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Mediator type of glucose microbial biosensor based on Aspergillus niger
Analytica Chimica Acta 356 (1997) 217±224
Mediator type of glucose microbial biosensor based on Aspergillus niger J. KatrlõÂka,c, R. BrandsÏteterb, J. SÏvorca,c, M. Rosenbergb, S. MiertusÏc,* a
b
Department of Analytical Chemistry, Slovak Technical University, RadlinskeÂho 9, 81237, Bratislava, Slovakia Department of Biochemical Technology, Slovak Technical University, RadlinskeÂho 9, 81237, Bratislava, Slovakia c POLY-tech, Area di Ricerca, Padriciano 99, 34012 Trieste, Italy Received 24 June 1997; received in revised form 2 September 1997; accepted 7 September 1997
Abstract Whole cells of Aspergillus niger CCM 8004 containing glucose oxidase (EC 1.1.3.4.) were used for the construction of an amperometric microbial mediated carbon paste biosensor. The microorganism was either placed on the surface of the electrode or incorporated directly into the carbon paste. The mediators were either dissolved in the buffer (hexacyanoferrate(III) and ferrocene) or loaded in the carbon paste (ferrocene). All methods resulted in effective glucose amperometric biosensors. The operational stability of the surface-layer modi®ed whole cell biosensor based on ferrocene incorporated into the carbon paste was at least one month, the upper linearity limit was 6 mM. The sensor was used for measuring the glucose content in real samples. # 1997 Elsevier Science B.V. Keywords: Biosensor; Amperometry; Glucose; Carbon paste; Microbial biosensor
1. Introduction The development of amperometric bioelectrodes is one of the major areas of interest in chemical sensors [1]. They are the most successful class of biosensors in widespread use. Many of them are based on the change of oxygen or hydrogen peroxide concentration during enzymatic reaction on the bioelectrode. In this case, the electrode response is dependent on the oxygen concentration in the buffer. The linearity range and the sensitivity might be improved by use of electroactive compounds, mediators, instead of oxygen. The ®rst mediated glucose biosensor based on 1,10 -dimethyl*Corresponding author. Tel.: +39 (40) 3756622; fax: +39 (40) 7797091; e-mail: [email protected] 0003-2670/97/$17.00 # 1997 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00524-2
ferrocene and glucose oxidase was presented by Cass et al. [2] in 1984. Presently, many systems that combine various types of mediators with enzymes have been described [3±8]. Some amperometric biosensors use microorganisms instead of puri®ed enzymes [9±13]. The use of microorganisms could bring some advantages which are mentioned in our previous work [14]. These whole cell sensors are usually based on the amperometric measurement of O2 consumption due to interaction between the substrate and the biocatalytic layer. It was reported in 1966 that Fe CN3ÿ 6 could be used as an electron acceptor for the metabolic oxidation of Dglucose by Escherichia coli [15]. However, the development of whole cell mediated bioelectrodes for analytical purposes has started only in the late 80's,
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probably due to dif®culties in combining microorganism, mediator and electrode in one system. The mediator has to be able to shuttle the electrons between the redox centres of enzymes in the microorganisms and the electrode surface, but details of the electrocatalytic reactions are not clear yet [16]. Among the whole cell mediated amperometric biosensors described, the ethanol yeast-based bioelectrode was based on the activity of alcohol dehydrogenase in yeasts and hexacyanoferrate(III) was used as an electron acceptor for enzymatically produced NADH [12]. Kulys et al. [17] have reported a mediated amperometric L-lactate biosensor using the yeast Hansenula anomala. A glucose bioelectrode based on the bacterium Gluconobacter industries and p-benzoquinone has been developed [16]. Yeast of Saccharomyces cerevisiae and Vitamin K3 were used for preparation of bioelectrode sensitive to ethanol and glucose [18]. Amperometric microbial bioelectrodes for lactate and succinate using ferrocene as a mediator were also reported [19]. Ferrocene was used as the mediator for hydrogen peroxide planttissue biosensor as well [20]. This paper describes the performance of both whole cell carbon paste electrodes for determination of glucose containing ferrocene as a mediator, and the biosensor based on dissolved mediators. Spores of Aspergillus niger were either immobilized on the surface of the electrode (platinum or carbon paste), or incorporated into the carbon paste together with the mediator (ferrocene). Both methods produced effective glucose amperometric biosensors. The preparation, characteristics and analytical performance of these electrodes are reported. 2. Experimental 2.1. Materials Glucose, fructose, sucrose, galactose, maltose, mannose, arabinose, mannitol, sorbitol and cholesterol were obtained from Sigma (St. Louis, MO, USA), Bacto-agar from Difco (USA). Lactozym1 (Novo Nordisk A/S, Bagsvaerd, Denmark, activity 3000 Lactozym units (LAU) per ml) was used for hydrolysis of lactose in whey. Other reagents were commercially available as analytical grade.
2.2. Microorganism The fungal strain Aspergillus niger CCM 8004 obtained from Czechoslovak Culture Collection of Microorganisms (Brno, Czech Rep.) was subcultured weekly on the agar medium (Czapek-Dox agar 50 g, Bacto-agar 1.5 g, distilled water to 1 l) at 288C. The cultivation medium (glucose 200 g, KCl 0.25 g, KH2PO4 0.25 g, MgSO4.7H2O 0.25 g, Ca(NO3)2.4H2O 1 g, (NH4)2SO4 1.1 g, distilled water to 1 l) was inoculated by spore suspension (2 106 spores/ml) and the culture was aerobically cultivated at 308C, pH 5.7 in the fermentor LF-2 (Vyvojove dilny CSAV, Prague, Czech Rep.). Samples for determination of the respiration activity and for determination of dry cell weight (DCW) were taken out every hour. 2.3. Determination of respiration activity and dry cell weight of the microorganism The respiration activity (RA) was determined as the moles of O2 consumed by 1 g (DCW) of biomass in 1 s. The respiration rate was measured by a Clark oxygen electrode at ÿ650 mV vs. Ag/AgCl ref. electrode. For DCW determination, a biomass sample (1± 20 cm3) was ®ltered through a dried membrane ®lter type HA, pore diameter 0.45 mm (Millipore, Bedford, MA, USA), washed with phosphate buffer pH 7.0 and dried at 508C for 6±8 h and weighed. 2.4. Preparation of the biocatalytic layer, carbon paste and sensors The cultivated mycelium with the maximum respiration activity was washed by phosphate buffer pH 7.0 and adjusted to the concentration of 5 g/l DCW. A 20 ml portion of this suspension was ®ltered through a 5 mm diameter membrane ®lter HA. The biosensor based on dissolved mediator was prepared by attachment of cultivated mycelium immobilized on the ®lter on top of the polished platinum electrode. This platinum electrode consisted of a platinum wire (diameter 1 mm) placed in a glass tube (o.d. 10 mm). The biocatalytic layer was covered with a nylon net and ®xed by a rubber O-ring. Carbon paste was prepared as follows: 0.5 g of graphite powder (Elektrokarbon Topolcany, Slovakia)
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were modi®ed by 1.5 ml of solution of ferrocene in acetonitril (®nal concentration of ferrocene in the paste was 3 or 5% (w/w)). The mixture was then activated in an ultrasonic bath for about 20 min and dried using an IR lamp. Then 0.1 ml of paraf®n oil (Slovakofarma Hlohovec, Slovakia) were then added and the ®nal paste was prepared by manual mixing. A portion of the modi®ed carbon paste was packed at the top of a 2 ml plastic syringe with the tip cut off (2 mm i.d.). A copper wire was inserted through the opposite end to establish electrical contact. The active surface of the electrode was polished with ®ne emery paper and the biocatalytic layer was attached as described above. Alternatively, the microorganism was directly incorporated into the carbon paste. The mycelium of A. niger was washed with distilled water, homogenised by vigorous shaking and dried at 378C until constant weight was reached. Then, the dry mycelium was mixed with ferrocene-modi®ed carbon powder (amount of microorganism was 5 or 15% (w/w)) before the paraf®n oil was added. After mixing, the paste was again packed into the plastic syringe and polished with emery paper. 2.5. Apparatus and procedure Electrochemical experiments were carried out on a polarographic analyzer PA-4 (Laboratorni pristroje, Prague, Czech Rep.) A three-electrode system was used with Ag/AgCl (saturated KCl) ref. electrode and a platinum wire as an auxiliary electrode. The measured current was converted into voltage and displayed on a XY 4106 recorder (Laboratorni pristroje, Prague, Czech Rep.). The determination of glucose with the microbial mediated biosensor was carried out at room temperature (208C) in a reaction vessel, equipped with a magnetic stirrer (400 rpm). The sensor was immersed in 0.2 M phosphate buffer pH7.0 (10 ml). The operational potential of the bioelectrode was 350 mV vs. ref. electrode. After the sensor current became constant, the sensor was ®rst calibrated by the addition of standard glucose solution and then used for the determination of glucose in samples. Current±time curves were recorded. The height of the recorded wave (current decrease) corresponds to the concentration of glucose.
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2.6. Hydrolysis of lactose by lactase Lactose in real samples was ®rst hydrolysed by Lactozym1. Hydrolysis was carried out at 408C in phosphate buffer pH 6.5, activity of Lactozym1 was 5 LAU/ml. Degree of hydrolysis tested on a model sample of lactose was 97% after 6 h. 3. Results and discussion 3.1. Optimization of cultivation of Aspergillus niger and working conditions of the biosensor The activity of the enzymes involved in the biocatalytic layer is one of the main factors that in¯uence the biosensor characteristics. The mycelium showing maximum respiration activity was used for biosensor preparation. The respiration activity of Aspergillus niger during cultivation is shown in Fig. 1. The increase of speci®c activity reached a maximum after 15±16 h of cultivation. The cultivation was stopped after 15 h and the biomass was treated as described above. Evaluation of the effect of such parameters as pH, temperature and type of buffer on biosensor response gave results consistent with those described in previous work [14]. Shortly, the response of the sensor was independent of pH in the range 5±8. The temperature pro®le reached a maximum at 508C. The sensitivity of the biosensor to glucose was not signi®cantly dependent on the type of tested buffers. All measurements were then performed at 308C in 0.2 M phosphate buffer, pH 7.0. 3.2. Microbial biosensor with the mediator in solution The electrochemical behaviour of the microbial biosensor based on the platinum electrode with dissolved mediator was studied by means of cyclic voltammetry. The mediator, ferrocene, was dissolved in the solution of hexacyanoferrate(III). Addition of the saturated solution of ferrocene (Fe(Cp)2) in ethanol into the buffer caused a turbidity due to precipitation of ferrocene (water solubility of ferrocene is very low). After addition of hexacyanoferrate(III) (concentration 0.5 mM), the turbidity disappeared because
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Fig. 1. Dependence of the respiration activity (RA) of mycelium Aspergillus niger on the cultivation time. Measured in citrate±phosphate buffer pH5.7 at 308C. The polarisation potential of Clark oxygen electrode was ÿ650 mV vs. Ag/AgCl ref. electrode.
more soluble of ferricinium (Fe Cp 2 )-ions are formed Eq. (1). 4ÿ Fe Cp2 Fe CN6 3ÿ ! Fe Cp 2 Fe CN6 (1)
Well de®ned peaks were observed between 200 and 300 mV vs. Ag/AgCl ref. electrode in the glucose free solution (Fig. 2, curve a). The anodic and cathodic peaks responded to oxidized and reduced forms of hexacyanoferrate, however, the cyclic voltammogram is also affected by the presence of ferricinium. The course of reaction (1) was con®rmed by photometric experiments. At a wavelength of 617 nm, absorption of ferricinium was observed. No other compound of reaction (1) absorbs at this wave length. In Fig. 2, curve b, the cyclic voltammogram of this system after addition of glucose with ®nal concentration 0.1 M is shown. Glucose is converted by glucose oxidase/¯avine adenine dinucleotide (GOx/FAD) of Aspergillus niger Eq. (2). Gox
b-d-glucose FAD ! gluconolactone FADH2 (2)
The mechanism of the reaction between the reduced FAD (FADH2) and the mediator is likely as follows: FADH2 reacts with ions of Fe Cp 2 with formation of ferrocene Eq. (3). The ferrocene formed then reacts with the excess hexacyanoferrate(III) and at the anodic side of the voltammogram, the oxidation wave of formed hexacyanoferrate(II) is observed. FADH2 2Fe Cp 2 ! FAD 2H 2Fe Cp2
(3)
By comparison of curves a and b in Fig. 2, the catalytic wave at the oxidation side and loss of the peak at the reduction side of the cyclic voltammogram as a result of the reaction FADH2 with ferricinium can be observed. The calibration curve was obtained at a constant potential of 350 mV vs. Ag/AgCl ref. electrode. The rate of the oxidation of hexacyanoferrate(II) on the electrode is proportional to the glucose concentration. This dependence is shown in Fig. 3. The concentration of hexacyanoferrate(III) in 10 ml of the buffer was 0.5 mM, with addition of 100 ml of the saturated ferrocene solution in ethanol. The calibration curve was linear up to 10 mM glucose.
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Fig. 2. Cyclic voltammograms of the microbial biosensor with mediators in solution and Aspergillus niger on the surface of the platinum electrode for glucose free solution (a) and for 0.1 M glucose solution (b) Supported electrolyte 0.2 M phosphate buffer (pH 7.0), at room temperature, scan rate 5 mV/s.
Fig. 3. Dependence of the steady-state current on the concentration of glucose for the microbial biosensor with mediators ferrocene and hexacyanoferrate(III) in solution and Aspergillus niger on the surface of platinum electrode. Measured in 0.2 M phosphate buffer (pH 7.0).
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3.3. Surface-layer modified biosensor based on the carbon paste electrode A surface-layer modi®ed carbon paste microbial biosensor containing ferrocene incorporated in the carbon paste was prepared. Three types of materials for the preparation of the paste (5% (w/w) of ferrocene) were tested: commercial product Nujol, medical vaseline Alba and paraf®n oil. The electrodes (without biocatalytic layer) were tested by cyclic voltammetry. In the cases of Nujol and medical vaseline Alba, considerable differences in potential between the anodic and cathodic current peaks (250±300 mV) were measured. This means that these systems are not suf®ciently reversible. This difference was about 100 mV for the paraf®n oil based electrode and this product was used for the preparation of the sensor. Similar experiments were done for 3% (w/w) concentration of ferrocene but almost the same results were obtained. The measurements with the carbon paste microbial biosensor were carried out in hexacyanoferrate(III) free solution. In this case, only reactions (2) and (3) occur in the solution and ferrocene is detected on the
electrode. The polarisation potential was 350 mV vs. Ag/AgCl ref. electrode. At this potential, ferricinium ions are produced at the electrode surface. Before the ®rst measurement, the electrode was held at a potential of 300 mV for 30 min. After several measurements, the surface of the sensor was renewed by polishing on ®lter paper, and a new biocatalytic layer was applied for the preparation of a new biosensor. The calibration curve (a) of the sensor is shown in Fig. 4. The dynamic range of the biosensor is about 13 mM glucose, the linear range is up to 6 mM glucose. The time needed for signal stabilization after glucose addition was 90±120 s. The operational stability of the sensor was at least one month. The bioelectrode continuously immersed in 0.2 M phosphate buffer (pH7.0) at 48C with a glucose content of 0.5 mM lost about 30% of the initial sensitivity after 30 days. 3.4. Bulk modified microbial mediated biosensor In the bulk modi®ed microbial bioelectrode, all components (mediator and A. niger) are incorporated
Fig. 4. Dependence of the steady-state current on the concentration of glucose for the surface-layer modified carbon paste microbial biosensor, loading of ferrocene 5% (w/w) (a) and for the bulk modified microbial biosensor, loading of ferrocene 5% (w/w) and of Aspergillus niger 15% (w/w) (b) Measured in 0.2 M phosphate buffer (pH 7.0).
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in the electrode body mixed with the carbon powder and paraf®n oil and therefore no biocatalytic layer is present on the surface of the sensor. The sensor then can be renewed just by polishing the electrode surface on ®lter paper without a need to create a new biocatalytic layer. The preparation of the sensor is described above. The prepared bioelectrode was ®rst immersed in the buffer for 15 min to rehydrate mycelium of A. niger. The biosensor was held for 30 min at a potential of 300 mV vs. Ag/AgCl ref. electrode before the measurement. The electrochemical processes are the same as for the surface-layer modi®ed microbial carbon paste biosensor. Biosensors containing 5% and 15% (w/w) of the mycelium were tested. The sensor with 5% (w/w) of mycelium was not stable enough, the sensitivity decreased rapidly after a few measurements, the response time was more than 4 min and a considerable drift of signal was observed. The calibration curve of the sensor with 15% (w/w) of the biomass was ®rst obtained directly after rehydration and polarisation of the biosensor. The dynamic range was about 40 mM glucose, the curve was linear up to 20 mM glucose. However, the sensitivity and dynamic range of the biosensor decreased during repeated measurements. After a few measurements (3±5), the response of the sensor stabilised and it was possible to reproduce the calibration curve with a dynamic range of 15 mM glucose. This dependency (b) was not linear (Fig. 4). Non-linearity, low operational stability and decrease of both sensitivity and dynamic range of the bulk modi®ed microbial biosensor is likely due to the leakage of mediator from the surface of the biosensor, which cannot be prevented by ferrocene concentration increased up to 5%. Ferricinium ions produced by electrochemical reaction of ferrocene, are much more water soluble than ferrocene. In the case of the surface-layer modi®ed biosensor, the biocatalytic layer forms a barrier against the escape of ferricinium into the solution. Another problem could be the non-homogenous distribution of the dried mycelium in the carbon paste. The amount of biomass is so high that it might cause the loss of the paste character of the electrode if not suf®cient mixing of the paste is achieved. Due to the irreproducibility of surface refreshment, caused by
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Table 1 Surface-layer modified biosensor analyses of glucose contents in the samples by method of the standard addition of glucose c (mM)
sr (%)
29 35 39 47 62 88
2.1 1.9 2.1 0.8 0.5 1.6
c - Arithmetic mean of determined concentrations (3 measurements) of glucose. sr - Standard relative deviation (n3).
non-homogeneous distribution of the dried mycelium, operational stability of bulk modi®ed biosensor cannot be accurately evaluated. Nevertheless, according to our empirical experiences, we suppose that the mycelium inactivation inside the paste is several times slowly compared to surface-modi®ed biosensor. 3.5. Analyses of samples The surface-layer modi®ed A. niger biosensor was used for monitoring the glucose content during xanthan fermentation by Xanthomonas campestris on milk whey and hydrolysis of lactose by Lactozym1. Initially, the determination of glucose in model samples of glucose was tested. The average percentage accuracy was 2.8% for 7 samples. Then, the concentration of glucose in samples after fermentation in the presence of Xanthomonas cells and hydrolysis of lactose was determined. The determination of glucose content in real samples was carried out by standard additions. The concentration of glucose in samples was in the range 29±88 mM. Triplicate measurements were performed on 250±400 ml of sample (depending on the content of glucose) added to 10 ml of phosphate buffer (pH 7.0). The results are summarized in Table 1. 4. Conclusions We have developed a new mediator type whole cell carbon paste biosensor for determination of glucose. Three types of bioelectrodes have been tested: (i) with
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mediators (hexacyanoferrate(III) and ferrocene) in solution, (ii) surface-layer modi®ed biosensor with ferrocene incorporated into the carbon paste and (iii) bulk modi®ed biosensor with both ferrocene and mycelium of Aspergillus niger incorporated into the carbon paste. Such parameters as pH and temperature pro®le and the type of buffer were evaluated. The performance is very similar to those for the whole cell A. niger amperometric biosensor based on a Clark oxygen electrode [14]. In the case of electrodes with dissolved mediators in solution, a two-mediators system was tested. Because of the poor water solubility of ferrocene (mediator for FAD) another mediator (hexacyanoferrate(III)) was added. Ferricinium is reduced to ferrocene in the presence of FADH2 and ferrocene is oxidated by hexacyanoferrate(III) which is then oxidated on the electrode. The carbon paste bioelectrodes were prepared by incorporation of the insoluble mediator (ferrocene) into the paste and then either the surface-layer modi®cation or bulk modi®cation with A. niger was done. The surface-layer modi®ed biosensor showed better features for analytical use (linearity, sensitivity) than the bulk modi®ed microbial one. The stability of the surface-layer modi®ed bioelectrode was at least one month. For glucose determination, the following conditions should be maintained: room temperature, 0.2 M phosphate buffer, pH 7.0. These conditions ensure suf®cient sensitivity, selectivity and stability of the biosensor. The sensor was used for the determination of glucose content in milk whey samples after hydrolysis of lactose. Experiments using microorganisms incorporated into the carbon powder and a solid binding matrix (SBM) based transducer [7,21] to improve mechanical properties of the sensor are in progress.
References [1] A.M. Yacynych, Immunochem. Assays and Biosensor Technology for the 1990s , in: R.M. Nakamura, Y. Kasahara, G.A. Rechnitz (Eds.), Washington, 1992, ch. 17, p. 335. [2] A.E.G. Cass, G. Francis, H.A.O. Hill, I.J. Higgins, W.J. Aston, E.W. Plotkin, L.D.L. Scott, A.P.F. Turner, Anal. Chem. 56 (1984) 667. [3] S.D. Sprules, J.P. Hart, R. Pittson, S.A. Wring, Electroanalysis 8 (1996) 539. [4] J. Wang, Anal. Chem. 67 (1995) 487R. [5] M. Lutz, E. Burestedt, J. EmneÂus, H. LideÂn, S. Gobhadi, L. Gorton, G. Marko-Varga, Anal. Chim. Acta 305 (1995) 8. [6] J. Kulys, L. Wang, H.E. Hansen, T. Buch-Rasmussen, J. Wang, M. Ozsoz, Electroanalysis 7 (1995) 92. [7] J. SÏvorc, S. MiertusÏ, J. KatrlõÂk, M. Stred'anskyÂ, Anal. Chem. 69 (1997) 2086. [8] D. Narasaiah, U. Spohn, L. Gorton, Anal. Lett. 29 (1996) 181. [9] A. BarlõÂkovaÂ, J. SÏvorc, S. MiertusÏ, Anal. Chim. Acta 247 (1991) 83. [10] J. SÏvorc, S. MiertusÏ, A. BarlõÂkovaÂ, Anal. Chem. 62 (1990) 1628. [11] K. Takayama, T. Kurosaki, T. Ikeda, T. Nagasawa, J. Electroanal. Chem. 381 (1995) 47. [12] W.W. Kubiak, J. Wang, Anal. Chim. Acta 221 (1989) 43. [13] H. Ukeda, N. Yamamoto, M. Sawamura, H. Kusunose, Anal. Sci. 11 (1995) 941. [14] J. KatrlõÂk, J. SÏvorc, M. Rosenberg, S. MiertusÏ, Anal. Chim. Acta 331 (1996) 225. [15] L.P. Hadjipetrou, T.G. Young, M.D. Liliy, J. Gen. Microbiol. 45 (1966) 479. [16] K. Takayama, T. Kurosaki, T. Ikeda, J. Electroanal. Chem. 356 (1993) 295. [17] J. Kulys, L. Wang, V. Razumas, Electroanalysis 4 (1992) 527. [18] K. Miki, T. Tsuchida, M. Kawagoe, H. Kinoshita, T. Ikeda, Denki Kagaku 62 (1994) 1249. [19] T. KalaÂb, P. SklaÂdal, Electroanalysis 6 (1994) 1004. [20] W. Oungpipat, P.W. Alexander, P. Southwellkeely, Anal. Chim. Acta 309 (1995) 35. [21] J. SÏvorc, S. MiertusÏ, M. Stred'anskyÂ, Patent WO-A-9702359, 1995.
Mediator type of glucose microbial biosensor based on Aspergillus niger J. KatrlõÂka,c, R. BrandsÏteterb, J. SÏvorca,c, M. Rosenbergb, S. MiertusÏc,* a
b
Department of Analytical Chemistry, Slovak Technical University, RadlinskeÂho 9, 81237, Bratislava, Slovakia Department of Biochemical Technology, Slovak Technical University, RadlinskeÂho 9, 81237, Bratislava, Slovakia c POLY-tech, Area di Ricerca, Padriciano 99, 34012 Trieste, Italy Received 24 June 1997; received in revised form 2 September 1997; accepted 7 September 1997
Abstract Whole cells of Aspergillus niger CCM 8004 containing glucose oxidase (EC 1.1.3.4.) were used for the construction of an amperometric microbial mediated carbon paste biosensor. The microorganism was either placed on the surface of the electrode or incorporated directly into the carbon paste. The mediators were either dissolved in the buffer (hexacyanoferrate(III) and ferrocene) or loaded in the carbon paste (ferrocene). All methods resulted in effective glucose amperometric biosensors. The operational stability of the surface-layer modi®ed whole cell biosensor based on ferrocene incorporated into the carbon paste was at least one month, the upper linearity limit was 6 mM. The sensor was used for measuring the glucose content in real samples. # 1997 Elsevier Science B.V. Keywords: Biosensor; Amperometry; Glucose; Carbon paste; Microbial biosensor
1. Introduction The development of amperometric bioelectrodes is one of the major areas of interest in chemical sensors [1]. They are the most successful class of biosensors in widespread use. Many of them are based on the change of oxygen or hydrogen peroxide concentration during enzymatic reaction on the bioelectrode. In this case, the electrode response is dependent on the oxygen concentration in the buffer. The linearity range and the sensitivity might be improved by use of electroactive compounds, mediators, instead of oxygen. The ®rst mediated glucose biosensor based on 1,10 -dimethyl*Corresponding author. Tel.: +39 (40) 3756622; fax: +39 (40) 7797091; e-mail: [email protected] 0003-2670/97/$17.00 # 1997 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00524-2
ferrocene and glucose oxidase was presented by Cass et al. [2] in 1984. Presently, many systems that combine various types of mediators with enzymes have been described [3±8]. Some amperometric biosensors use microorganisms instead of puri®ed enzymes [9±13]. The use of microorganisms could bring some advantages which are mentioned in our previous work [14]. These whole cell sensors are usually based on the amperometric measurement of O2 consumption due to interaction between the substrate and the biocatalytic layer. It was reported in 1966 that Fe CN3ÿ 6 could be used as an electron acceptor for the metabolic oxidation of Dglucose by Escherichia coli [15]. However, the development of whole cell mediated bioelectrodes for analytical purposes has started only in the late 80's,
218
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probably due to dif®culties in combining microorganism, mediator and electrode in one system. The mediator has to be able to shuttle the electrons between the redox centres of enzymes in the microorganisms and the electrode surface, but details of the electrocatalytic reactions are not clear yet [16]. Among the whole cell mediated amperometric biosensors described, the ethanol yeast-based bioelectrode was based on the activity of alcohol dehydrogenase in yeasts and hexacyanoferrate(III) was used as an electron acceptor for enzymatically produced NADH [12]. Kulys et al. [17] have reported a mediated amperometric L-lactate biosensor using the yeast Hansenula anomala. A glucose bioelectrode based on the bacterium Gluconobacter industries and p-benzoquinone has been developed [16]. Yeast of Saccharomyces cerevisiae and Vitamin K3 were used for preparation of bioelectrode sensitive to ethanol and glucose [18]. Amperometric microbial bioelectrodes for lactate and succinate using ferrocene as a mediator were also reported [19]. Ferrocene was used as the mediator for hydrogen peroxide planttissue biosensor as well [20]. This paper describes the performance of both whole cell carbon paste electrodes for determination of glucose containing ferrocene as a mediator, and the biosensor based on dissolved mediators. Spores of Aspergillus niger were either immobilized on the surface of the electrode (platinum or carbon paste), or incorporated into the carbon paste together with the mediator (ferrocene). Both methods produced effective glucose amperometric biosensors. The preparation, characteristics and analytical performance of these electrodes are reported. 2. Experimental 2.1. Materials Glucose, fructose, sucrose, galactose, maltose, mannose, arabinose, mannitol, sorbitol and cholesterol were obtained from Sigma (St. Louis, MO, USA), Bacto-agar from Difco (USA). Lactozym1 (Novo Nordisk A/S, Bagsvaerd, Denmark, activity 3000 Lactozym units (LAU) per ml) was used for hydrolysis of lactose in whey. Other reagents were commercially available as analytical grade.
2.2. Microorganism The fungal strain Aspergillus niger CCM 8004 obtained from Czechoslovak Culture Collection of Microorganisms (Brno, Czech Rep.) was subcultured weekly on the agar medium (Czapek-Dox agar 50 g, Bacto-agar 1.5 g, distilled water to 1 l) at 288C. The cultivation medium (glucose 200 g, KCl 0.25 g, KH2PO4 0.25 g, MgSO4.7H2O 0.25 g, Ca(NO3)2.4H2O 1 g, (NH4)2SO4 1.1 g, distilled water to 1 l) was inoculated by spore suspension (2 106 spores/ml) and the culture was aerobically cultivated at 308C, pH 5.7 in the fermentor LF-2 (Vyvojove dilny CSAV, Prague, Czech Rep.). Samples for determination of the respiration activity and for determination of dry cell weight (DCW) were taken out every hour. 2.3. Determination of respiration activity and dry cell weight of the microorganism The respiration activity (RA) was determined as the moles of O2 consumed by 1 g (DCW) of biomass in 1 s. The respiration rate was measured by a Clark oxygen electrode at ÿ650 mV vs. Ag/AgCl ref. electrode. For DCW determination, a biomass sample (1± 20 cm3) was ®ltered through a dried membrane ®lter type HA, pore diameter 0.45 mm (Millipore, Bedford, MA, USA), washed with phosphate buffer pH 7.0 and dried at 508C for 6±8 h and weighed. 2.4. Preparation of the biocatalytic layer, carbon paste and sensors The cultivated mycelium with the maximum respiration activity was washed by phosphate buffer pH 7.0 and adjusted to the concentration of 5 g/l DCW. A 20 ml portion of this suspension was ®ltered through a 5 mm diameter membrane ®lter HA. The biosensor based on dissolved mediator was prepared by attachment of cultivated mycelium immobilized on the ®lter on top of the polished platinum electrode. This platinum electrode consisted of a platinum wire (diameter 1 mm) placed in a glass tube (o.d. 10 mm). The biocatalytic layer was covered with a nylon net and ®xed by a rubber O-ring. Carbon paste was prepared as follows: 0.5 g of graphite powder (Elektrokarbon Topolcany, Slovakia)
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were modi®ed by 1.5 ml of solution of ferrocene in acetonitril (®nal concentration of ferrocene in the paste was 3 or 5% (w/w)). The mixture was then activated in an ultrasonic bath for about 20 min and dried using an IR lamp. Then 0.1 ml of paraf®n oil (Slovakofarma Hlohovec, Slovakia) were then added and the ®nal paste was prepared by manual mixing. A portion of the modi®ed carbon paste was packed at the top of a 2 ml plastic syringe with the tip cut off (2 mm i.d.). A copper wire was inserted through the opposite end to establish electrical contact. The active surface of the electrode was polished with ®ne emery paper and the biocatalytic layer was attached as described above. Alternatively, the microorganism was directly incorporated into the carbon paste. The mycelium of A. niger was washed with distilled water, homogenised by vigorous shaking and dried at 378C until constant weight was reached. Then, the dry mycelium was mixed with ferrocene-modi®ed carbon powder (amount of microorganism was 5 or 15% (w/w)) before the paraf®n oil was added. After mixing, the paste was again packed into the plastic syringe and polished with emery paper. 2.5. Apparatus and procedure Electrochemical experiments were carried out on a polarographic analyzer PA-4 (Laboratorni pristroje, Prague, Czech Rep.) A three-electrode system was used with Ag/AgCl (saturated KCl) ref. electrode and a platinum wire as an auxiliary electrode. The measured current was converted into voltage and displayed on a XY 4106 recorder (Laboratorni pristroje, Prague, Czech Rep.). The determination of glucose with the microbial mediated biosensor was carried out at room temperature (208C) in a reaction vessel, equipped with a magnetic stirrer (400 rpm). The sensor was immersed in 0.2 M phosphate buffer pH7.0 (10 ml). The operational potential of the bioelectrode was 350 mV vs. ref. electrode. After the sensor current became constant, the sensor was ®rst calibrated by the addition of standard glucose solution and then used for the determination of glucose in samples. Current±time curves were recorded. The height of the recorded wave (current decrease) corresponds to the concentration of glucose.
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2.6. Hydrolysis of lactose by lactase Lactose in real samples was ®rst hydrolysed by Lactozym1. Hydrolysis was carried out at 408C in phosphate buffer pH 6.5, activity of Lactozym1 was 5 LAU/ml. Degree of hydrolysis tested on a model sample of lactose was 97% after 6 h. 3. Results and discussion 3.1. Optimization of cultivation of Aspergillus niger and working conditions of the biosensor The activity of the enzymes involved in the biocatalytic layer is one of the main factors that in¯uence the biosensor characteristics. The mycelium showing maximum respiration activity was used for biosensor preparation. The respiration activity of Aspergillus niger during cultivation is shown in Fig. 1. The increase of speci®c activity reached a maximum after 15±16 h of cultivation. The cultivation was stopped after 15 h and the biomass was treated as described above. Evaluation of the effect of such parameters as pH, temperature and type of buffer on biosensor response gave results consistent with those described in previous work [14]. Shortly, the response of the sensor was independent of pH in the range 5±8. The temperature pro®le reached a maximum at 508C. The sensitivity of the biosensor to glucose was not signi®cantly dependent on the type of tested buffers. All measurements were then performed at 308C in 0.2 M phosphate buffer, pH 7.0. 3.2. Microbial biosensor with the mediator in solution The electrochemical behaviour of the microbial biosensor based on the platinum electrode with dissolved mediator was studied by means of cyclic voltammetry. The mediator, ferrocene, was dissolved in the solution of hexacyanoferrate(III). Addition of the saturated solution of ferrocene (Fe(Cp)2) in ethanol into the buffer caused a turbidity due to precipitation of ferrocene (water solubility of ferrocene is very low). After addition of hexacyanoferrate(III) (concentration 0.5 mM), the turbidity disappeared because
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Fig. 1. Dependence of the respiration activity (RA) of mycelium Aspergillus niger on the cultivation time. Measured in citrate±phosphate buffer pH5.7 at 308C. The polarisation potential of Clark oxygen electrode was ÿ650 mV vs. Ag/AgCl ref. electrode.
more soluble of ferricinium (Fe Cp 2 )-ions are formed Eq. (1). 4ÿ Fe Cp2 Fe CN6 3ÿ ! Fe Cp 2 Fe CN6 (1)
Well de®ned peaks were observed between 200 and 300 mV vs. Ag/AgCl ref. electrode in the glucose free solution (Fig. 2, curve a). The anodic and cathodic peaks responded to oxidized and reduced forms of hexacyanoferrate, however, the cyclic voltammogram is also affected by the presence of ferricinium. The course of reaction (1) was con®rmed by photometric experiments. At a wavelength of 617 nm, absorption of ferricinium was observed. No other compound of reaction (1) absorbs at this wave length. In Fig. 2, curve b, the cyclic voltammogram of this system after addition of glucose with ®nal concentration 0.1 M is shown. Glucose is converted by glucose oxidase/¯avine adenine dinucleotide (GOx/FAD) of Aspergillus niger Eq. (2). Gox
b-d-glucose FAD ! gluconolactone FADH2 (2)
The mechanism of the reaction between the reduced FAD (FADH2) and the mediator is likely as follows: FADH2 reacts with ions of Fe Cp 2 with formation of ferrocene Eq. (3). The ferrocene formed then reacts with the excess hexacyanoferrate(III) and at the anodic side of the voltammogram, the oxidation wave of formed hexacyanoferrate(II) is observed. FADH2 2Fe Cp 2 ! FAD 2H 2Fe Cp2
(3)
By comparison of curves a and b in Fig. 2, the catalytic wave at the oxidation side and loss of the peak at the reduction side of the cyclic voltammogram as a result of the reaction FADH2 with ferricinium can be observed. The calibration curve was obtained at a constant potential of 350 mV vs. Ag/AgCl ref. electrode. The rate of the oxidation of hexacyanoferrate(II) on the electrode is proportional to the glucose concentration. This dependence is shown in Fig. 3. The concentration of hexacyanoferrate(III) in 10 ml of the buffer was 0.5 mM, with addition of 100 ml of the saturated ferrocene solution in ethanol. The calibration curve was linear up to 10 mM glucose.
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Fig. 2. Cyclic voltammograms of the microbial biosensor with mediators in solution and Aspergillus niger on the surface of the platinum electrode for glucose free solution (a) and for 0.1 M glucose solution (b) Supported electrolyte 0.2 M phosphate buffer (pH 7.0), at room temperature, scan rate 5 mV/s.
Fig. 3. Dependence of the steady-state current on the concentration of glucose for the microbial biosensor with mediators ferrocene and hexacyanoferrate(III) in solution and Aspergillus niger on the surface of platinum electrode. Measured in 0.2 M phosphate buffer (pH 7.0).
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3.3. Surface-layer modified biosensor based on the carbon paste electrode A surface-layer modi®ed carbon paste microbial biosensor containing ferrocene incorporated in the carbon paste was prepared. Three types of materials for the preparation of the paste (5% (w/w) of ferrocene) were tested: commercial product Nujol, medical vaseline Alba and paraf®n oil. The electrodes (without biocatalytic layer) were tested by cyclic voltammetry. In the cases of Nujol and medical vaseline Alba, considerable differences in potential between the anodic and cathodic current peaks (250±300 mV) were measured. This means that these systems are not suf®ciently reversible. This difference was about 100 mV for the paraf®n oil based electrode and this product was used for the preparation of the sensor. Similar experiments were done for 3% (w/w) concentration of ferrocene but almost the same results were obtained. The measurements with the carbon paste microbial biosensor were carried out in hexacyanoferrate(III) free solution. In this case, only reactions (2) and (3) occur in the solution and ferrocene is detected on the
electrode. The polarisation potential was 350 mV vs. Ag/AgCl ref. electrode. At this potential, ferricinium ions are produced at the electrode surface. Before the ®rst measurement, the electrode was held at a potential of 300 mV for 30 min. After several measurements, the surface of the sensor was renewed by polishing on ®lter paper, and a new biocatalytic layer was applied for the preparation of a new biosensor. The calibration curve (a) of the sensor is shown in Fig. 4. The dynamic range of the biosensor is about 13 mM glucose, the linear range is up to 6 mM glucose. The time needed for signal stabilization after glucose addition was 90±120 s. The operational stability of the sensor was at least one month. The bioelectrode continuously immersed in 0.2 M phosphate buffer (pH7.0) at 48C with a glucose content of 0.5 mM lost about 30% of the initial sensitivity after 30 days. 3.4. Bulk modified microbial mediated biosensor In the bulk modi®ed microbial bioelectrode, all components (mediator and A. niger) are incorporated
Fig. 4. Dependence of the steady-state current on the concentration of glucose for the surface-layer modified carbon paste microbial biosensor, loading of ferrocene 5% (w/w) (a) and for the bulk modified microbial biosensor, loading of ferrocene 5% (w/w) and of Aspergillus niger 15% (w/w) (b) Measured in 0.2 M phosphate buffer (pH 7.0).
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in the electrode body mixed with the carbon powder and paraf®n oil and therefore no biocatalytic layer is present on the surface of the sensor. The sensor then can be renewed just by polishing the electrode surface on ®lter paper without a need to create a new biocatalytic layer. The preparation of the sensor is described above. The prepared bioelectrode was ®rst immersed in the buffer for 15 min to rehydrate mycelium of A. niger. The biosensor was held for 30 min at a potential of 300 mV vs. Ag/AgCl ref. electrode before the measurement. The electrochemical processes are the same as for the surface-layer modi®ed microbial carbon paste biosensor. Biosensors containing 5% and 15% (w/w) of the mycelium were tested. The sensor with 5% (w/w) of mycelium was not stable enough, the sensitivity decreased rapidly after a few measurements, the response time was more than 4 min and a considerable drift of signal was observed. The calibration curve of the sensor with 15% (w/w) of the biomass was ®rst obtained directly after rehydration and polarisation of the biosensor. The dynamic range was about 40 mM glucose, the curve was linear up to 20 mM glucose. However, the sensitivity and dynamic range of the biosensor decreased during repeated measurements. After a few measurements (3±5), the response of the sensor stabilised and it was possible to reproduce the calibration curve with a dynamic range of 15 mM glucose. This dependency (b) was not linear (Fig. 4). Non-linearity, low operational stability and decrease of both sensitivity and dynamic range of the bulk modi®ed microbial biosensor is likely due to the leakage of mediator from the surface of the biosensor, which cannot be prevented by ferrocene concentration increased up to 5%. Ferricinium ions produced by electrochemical reaction of ferrocene, are much more water soluble than ferrocene. In the case of the surface-layer modi®ed biosensor, the biocatalytic layer forms a barrier against the escape of ferricinium into the solution. Another problem could be the non-homogenous distribution of the dried mycelium in the carbon paste. The amount of biomass is so high that it might cause the loss of the paste character of the electrode if not suf®cient mixing of the paste is achieved. Due to the irreproducibility of surface refreshment, caused by
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Table 1 Surface-layer modified biosensor analyses of glucose contents in the samples by method of the standard addition of glucose c (mM)
sr (%)
29 35 39 47 62 88
2.1 1.9 2.1 0.8 0.5 1.6
c - Arithmetic mean of determined concentrations (3 measurements) of glucose. sr - Standard relative deviation (n3).
non-homogeneous distribution of the dried mycelium, operational stability of bulk modi®ed biosensor cannot be accurately evaluated. Nevertheless, according to our empirical experiences, we suppose that the mycelium inactivation inside the paste is several times slowly compared to surface-modi®ed biosensor. 3.5. Analyses of samples The surface-layer modi®ed A. niger biosensor was used for monitoring the glucose content during xanthan fermentation by Xanthomonas campestris on milk whey and hydrolysis of lactose by Lactozym1. Initially, the determination of glucose in model samples of glucose was tested. The average percentage accuracy was 2.8% for 7 samples. Then, the concentration of glucose in samples after fermentation in the presence of Xanthomonas cells and hydrolysis of lactose was determined. The determination of glucose content in real samples was carried out by standard additions. The concentration of glucose in samples was in the range 29±88 mM. Triplicate measurements were performed on 250±400 ml of sample (depending on the content of glucose) added to 10 ml of phosphate buffer (pH 7.0). The results are summarized in Table 1. 4. Conclusions We have developed a new mediator type whole cell carbon paste biosensor for determination of glucose. Three types of bioelectrodes have been tested: (i) with
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mediators (hexacyanoferrate(III) and ferrocene) in solution, (ii) surface-layer modi®ed biosensor with ferrocene incorporated into the carbon paste and (iii) bulk modi®ed biosensor with both ferrocene and mycelium of Aspergillus niger incorporated into the carbon paste. Such parameters as pH and temperature pro®le and the type of buffer were evaluated. The performance is very similar to those for the whole cell A. niger amperometric biosensor based on a Clark oxygen electrode [14]. In the case of electrodes with dissolved mediators in solution, a two-mediators system was tested. Because of the poor water solubility of ferrocene (mediator for FAD) another mediator (hexacyanoferrate(III)) was added. Ferricinium is reduced to ferrocene in the presence of FADH2 and ferrocene is oxidated by hexacyanoferrate(III) which is then oxidated on the electrode. The carbon paste bioelectrodes were prepared by incorporation of the insoluble mediator (ferrocene) into the paste and then either the surface-layer modi®cation or bulk modi®cation with A. niger was done. The surface-layer modi®ed biosensor showed better features for analytical use (linearity, sensitivity) than the bulk modi®ed microbial one. The stability of the surface-layer modi®ed bioelectrode was at least one month. For glucose determination, the following conditions should be maintained: room temperature, 0.2 M phosphate buffer, pH 7.0. These conditions ensure suf®cient sensitivity, selectivity and stability of the biosensor. The sensor was used for the determination of glucose content in milk whey samples after hydrolysis of lactose. Experiments using microorganisms incorporated into the carbon powder and a solid binding matrix (SBM) based transducer [7,21] to improve mechanical properties of the sensor are in progress.
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