Analytica Chimica Acta 361 (1998) 4553

Biosensor for L-lactate determination as an index of E. coli number in crude culture medium
Viviane Casimiri*, Claude Burstein
Paris 7, Denis Diderot Tour 54, Laboratoire de Technologies des Enzymes et des Biomembranes Universite tage. 2, place Jussieu, 75251 Paris Cedex 05, France 5e e Received 25 July 1997; received in revised form 10 December 1997; accepted 16 December 1997

Abstract In a culture medium E. coli produces L-lactate as a metabolite. L-lactate enzyme sensors were assembled and applied to a crude E. coli growth medium for L-lactate determination. The amount of L-lactate was used as an index of the cell number. In the standard solution, L-lactate oxidase (LOD) sensor gave a linear response from 5 to 300 mM L-lactate. Determination of L-lactate in bacteria cultures of different absorbance (A) was possible from an absorbance at 600 nm, A6000.01 and higher. L-lactate measurements were not modied when the LOD electrode was associated with L-lactate dehydrogenase (LDH) in solution. In contrast, the bi-enzyme sensor consisting of LOD coimmobilized with LDH on the electrode, lowered the detection limit to 30 nM L-lactate by amplication of measurements via coupled reactions and substrate recycling. Responses were obtained in growth medium from A0.0005 (5105 bacteria ml1). In comparison, the standard spectrophotometer cell estimation was not reliable below A0.02. The L-lactate detection limit can be decreased further by incubating bacteria with glucose for 1 to 2 h. Measurements of L-lactate by oxygen consumption could be performed in the presence of bacteria and growth medium components. The recycling enzymatic electrode kept 80% of its initial activity with storage overnight in a buffer, at 48C, during 2 weeks. Such sensors permit fast and sensitive procedures for L-lactate and E. coli assays. The procedure does not disturb the developing cultures as relatively small sample volumes from 1 to 500 ml and no cells are needed for L-lactate determination. # 1998 Elsevier Science B.V.
Keywords: L-lactate biosensor; L-lactate oxidase (LOD); L-lactate dehydrogenase (LDH); L-lactate recycling; L-lactate in cell culture; E. coli determination; Oxygen electrode

1. Introduction Bacterial cultures have been used over the past decades for the study or manufacture of a large range of products. Lactate, a major metabolite of bacterial energy metabolism, is produced in high amounts during both anaerobic fermentation of glucose and
*Corresponding author. Fax: (33) 01 44 27 57 04. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00011-7

other carbohydrates, and by aerobic pathways [1]. Studies with E. coli indicate that, the lactate concentration on the two sides of the cytoplasmic membrane is regulated by energy changes through the respiratory chain or lactate transport system that respond to glycolytic activity [24]. Several cell types have energy requirements corresponding to their proliferation rate [57]. Under low growth rate conditions, Llactate from lactose transformation remains the essen-

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tial metabolite of lactic fermentation of Lactococcus lactis [8]. The metabolite is mainly produced in a growth phase dependent mode in Streptococci A and C [9]. From these and other studies [10], the lactate excreted in the medium by a bacterial metabolism was associated with bacterial growth conditions and growth phases. Despite considerable work that has been done on lactate generation and control, analytical systems suitable for reliable and fast detection in biological media are still required. There has been increasing interest in the eld of biosensors for lactate analysis [7]. Biochemical immobilization of enzymes, stabilizes their activity and allows specic analysis; however, complex processes for immobilization or measurement may restrict their utilization for lactate evaluation [11,12]. L-lactate oxidase (LOD), immobilized alone, or coimmobilized with L-lactate dehydrogenase (LDH), has provided specic analysis via lactate oxidation and oxygen consumption, at low substrate concentrations [1214]. Apart from the complexity of bioprocesses, in cell population analysis, several problems inherent to the nature of the bacteria and culture conditions exist. Conventional microbiological methods, such as, plate counting techniques require up to 2 weeks for bacterial detection [15]. Absorbance evaluation is not reliable at low bacterial concentration [16], and the presence of numerous compounds in biological media may interfere with the analysis. Other studies tried to establish relationships between bacterial density [17], and a cellular compound as chemical marker, including ATP [18,19], to improve the sensitivity. The applications often require preliminary preparation or are not suitable for analysis in complex solutions. Antibody-based immunoassays rely on several parameters and on antibody availability. Compared with chemical determinations, biosensor analysis has the possibility for selective, sensitive and simple procedures for analysis in complex solutions, as required in different elds of biotechnology. In this work, L-lactate excreted by E. coli was studied with enzymatic L-lactate biosensor and amperometric monitoring of oxygen consumed in the reactions, for sensitive and convenient determinations in crude culture medium. To study media with low substrate concentration, enzymatic amplication of L-lactate

determination by recycling was used. The L-lactate concentration measured in growth medium was used as a parameter of cell density. 2. Experimental 2.1. Reagents LOD from Pediococcus species, LDH from rabbit muscle (EC 1.1.1.27), glucose oxidase from Aspergillus niger (EC 1.1.3.4), and substrates were supplied by Sigma. 2.2. Enzyme immobilization Immobilization of enzymes LOD and LDH on covalent binding membranes (UltraBind US-450 from Gelman Sciences Ann Arbor, USA) followed basically the immobilization procedure previously described [13,14]. The membrane presents aldehyde groups that react covalently with the accessible amino groups of the protein (NH2 at the " position of lysine). The oxygen electrode was covered with one layer of a hydrophobic polypropylene gas permeable lm and by the binding membrane. An aliquot of 3 ml of enzyme solution at 48C containing 2 IU LOD or a mixture of 2 IU LOD and 1 IU LDH, in 0.05 M phosphate buffer, pH 7.0, were dropped on the membrane and dried rapidly under a stream of cold air. Further treatment during 2 min with 0.05% glutaraldehyde in cold distilled water increased cross-linking of the enzymes. The enzyme electrodes thus prepared were washed twice for 2 min in a working buffer, 0.1 M sodium phosphate, pH 7.0. The amount of enzyme remaining active after immobilization was 1.8 IU LOD and 0.4 IU LDH. The immobilization procedure can be performed in less than 10 min and the sensor is functional 30 min after equilibration in stirred buffer. When not used, the enzyme membranes were stored in the buffer at 48C. 2.3. Apparatus and oxygen consumption measurement Variations of oxygen partial pressure during reactions were measured amperometrically at 0.7 V

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using a Clark electrode (silver anode and platinum cathode) of 1 cm diameter with an active surface area of 0.2 mm diameter. 100% oxygen was adjusted on the recorder with the buffer saturated with air, which has an oxygen concentration of 240 mM at 308C [13]. In order to adjust rapidly the zero oxygen, 1 IU of glucose oxidase and a saturated glucose solution (100 mM) were used to obtain the maximal velocity [14]. Assays were performed in a 2 ml electrochemical cell, the solution being constantly mixed and oxygenated with a magnetic stirrer. The current from the electrode was recorded on a Kipp and Zonen BD 111 recorder from 2.5 to 10 mV. Two nA correspond to 1 nmol of oxygen. Results obtained from different sensors were similar. The mean values evaluated from 57 measurements for each sensor differed in less than 5%. 2.4. L-lactate assay procedures The enzyme electrode was immersed in 2 ml of 0.1 M phosphate buffer, pH 7.0, at 308C in the cell. Assays were started by adding different concentrations of substrates. After each assay, the enzyme electrode was washed 3 times in the mixing cell with 2 ml buffer for 2 min to ensure oxygen saturation. 2.5. E. coli preparation E. coli (strain GC44 68) cultures were grown in 100 ml minimal synthetic medium no. 863 (from J. Monod Institut, Paris 5, France) with 0.4% glucose, pH 7.0, in current use for E. coli cultures [20], and agitated in a shaking water bath at 378C. E. coli cells were harvested during the exponential growth phase, and the crude suspension used directly or centrifuged 2 min at 10.000 RPM to separate bacteria. The different preparations, supernatant, pellet and suspended bacteria in culture media could be kept frozen at 808C in 17% glycerol until used. Spectrophotometric measurements of bacterial density were performed at absorbance (A)600 nm. A6001.0 is considered in the literature to correspond to a concentration of 1109 E. coli ml1 cultured under similar conditions [20].

2.6. L-lactate determination in E. coli culture medium Cell suspension or growth medium supernatant were warmed to 308C just before assays. Appropriate amounts were added to the sensor chamber and measurements made as described above. 2.7. Selectivity 2.7.1. Bacterial respiration When monitoring bacterial suspensions, endogenous bacterial respiration in concentrated culture through electroactive processes may interfere with L-lactate measurements. In these circumstances, endogenous bacterial O2 consumption was evaluated with the electrode free of enzymes before experimentation. Growth medium clear of bacteria gave no signal or a very low signal. Bacterial respiration was subtracted from measurements, when necessary. With samples diluted below A0.05, endogenous respiratory activity was under 5% of L-lactate oxidation, which did not signicantly inuence the measurements. 2.7.2. Medium components Culture medium components may induce relatively low unspecic oxidations, also seen in studies with carbon paste electrodes [21] in the presence or absence of enzymes. In other systems with microorganisms [22], interferences from components such as salts may have to be determined by separate measurements. In our assays, dilution of the samples below A0.25 eliminated interfering signals from medium components. 2.7.3. Sample volume Determinations with different volumes of buffer, crude cell suspension or supernatant indicated that, additions above 500 ml in the 2 ml chamber create disturbances in measurements. Prior oxygenation of samples above a volume of 50 ml eliminates differences in oxygen content between buffer and sample. 3. Results and discussion
L-lactate produced by E. coli metabolism in culture medium was determined with different sensors and

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used as a parameter of cell density. The different approaches for L-lactate measurements were based on LOD immobilized at the electrode surface; for low concentrations of substrate, immobilized LOD was associated with LDH on the sensor membrane, or with LDH free in solution. Possible interfering oxidations were measured with sensors made with or without enzymes on the membrane. 3.1. Bacterial concentration determined with optical density The limits of A600 measurements on crude bacterial suspensions were determined. As seen in Fig. 1, in our conditions spectrophotometer cell estimations were not reliable under A0.02. Values were reproducible within 5% of the mean value. It is well documented in the literature that A1.0 estimated in similar cultures conditions correspond approximately to 1109 bacteria ml1 [20]. Therefore, it was assumed that spectrophotometric cell estimation was not possible below 2107 bacteria ml1 in the present experiments. Furthermore, the absorbance method requires transparent and dened media to get accurate results.

3.2. L-lactate oxidase biosensor In the L-lactate biosensor based on immobilized LOD, the enzyme catalyses the reaction:
l-LOD l-lactate O2 ! pyruvate H2 O2

Fig. 2 shows a typical calibration curve for this biosensor. The system has micromolar sensitivity for L-lactate. The linear range was between 5 and 300 mM, with a correlation coefcient of 0.997. The sensor stability, the immobilization efciency and the sensitivity of determination depend on several factors. Variations of parameters already considered in earlier work [14,23], shows that the enzyme concentration determines the sensor sensitivity. 3.3. L-lactate in bacterial culture medium with the LOD sensor
L-lactate

The LOD sensor described above was used to detect in E. coli culture medium. Results indicated the possibility to measure L-lactate excreted in the

Fig. 1. Evaluation of E. coli number with a spectrophotometer at 600 nm.

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Fig. 2. Calibration graph of the LOD sensor. The signal is provided by O2 consumed in the reaction chamber after mMolar L-lactate addition. Inset: response to mMM L-lactate concentrations.

turbid growth medium, in the presence of suspended E. coli, in contrast to spectrophotometric L-Lactate determinations [7]. The sensor applied to crude suspensions of various absorbances and from different batches showed with increasing dilutions a linear decrease of O2 consumed per minute, from A0.25 to the limit of A0.01 (107 bacteria ml1) (Fig. 3). Values were statistically signicant at each cell density. The detection limit measured correspond to 5 mM L-lactate. In the literature, L-lactate in cell culture medium was associated with cell proliferation in studies of different types of cell [59]. As seen above, cell dependent responses with L-lactate were also obtained under our conditions. Therefore, the present LOD sensor can be used to assay bacteria by correspondence to the L-lactate concentration in culture medium. Determinations of L-lactate carried out in both bacterial suspensions below A0.05, and the corresponding growth medium without bacteria, seen in Fig. 3, were found to be similar. Consequently, the procedure gives the interesting possibility of rapid

Fig. 3. &: O2 consumed by L-lactate oxidation of E. coli suspended in crude culture medium determined with the LOD sensor. Bacterial suspensions from different batches at various absorbances, were diluted with buffer, then added to the reaction chamber. : Results obtained with growth medium deprived of bacteria by centrifugation.

bacterial number determinations without harvesting the cultured cells.

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3.4. L-lactate oxidase/L-lactate dehydrogenase sensor 3.4.1. Coimmobilized enzymes One way to increase the sensitivity of the L-lactate biosensor with LOD is to coimmobilize the enzyme LDH on the electrode [11,13,14,24]. L-lactate is recycled between the two enzymes according to the coupled reactions:
l-LOD l-lactate O2 ! Pyruvate H2 O2 l-LDH Pyruvate NADH H ! l-lactate NAD

The sensitivity provided by substrate regeneration is revealed via the oxidase reaction. In our system, the recycling of a low amount of L-lactate by enzymatic reactions leads to amplication of the signal as well as to its persistence [13,14]. L-lactate recycling is initiated by NADH. The responses were dependent on the NADH concentration. With coimmobilized LODLDH and 10 mM pyruvate, the apparent Km obtained for NADH was 1 mM. Interfering oxidation induced by NADH may disturb low substrate measurements. This was overcome by rst injecting the NADH to allow disappearance of the interfering signal and establishment of the base-line before substrate addition. With 0.5 mM NADH, a good increase in the L-lactate response was recorded while direct electrochemical interfering oxidation was reduced. The bi-enzyme electrode allowed an L-lactate concentration as low as 30 nM to be measured, and a linear response up to 1 mM was obtained. Regression analysis gave a correlation coefcient of 0.996. Limits of the detection are shown in Fig. 4. As observed in Table 1(C), coimmobilization of enzymes provided a measurement of analyte in the nanomolar range with

Fig. 4. Calibration graph of the coimmobilized LODLDH sensor. Response to nM L-lactate concentrations.

at least 100-fold amplication of sensitivity over the LOD electrodes (Table 1(A) and (B)). Several parameters are involved for high enzymatic amplication of L-lactate measurements. With coimmobilized LOD and LDH, variations of the limit of detection were essentially subject to the enzyme ratio sensed by the electrode [14,26]. High enzyme loading increases the sensor signal and stability in the present system [14], however, selectivity may be reduced [2426] in comparison with lower enzyme loading. From these studies, enzyme concentrations and ratio for sensitive measurements and low background were selected. 3.4.2. Study with LDH in solution Our preliminary studies on recycling L-lactate were carried out with LOD immobilized on the electrode and LDH in solution in the reaction chamber. The data gave a linear response from 3 to 200 mM for L-lactate

Table 1 Characteristics of L-lactate sensors using immobilized LOD, alone (A), or coupled to LDH in solution (B), or coimmobilized to LDH on the electrode (C) Enzymes Immobilized LOD (A) Immobilized LOD free LDH (B) Coimmobilized LODLDH (C) Detection limit (mM) 5.0 3.0 0.03 Linearity up to (mM) 300 200 1.0 Apparent Km for L-lactate (mM) 1200 700 3.0

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Fig. 5. O2 consumed by recycling L-lactate from E. coli in crude culture medium vs. absorbance and measured by analyte recycling with a coimmobilized LODLDH sensor. NADH cofactor 0.5 mM, then micro-samples of bacteria suspension from different calculated absorbance were added in the reaction chamber. Inset: O2 limit of detection.

determination (Table 1(B)). Compared to the sensor with only LOD (Table 1(A)), the slight increase in sensitivity was not signicant. The sensitivity of determination was not amplied as observed with coimmobilization of LOD and LDH on the electrode (Table 1(C)). However, coupled reactions and the presence of a recycling system were indicated by the response to pyruvate obtained with NADH and the occurrence of a persisting signal. Limited transport of free enzyme to the electrode or changes in enzyme catalytic activity with immobilization may account for the difference in sensitivity. 3.4.3. Stability In continuous use with 20 assays per day, the enzymatic electrode with coimmobilized LODLDH remained at 80% of its initial activity for 2 weeks, when stored overnight in buffer at 48C. In this type of sensors, stability is essentially dependent upon enzyme inactivation or release [7]. High enzyme immobilization stabilizes similar LODLDH systems for more than 2 months [14]. With storage at room temperature, the biosensor response was stable for

24 h, then lost 50% of its activity, the following 24 h by decrease of the LDH response. 3.5. L-lactate in E. coli culture medium with the coimmobilized LOD LDH sensor The sensor was used for sensitive determination of concentration to assay dilute bacterial suspensions. The method is based on the amplied detection of L-lactate produced by bacteria metabolism and signaled by the O2 consumed in enzyme-substrate reactions. The cell number is obtained by correspondence of cell L-lactate with cell number calibration curve. As shown in Fig. 5, a cell number dependent response was obtained. The electrode response was linear up to A0.010, with a signicant increase in values with absorbance in the linear range. The limit for detectable O2 consumption by reactions was obtained with samples at A0.0005 (5105 bacteria ml1). This corresponds to a 40-fold decrease in the limit of bacterial detection, when compared to spectrophotometric evaluation. As little as 1 ml from
L-lactate

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Table 2 Effect of glucose addition on measurement of L-lactate produced by bacteria in the limit of detection a Incubation (308C) h 0 1 2
a

4. Conclusion Accurate L-lactate determination in biological uid is of importance in various elds of research including medicine, food processing-chemistry and environment. E. coli produces L-lactate in signicant amounts during both aerobic and anaerobic glycolysis [1,10]. The system described in this paper, may meet the requirements for sensitive and convenient monitoring of the L-lactate produced by bacteria. It allows determinations in a large range of L-lactate concentration, from nanomolar to millimolar in the growth medium. L-lactate may be considered as a parameter of E. coli assaying to detect from 5105 bacteria ml1 (A0.0005) or lower, if using prior incubations with glucose. Standard spectrophotometric measurements have a limit at A0.02. A difculty in culture medium analysis is the presence of a variety of potentially interfering compounds. This method with determinations via oxygen pressure is compatible with the presence of several excreted products in medium and turbidity of samples. Moreover, errors of the system would be reduced as only microsamples are needed at bacterial concentration higher than A0.001. Growth media from which E. coli has been removed, gave results similar to the original crude suspension at absorbance below 0.05. As a consequence, the sensor permits L-lactate assay and cell number evaluation without disturbance of different types of developing cultures, in particular eukaryotic cell monolayers. Furthermore, it is possible to work with culture medium from relatively high bacterial concentrations, without interferences of respiratory oxidations. The procedure is fast with high sensitivity at low concentration of L-lactate and bacteria. It may be of interest when studying the growth of microorganisms, animal or plant cells, to associate with standard evaluations, L-lactate determinations in the medium.

Glucose added (%) nmol O2 min1 0.01 0 0 12 1 0 42 72

Bacteria absorbance0.0005 (diluted cultures).

A1.0 bacterial growth medium was sufcient to perform the measurements at the limit of detection level. Table 2 shows the possibility to decrease further the L-lactate limit of detection by incubating before measurement, bacteria in media with glucose. Bacteria at concentration in the detection limit range were suspended and incubated in culture media deprived of glucose. In these conditions, L-lactate was not detectable up to 2 h. Glucose addition increases the electrode response by increasing both bacteria metabolism and L-lactate produced in the media [1]. O2 consumption by enzymatic oxidation reactions of L-lactate was enhanced and detectable by the sensor. This procedure may facilitate correlation between L-lactate and E. coli, when working in the detection limit range. Assays with the recycling system may be characterized by the reactivity to both substrates, L-lactate and pyruvate. The presence of NADH increases L-lactate measurements and permits a response to pyruvate. In some particular aerobic processes, oxidized products of substrate catabolism such as pyruvate may accumulate in the medium [1,10]. In circumstances, where both substrates occur in culture medium, the sensor has the ability to measure their total concentration. For specic assays, L-lactate could be determined with the sensor after pyruvate removal by different simple procedures such as enzymatic treatment [27,13]. Another possibility is to assay micromolar pyruvate concentrations, with a pyruvate oxidase biosensor [28]. Using properties of the present LODLDH sensor, an alternative is to measure L-lactate in the micromolar range via the oxidase or L-lactate with pyruvate at nanomolar level through the coupled enzymes, depending of NADH addition.

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