivation of Gluconobacter oxydans The strain of G. oxydans CCM 1783 (/ATCC 621) was maintained on the agar containing (g l 1): Dglucose, 100; yeast extract (Oxoid Ltd., Basingstoke, UK), 10; calcium carbonate, 20; agar (Oxoid Ltd., Basingstoke, UK), 20; and transferred monthly (Tkac et al., 2000a). The cell biomass was prepared by aerobic cultivation at 28 8C on a rotary shaker in 500 ml flasks filled with 100 ml of media. The growth medium contained (g l 1): carbon source, 5; yeast extract, 5. The culture, inoculated from the slant agar, was incubated until reaching the late exponential phase (preferentially 12 h), when the cells contained the most active PQQ-dependent alcohol dehydrogenase in the highest yield (Shinagawa et al., 1989). Then the cells from one cultivation flask were collected by centrifugation (10 min, 3500 /g ), resuspended in 2 ml of cold and sterile 0.9% NaCl solution containing 2 mM CaCl2 and this procedure was repeated three times to assure a cell suspension free from fermentation broth. The biomass concentration was expressed as the dry weight matter of cells determined by drying to a constant weight at 105 8C. 2.3. Apparatus
2. Material and methods
Biosensor measurements were carried out on an Amperometric Detector ADLC2 (Laboratorni pristroje, Prague, Czech Republic) using a glassy carbon electrode as the working (diameter 6 mm) and a saturated calomel electrode (SCE) as the reference electrode. Cyclic voltammetry experiments were performed using the GCE as the working electrode, a platinum wire as the auxiliary electrode and a SCE as the reference. In both cases data were collected using a PC.
2.1. Reagents
2.4. Biosensor preparation
CA (approx. 40% of acetyl groups). Dialysis membrane (cut off 12 000), sodium alginate from Macrocystis perifea were purchased from Sigma (St. Louis, USA). As sugar source D-glucose, glycerol, D-mannitol, Dsorbitol, galactitol (dulcitol), D-ribose, D-gluconate, Larabinitol, L-sorbose and D-fructose were used for
On the surface of the GCE, 10 ml of a 1 gDW l 1 of G. oxydans suspension in McIlvaine buffer pH 6.0 containing 0.1 M KCl and 2 mM CaCl2 (if not stated otherwise) were spread together with 10 ml of a 1 mM ferricyanide solution in distilled water and then the water was allowed to evaporate at 25 8C using a fan (15 min).
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Then 25 ml of a 1% CA solution (if not stated otherwise) was spread and the solvents were allowed to evaporate (40 min, 25 8C). The CA solution was prepared by dissolving a desired amount of CA powder, preferentially 1 g, in a mixture of 55 ml of acetone and 45 ml of cyclohexanone (Tkac et al., 2001). The resulting thickness of the CA membrane on the surface of cell modified glassy carbon electrode was estimated to be about 12 mm (Yamamoto et al., 2000). Before measurement, the sensor was equilibrated for 30 min in McIlvaine buffer pH 6.0 containing 0.1 M KCl, 2 mM CaCl2 and 10 mM ferricyanide at 289/0.2 8C. All experiments were done in a 20 ml thermostated vessel at 289/0.2 8C containing 10 ml of measurement buffer using magnetic stirring (300 rpm) at a working potential of /300 mV, if not mentioned otherwise. 2.5. Fermentation experiments The distillery strain of Saccharomyces cerevisiae GY2 (from the Collection of Microorganisms of the Department of Biochemical Technology, Faculty of Chemical Technology, Bratislava, Slovak Republic) was propagated in 100 ml of liquid medium containing glucose, 30 g l1; yeast extract, 5 g l 1 and peptone (Difco, Detroit, USA), 10 g l 1 on a rotary shaker at 28 8C. The cells were collected by centrifugation (10 min, 3500 /g) after reaching the late exponential phase. The suspension containing the biomass from five cultivation flasks in 300 ml of sterile NaCl (9 g l 1) solution was used to prepare a 2% sodium alginate solution. This solution was dropped with an injection needle into 2% CaCl2. The diameter of the gel beads was about 1.5 mm. The beads were placed in 700 ml of liquid medium containing glucose, 100 g l1; yeast extract, 5 g l 1; and peptone 10 g l 1. After 12 h of aerobic cultivation at 309/0.5 8C, the beads were transferred into 700 ml of fresh medium containing glucose, 200 g l 1; yeast extract, 5 g l1; peptone, 10 g l 1; (NH4)2SO4, 10 g l 1; KH2PO4, 4 g l 1; MgSO4 ×/ 7H2O, 10 g l 1; and CaCl2, 0.2 g l 1. Batch ethanol fermentation was carried out at 309/0.5 8C using S. cerevisiae cells immobilized in the alginate beads under anaerobic conditions. The ethanol concentration was monitored during 10 h by the biosensor and by a reference analytical method (HPLC). 2.6. HPLC measurements Ethanol was determined by HPLC (Waters, Milford, USA) using a column (TESSEK Steel 8 /250 mm) packed with Ostion LGKS 0800 ion exchange particles in H form (TESSEK, Prague, Czech Republic) with differential refractometric detection at 25 8C (Waters R403, Milford, USA). The column was used at 50 8C with a flow rate of 0.5 ml min1 of 1.25 mM H2SO4 (in
1127
deionised water) used as the mobile phase. For detection a sample volume of 5 ml was injected.
3. Results and discussion 3.1. Influence of the physiological state of G. oxydans on the substrate oxidation 3.1.1. Effect of carbon source It was found that the carbon source used in the cultivation medium for biomass production influenced very strongly the sensitivity of the G. oxydans sensor toward glucose and ethanol. Among the ten different carbon sources used for cultivation (see Section 2.1), only two had a positive effect on the ethanol/glucose (EtOH/Glc) sensitivity ratio. Although the EtOH/Glc sensitivity ratio for cells cultivated on sorbitol was similar to that of glycerol, for further work a cultivation medium containing glycerol as carbon source was used because of the shorter time needed for biomass production of G. oxydans . 3.1.2. Effect of cultivation time For evaluation of the influence of the physiological state on the glucose and ethanol oxidation rates, a biosensor with G. oxydans cells immobilized on the GCE behind a dialysis membrane was used. It was found that with an increase of the cultivation time from 10 to 16 h, the EtOH/Glc sensitivity ratio measured by the biosensor decreased from 14.5 to 5.0 and the ethanol/glycerol (EtOH/Gly) sensitivity ratio also decreased from 326.7 to 44.0 (Fig. 1). On the other hand, the linear range of the biosensor was wider, when the cells were collected after 12 h of cultivation. Thus, a cultivation time of 12 h for bacterial biomass production was used for further work. 3.2. Influence of physico/chemical parameters on the biosensor response 3.2.1. Effect of pH and buffer When comparing the effect of pH on the response of the whole cell biosensor with G. oxydans immobilized behind a dialysis membrane for glucose and ethanol it was found that the response for glucose was highly sensitive having a sharp pH optimum at pH 5.5, whereas, that for ethanol was not affected significantly by pH (5.0 /7.0) with an optimum at 6.0. This should reflect the difference in pH optima between aldose and alcohol dehydrogenase, the enzymes responsible for glucose and ethanol oxidation inside intact cells of G. oxydans . When a CA membrane was used for enhancement of the biosensor selectivity, the sensor sensitivity toward ethanol was not affected by pH in the studied range (5.0 /7.0).
1128
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
its inactive apo-form, while phosphate buffer caused hardly any apoenzyme formation (Shinagawa et al., 1989). The maximum holoenzyme occurred at pH 6.0 in the presence of Ca2 (Shinagawa et al., 1989). Thus, for further work a McIlvaine buffer was chosen containing 2 mM CaCl2 and with the pH set to 6.0, resulting in high operational stability (see Section 3.4.4). 3.2.2. Effect of the working potential The effect of the working potential on the sensor performance was studied in the range between 0 and 340 mV. The biosensor sensitivity increased with increasing the working potential up to 220 and then slightly decreased (Fig. 2). At a working potential of 250 mV (cyclic voltammetry study), an anodic current raised after ethanol addition, indicating efficient ethanol oxidation by the biosensor (Fig. 3). For further work the working potential was set to/300 mV. 3.3. Influence of other parameters on the biosensor performance
Fig. 1. The effect of cultivation time on the oxidation of ethanol, glucose and glycerol by G. oxydans cells. First, the sensitivity on additions of ethanol, glucose or glycerol was expressed in nA mM 1 and then the sensitivity to EtOH was expressed as a ratio to the glucose sensitivity (EtOH/Glc) or to glycerol sensitivity (EtOH/Gly) of the biosensor.
It is well known that Ca2 ions are essential for proper function of PQQ-dependent alcohol dehydrogenases (Jongejan et al., 2000). Since phosphate buffer may chelate Ca2 from the active site of the enzyme causing an inactivation, a sodium acetate buffer has been used for the preparation of the ethanol biosensor based on the PQQ-dependent dehydrogenase isolated from G. oxydans (Ramanavicius et al., 1999). On the other hand, acetate is a product of ethanol oxidation by the cells (oxidation by PQQ-dependent alcohol and aldehyde dehydrogenases); therefore, an inhibition of ethanol oxidation by G. oxydans in the presence of sodium acetate buffer is expected. Moreover, incubation of ADH with acetate buffer converted 90% of ADH into
3.3.1. Effect of the amount of CA For investigation of the effect of the amount of CA on the biosensor performance, four membranes were tested prepared by casting 25 ml of CA solution (0.5, 1, 2 and 4% of CA) on the modified GCE electrode. With increasing the thickness of the CA membrane, the sensitivity of the sensor for ethanol decreased rapidly from 5660 to 22 nA mM 1 and the response time increased linearly from 13 to 51 s (Table 1). For further work a CA membrane prepared from 1% solution was used, because spreading of 0.5% solution of CA produced an unstable sensor with high background noise. During 8.5 h of measurement the response time of the sensor on standard ethanol addition was practically the same, indicating stable diffusional parameters of the membrane in course of measurements (see Section 3.4.4). 3.3.2. Effect of the amount of biomass The biosensor sensitivity increased with increasing the amount of G. oxydans cells on the electrode in the range tested from 1 to 30 mgDW of G. oxydans per electrode. The response time decreased from 40 to 10 s, when the cell amount increased from 1 to 3 mgDW of G. oxydans per electrode. This is probably caused by kinetic limitations of ethanol oxidation, when 1 mgDW of G. oxydans was applied on the GCE resulting in a longer response time. The response time was the same for the biosensor containing either 3 or 10 mgDW of G. oxydans per electrode and then increased (Table 2). This is probably caused by a diffusion controlled process, because of higher cell loading. Spreading of 10 mgDW of G. oxydans per electrode was used for further work as
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
1129
Fig. 2. The influence of the working potential on the sensor performance (A). A typical sensor response for successive addition of 10 mM ethanol measured at a working potential of /300 mV at 289/0.2 8C (B). A glassy carbon electrode covered with 10 mgDW of G. oxydans and 10 nmol of ferricyanide. The cell-modified electrode was covered with 25 ml of 1% CA solution. Measurements were performed in stirred (250 rpm) McIlvaine buffer pH 6.0 containing 0.1 M KCl and 10 mM ferricyanide at 289/0.2 8C.
Table 2 The effect of the amount of G. oxydans cells spread on the GCE on the sensitivity and response time of the ethanol sensor (10 nmol of ferricyanide and 20 ml of 1% CA solution on the GCE) Amount of G. oxydans (mg)
Sensitivity (nA mM 1)
Response time (s)
1 3 10 30
120 520 1140 2100
40 10 10 25
a compromise between the sensitivity and the response time.
Fig. 3. Cyclic voltammetry of the whole cell ethanol biosensor before and after ethanol addition (1 mM). Sweep rate 50 mV s 1, for other conditions used see Fig. 2.
Table 1 The effect of the amount of CA spread on the GCE on the sensitivity and response time of the ethanol sensor (10 nmol of ferricyanide and 10 mgDW of G. oxydans on the GCE) Concentration of CA (%)
Sensitivity (nA mM 1)
Response time (s)
0.5 1 2 4
5660 1140 350 22
13 16 31 51
3.3.3. Effect of the amount of ferricyanide It was observed that even ferricyanide penetrates slightly through the CA membrane. Thus, a decrease of the sensor sensitivity to 53% of the initial value within 1 h occurred. When 1 ml of 100 mM ferricyanide solution was added to the measurement solution, restoration of the initial sensitivity was observed and the signal was stable. Hence, the measurement buffer used further contained 10 mM ferricyanide. 3.4. Characterization of the ethanol biosensor 3.4.1. Discrimination of a cellulose acetate membrane between ethanol and glucose It was confirmed that even a CA membrane spread on a G. oxydans layer hampers the diffusion of glucose
1130
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Fig. 4. The microbial biosensor response for ethanol (10 mM) addition or mixture of ethanol (10 mM) and glucose (100 mM) addition using a dialysis and a CA membrane (A). The response time of the biosensor for glucose or ethanol additions using a dialysis or a CA membrane coverage (B). For other conditions used see Fig. 2.
through the membrane, whereas, ethanol diffusion was possible. The response time for ethanol addition was the same using a dialysis membrane or a CA membrane (Fig. 4). The response time for glucose of the sensor covered by a dialysis membrane was 26 s, twice longer than that for ethanol detection. The response time of 430 s for glucose, when the CA membrane was applied on the modified GCE, indicating limited diffusion of glucose (Fig. 4). In order to prove a discrimination effect of the CA membrane between ethanol and glucose, standard solutions of ethanol (10 mM) or a mixture of glucose (100 mM) and ethanol (10 mM) were analyzed by biosensors with surface coverage either by a dialysis membrane or by the size-exclusion CA membrane. Detection of ethanol by the CA modified biosensor was not influenced by the presence of glucose, which is in contrast to the biosensor covered by a dialysis membrane (Fig. 4). The EtOH/Glc sensitivity ratio increased from 9.3 (a dialysis membrane application) to 541.1, when a CA was used. This membrane also prevents diffusion of glycerol, as the EtOH/Gly sensitivity ratio increased from 183.4 to 1366.6, under the same conditions. 3.4.2. Selectivity of the sensor toward alcohols Alcohol oxidase or alcohol dehydrogenases are able to oxidize several alcohols including ethanol. Moreover, alcohol oxidase is also able to oxidize methanol even more effectively than ethanol. The higher methanol response is attributed to the reaction product, formaldehyde, which is a substrate for alcohol oxidase, whereas, aldehydes formed from higher alcohols are not (Vijayakumar et al., 1996). The effect of several alcohols on the sensor sensitivity and response time was carefully investigated and the sensitivity of our ethanol
biosensor toward different alcohols is compared with that of previously reported biosensors based on alcohol oxidase and alcohol dehydrogenases (Table 3). The CA membrane is not an important diffusion barrier for ethanol because the same response time was achieved using a CA and a dialysis membrane (Table 3). The CA membrane, due to the hydrophobic nature is more permeable for higher alcohols than for ethanol. The response time for the CA modified electrode is shorter for propanol (7 s) and also shorter for butanol (11 s) than that for ethanol (13 s). When a dialysis membrane for G. oxydans retention was used, the response time for ethanol, propanol and butanol were practically the same (around 13 /15 s). Isopropanol does not diffuse well through the CA membrane, when compared with the dialysis membrane, with doubled response time (62 s respectively 30 s). This behavior can be explained by restricted diffusion of isopropanol through the CA membrane. Alcohol oxidases from various sources oxidize methanol, but their ability to oxidize higher alcohols decreases with increasing molecular weight. Moreover, these enzymes slightly oxidize branched alcohols. In the case AOD from Candida boindii was covalently immobilized on graphite particles mixed into carbon paste, isopropanol was oxidized with the same efficiency as propanol (Johansson et al., 1993). PQQ-dependent alcohol dehydrogenase (Acetobacter aceti or G. oxydans) oxidizes propanol and butanol almost as effectively as ethanol, but not methanol and only slightly branched alcohols. The affinity of PQQ-dependent alcohol dehydrogenase purified from Comamonas testosteroni for aliphatic and secondary alcohols increases with the chain length of the substrate. NAD-dependent alcohol dehydrogenase does not catalyze the oxidation of methanol, but higher and
C. boindii (Patel et al., 2001; Vijayakumar et al., 1996); P. pastoris (Lubrano et al., 1991; Patel et al., 2001; Vijayakumar et al., 1996); H. polymorpha (Karyakin et al., 1996; Patel et al., 2001); NADdependent ADH (Chi and Dong, 1994; Lobo Castanon et al., 1997; Sprules et al., 1996); PQQ-dependent ADH (acetic acid bacteria-G. oxydans or A. aceti ) (Kitagawa et al., 1987a,b, 1989); PQQdependent ADH (C. testosteroni ) (Somers et al., 1998; Stigter et al., 1996, 1997); RT, response time (90% of steady state response); Sensit., sensitivity expressed in nA mM 1, then the sensitivity to addition of ethanol was set to 100%; an/5; b; n /25; ca response time in the case of direct electron transfer of PQQ-dependent ADH (Ikeda et al., 1993); / not determined or not known.
/ / 20 /60 0.8 /2.2 / 0.06 / 0.005 /0.006 / 22 / / / 0 /1 / 15 /45 100 3c, 60 /300 / 39 /90 / / 41 /76 / / 16 /78 / / 0 /35 / Methanol Ethanol Propanol Butanol 2-propanol 2-butanol
0a 0a 13.09/0.8b 100.09/1.7b 7.39/0.6a 151.79/3.4a 11.29/0.6a 85.69/1.6a a 0.49/0.2a 62.39/3.1 / /
0a B/0.1a / 13.29/0.5b 100.09/1.5b 26 15.39/0.6a 108.19/1.7a / 13.19/0.7a 99.19/1.3a / a 30.49/1.9 5.09/0.5a / / / /
113 /128 / 117 /160 / 100 10 /19 100 12 4 /19 / 5 /29 / 3 /16 / 4 /16 / 0 /2 / 0 /8 / 0 /2 / 0 /2 /
120 /690 100 5 /18 0 /4 0 /1 0 /1
0 100 69 /130 73 /160 0 0
Kapp m [mM] RT [s] Sensit. [%] Sensit. RT [%] [s] RT [s] Sensit. [%] RT [s] RT [s] RT [s]
Sensit. [%]
RT [s]
Sensit. [%]
RT Sensit. [s] [%]
Sensit. [%]
PQQ (acetic acid bacteria) NAD Pichia pastoris CA Membrane
Dialysis membrane
C. boindii
Hansenula polymorpha
Alcohol dehydrogenase Alcohol oxidase This work
Table 3 Substrate specificity and response time of ethanol biosensors based on alcohol oxidase; NAD- and PQQ-dependent alcohol dehydrogenase; and G. oxydans cells
PQQ (C. testosteroni )
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
1131
branched alcohols are oxidized (Table 3). From this point of view, PQQ-dependent alcohol dehydrogenase isolated from acetic bacteria or kept within intact G. oxydans cells can be considered as the most selective. 3.4.3. Basic biosensor parameters When applying all the optimal conditions, the basic characteristics of the ethanol biosensor were evaluated. The sensitivity of the sensor was very high (3.5 mA mM 1) resulting in a low detection limit (0.85 mM, S/ N /3). The linear response range was wide (2 /270 mM) and the response fast (13.0 s, n /30, 90% of steady state, Fig. 2B). A comparison of response time of presented ethanol biosensor with those already published is shown in Table 3. The relative standard deviation for measurement of 10 mM ethanol solution was within 1.79% (n/ 30). Moreover, the biosensor is very cheap; it is possible to construct approximately 2000 sensors from one cultivation flask of G. oxydans suspension. Immobilizing G. oxydans cells on mass-produced electrodes prepared by ink- or screen-printing techniques, the price of one sensor will be negligible. 3.4.4. Operational stability During examination of the operational stability of the ethanol biosensor it was found that the sensor sensitivity decreased to 75% of its initial sensitivity within 4.5 h, when a cell suspension was prepared in phosphate buffer not containing Ca2. When the cells were resuspended in McIlvaine buffer containing 2 mM CaCl2, the sensitivity remained unchanged for 8.5 h of continuous use (100.49/1.8%, initial sensitivity /2.9 mA mM 1, n /16, Fig. 5). The operational stability of the biosensor during measurement of real samples was very good indicating effective anti-fouling effect of the membrane to compounds present in yeast extract and peptone, which are part of the fermentation medium. 3.4.5. Storage stability The sensor sensitivity examined during biosensor storage at 4 8C in McIlvaine buffer containing ferricyanide decreased to 7% of its initial value within 6 days. The low stability can be ascribed either to the uncoupling of the respiratory chain by ferricyanide or by cell starvation during storage. If no artificial redox compound was used for G. oxydans sensor preparation, the storage stability of G. oxydans sensor was good (50% of initial sensitivity after 8 days storage at 20 8C) (Reshetilov et al., 2001). 3.5. Analysis of real samples Ethanol fermentation has been realized under anaerobic conditions with S. cerevisiae cells immobilized in alginate gel prepared by ionotropic gelation in 2% calcium chloride. Ionotropic gelation allows immobili-
1132
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Fig. 5. Operational stability of the whole cell ethanol biosensor measured in k 0.1 M phosphate buffer pH 6.0 containing 0.1 M KCl and 10 mM of ferricyanide; in I McIlvaine buffer pH 6.0 containing 0.1 M KCl, 2 mM CaCl2 and 10 mM ferricyanide m in McIlvaine buffer pH 6.0 containing 0.1 M KCl, 2 mM CaCl2 and 10 mM ferricyanide during measurement of real samples. For other conditions used see Fig. 2.
zation of cells under mild condition with high cell loading. This method of immobilization preserves a high viability of the immobilized cells (Gemeiner et al., 1996). Samples taken from the S. cerevisiae cultivation were analyzed by the biosensor in the order; standardsample-standard. The sensitivity of the sensor on standard ethanol addition before and after sample measurement was averaged and applied for the determination of ethanol in the sample. Each sample was analyzed by the biosensor at least three times and the R.S.D. varied in the range 0.50 /4.19% with an average R.S.D. of 2.63%. During the measurement of real samples the sensitivity for the ethanol standard remained stable for 8.5 h of continuous operation, thus, revealing an excellent operational stability. During this time 150 samples or ethanol standards were analyzed, including the time needed for washing and equilibration of the sensor signal. The microbial biosensor with enhanced selectivity was successfully applied in an offline monitoring of S. cerevisiae batch fermentation, even in the presence of 200 g l 1 glucose in the initial stage. Only a mild influence of the highly excessive amount of glucose on ethanol detection with the microbial biosensor was observed (Fig. 6). The results obtained with the biosensor were in very good agreement with the HPLC measurements (Fig. 6), cHPLC /0.996*cBiosensor/0.143 (R2 /0.998, n /20).
4. Conclusion The presented microbial biosensor represents sensitive and selective alternative for ethanol detection even in the presence of high excess of glucose. The biosensor has a low detection limit combined with a fast response time. The high operational stability of the sensor makes it very promising for on-line monitoring, but the storage stability should be improved. The selectivity of the ethanol biosensor toward different alcohols was better compared to published ethanol biosensors based on alcohol oxidase, PQQ- and NAD-dependent alcohol dehydrogenases. Reliability of biosensor assay was confirmed by comparison with reference analytical method, performed on real samples. Moreover, the application of a permselective CA membrane might serve as a model for developing other redox sensor systems either with immobilized microbial cells or enzymes.
Acknowledgements The authors thank L. Kristofikova for the HPLC measurements. This work was supported by following the VEGA grants: 1/6252/99, 1/7347/20 and 2/1047/21 and by the Swedish Research Council (VR).
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Fig. 6. Correlation between biosensor and HPLC assays of ethanol during batch fermentation with immobilized S. cerevisiae cells. Bars at each point represent R.S.D. of sample measurement by both methods of analysis (A). Residuals between ethanol concentrations obtained by both methods of analysis (B).
References Aravanis, A.M., DeBusschere, B.D., Chruscinski, A.J., Gilchrist, K.H., Kobilka, B.K., Kovacs, G.T.A., 2001. A genetically engineered cell-based biosensor for functional classification of agents. Biosens. Bioelectron. 16, 571 /577. Chi, Q., Dong, S., 1994. Electrocatalytic oxidation of reduced nicotinamide coenzymes at Methylene Green-modified electrodes and fabrication of amperometric alcohol biosensors. Anal. Chim. Acta 285, 125 /133. Daunert, S., Barrett, G., Feliciano, J.S., Shetty, R.S., Shrestha, S., Smith-Spencer, W., 2000. Genetically engineered whole-cell sensing systems: Coupling biological recognition with reporter genes. Chem. Rev. 100, 2705 /2738. D’Souza, S.F., 2001. Microbial biosensors. Biosens. Bioelectron. 16, 337 /353. Gemeiner, P., Rexova-Benkova, L., Svec, F., Norrlow, O., 1996. Immobilized Biosystems. Theory and Practical Applications. Blackie Academic & Professional with Chapman & Hall, Glasgow. Gonchar, M.V., Maidan, M.M., Moroz, O.M., Woodward, J.R., Sibirny, A.A., 1998. Microbial O2 and H2O2-electrode sensors for alcohol assays based on the use of permeabilized mutant yeast cells as the sensitive bioelements. Biosens. Bioelectron. 13, 945 / 952.
1133
Gupta, A., Singh, V.K., Qazi, G.N., Kumar, A., 2001. Gluconobacter oxydans : its biotechnological applications. J. Mol. Microb. Biotechnol. 3, 445 /456. Hikuma, M., Kubo, T., Yasuda, T., Karube, I., Suzuki, S., 1979. Microbial electrode sensor for alcohols. Biotechnol. Bioeng. 21, 1845 /1853. Johansson, K., Jo¨nsson-Pettersson, G., Gorton, L., Marko-Varga, G., Cso¨regi, E., 1993. A reagentless amperometric biosensor for alcohol detection in column liquid-chromatography based on coimmobilized peroxidase and alcohol oxidase in carbon-paste. J. Biotechnol. 31, 301 /316. Jongejan, A., Machado, S.S., Jongejan, J.A., 2000. The enantioselectivity of quinohaemoprotein alcohol dehydrogenases: mechanistic and structural aspects. J. Mol. Catal. B: Enzyme 8, 121 /163. Karyakin, A.A., Karyakina, E.E., Gorton, L., 1996. Prussian-Bluebased amperometric biosensors in flow-injection analysis. Talanta 43, 1597 /1606. Kitagawa, Y., Ameyama, M., Nakashima, K., Tamiya, E., Karube, I., 1987a. Amperometric alcohol sensor based on an immobilized bacteria cell-membrane. Analyst 112, 1747 /1749. Kitagawa, Y., Tamiya, E., Karube, I., 1987b. Microbial-FET alcohol sensor. Anal. Lett. 20, 81 /96. Kitagawa, Y., Kitabatake, K., Kubo, I., Tamiya, E., Karube, I., 1989. Alcohol sensor based on membrane-bound alcohol dehydrogenase. Anal. Chim. Acta 218, 61 /68. Lobanov, A.V., Borisov, I.A., Gordon, S.H., Greene, R.V., Leathers, T.D., Reshetilov, A.N., 2001. Analysis of ethanol glucose mixtures by two microbial sensors: Application of chemometrics and artificial neural networks for data processing. Biosens. Bioelectron. 16, 1001 /1007. Lobo Castanon, M.J., Miranda Ordieres, A.J., Tunon Blanco, P., 1997. Amperometric detection of ethanol with poly-(o-phenylenediamine)-modified enzyme electrodes. Biosens. Bioelectron. 12, 511 /520. Lubrano, G.J., Faridnia, M.H., Palleschi, G., Guilbault, G.G., 1991. Amperometric alcohol electrode with extended linearity and reduced interferences. Anal. Biochem. 198, 97 /103. Matsushita, K., Toyama, H., Yamada, M., Adachi, O., 2002. Quinoproteins: structure, functions and biotechnological applications. Appl. Microb. Biotechnol. 58, 13 /22. Patel, N.G., Meier, S., Cammann, K., Chemnitius, G.-C., 2001. Screen-printed biosensors using different alcohol oxidases. Sens. Actuat. B. 75, 101 /110. Plegge, V., Slama, M., Suselbeck, B., Wienke, D., Spener, F., Knoll, M., Zaborosch, C.H., 2000. Analysis of ternary mixtures with a single dynamic microbial sensor and chemometrics using a nonlinear multivariate calibration. Anal. Chem. 72, 2937 /2942. Racek, J., 1994. Cell-Based Biosensors. Technomic Publishing Co, Lancaster. Ramanavicius, A., Habermu¨ller, K., Cso¨regi, E., Laurinavicius, V., Schuhmann, W., 1999. Polypyrrole entrapped quinohemoprotein alcohol dehydrogenase. Evidence for direct electron transfer via conducting-polymer chains. Anal. Chem. 71, 3581 /3586. Reshetilov, A.N., Lobanov, A.V., Morozova, N.O., Gordon, S.H., Greene, R.V., Leathers, T.D., 1998. Detection of ethanol in a twocomponent glucose/ethanol mixture using a nonselective microbial sensor and a glucose enzyme electrode. Biosens. Bioelectron. 13, 787 /793. Reshetilov, A.N., Trotsenko, J.A., Morozova, N.O., Iliasov, P.V., Ashin, V.V., 2001. Characteristics of Gluconobacter oxydans B1280 and Pichia methanolica MN4 cell based biosensors for detection of ethanol. Proc. Biochem. 36, 1015 /1020. Riedel, K., 1991. Biochemical fundamentals and improvement of the selectivity of microbial sensors */a minireview. Bioelectrochem. Bioenerg. 25, 19 /30. Riedel, K., 1998. Enzyme and Microbial Biosensors: Techniques and Protocols. Humana Press, Totowa.
1134
J. Tkac et al. / Biosensors and Bioelectronics 18 (2003) 1125 /1134
Shinagawa, E., Matsushita, K., Adachi, O., Ameyama, M., 1989. Formation of the apo-form of quinoprotein alcohol-dehydrogenase from Gluconobacter-suboxydans . Agric. Biol. Chem. 53, 1823 / 1828. Shrestha, S., Shetty, R.S., Ramanathan, S., Daunert, S., 2001. Simultaneous detection of analytes based on genetically engineered whole cell sensing systems. Anal. Chim. Acta 444, 251 /260. Sode, K., 2000. Water-soluble Acinetobacter pyrroloquinoline quinone glucose dehydrogenase mutants with improved glucose affinity for use in glucose assay and sensor. PCT Int. Appl., 40. Somers, W.A.C., Stigter, E.C.A., van Hartingsveldt, W., van der Lugt, J.P., 1998. Enantioselective oxidation of secondary alcohols at a quinohaemoprotein alcohol dehydrogenase electrode. Appl. Biochem. Biotechnol. 75, 151 /162. Sprules, S.D., Hartley, I.C., Wedge, R., Hart, J.P., Pittson, R., 1996. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages. Anal. Chim. Acta 329, 215 /221. Stigter, E.C.A., de Jong, G.A.H., Jongejan, J.A., Duine, J.A., van der Lugt, J.P., Somers, W.A.C., 1996. Electron transfer between a quinohemoprotein alcohol dehydrogenase and an electrode via a redox polymer network. Enzyme Microb. Tech. 18, 489 /494. Stigter, E.C.A., de Jong, G.A.H., Jongejan, J.A., Duine, H.A., van der Lugt, J.P., Somers, W.A.C., 1997. Electron transfer and stability of a quinohaemoprotein alcohol dehydrogenase electrode. J. Chem. Tech. Biotechnol. 68, 110 /116.
Svitel, J., Curilla, O., Tkac, J., 1998. Microbial cell-based biosensor for sensing glucose, sucrose or lactose. Biotechnol. Appl. Biochem. 27, 153 /158. Svorc, J., Miertus, S., Barlikova, A., 1990. Hybrid biosensor for the determination of lactose. Anal. Chem. 62, 1628 /1631. Tkac, J., Gemeiner, P., Svitel, J., Benikovsky, T., Sturdik, E., Vala, V., Petrus, L., Hrabarova, E., 2000a. Determination of total sugars in lignocellulose hydrolysate by a mediated Gluconobacter oxydans biosensor. Anal. Chim. Acta 420, 1 /7. Tkac, J., Svitel, J., Novak, R., Sturdik, E., 2000b. Triglyceride assay by amperometric microbial biosensor: Sample hydrolysis and kinetic approach. Anal. Lett. 33, 2441 /2452. Tkac, J., Vostiar, I., Sturdik, E., Gemeiner, P., Mastihuba, V., Annus, J., 2001. Fructose biosensor based on D-fructose dehydrogenase immobilized on a ferrocene-embedded cellulose acetate membrane. Anal. Chim. Acta 439, 39 /46. Vijayakumar, A.R., Cso¨regi, E., Heller, A., Gorton, L., 1996. Alcohol biosensors based on coupled oxidase-peroxidase systems. Anal. Chim. Acta 327, 223 /234. Wilson, G.S., The´venot, D.R., 1990. Biosensors. A practical approach. Oxford University Press, Oxford. Yamamoto, K., Ohgaru, T., Torimura, M., Kinoshita, H., Kano, K., Ikeda, T., 2000. Highly-sensitive flow injection determination of hydrogen peroxide with a peroxidase-immobilized electrode and its application to clinical chemistry. Anal. Chim. Acta 406, 201 /207.