Biosensors & Bioelectronics 16 (2001) 1001 1007 www.elsevier.

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Analysis of ethanolglucose mixtures by two microbial sensors: application of chemometrics and articial neural networks for data processing
Alexei V. Lobanov a, Ivan A. Borisov a, Sherald H. Gordon b, Richard V. Greene c, Timothy D. Leathers b,*, Anatoly N. Reshetilov d
b a Chair of Biotechnology and En6ironmental Protection, Pushchino State Uni6ersity, Pushchino, Moscow Region 142290, Russia Biopolymer Research Unit, National Center for Agricultural Utilization Research, USDA, ARS, 1815 North Uni6ersity Street, Peoria, IL 61604, USA c Ofce of International Programs, ARS, USDA, 5601 Sunnyside A6enue, Belts6ille, MD 20705, USA d G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia

Received 6 July 2000; received in revised form 28 March 2001; accepted 10 April 2001

Abstract Although biosensors based on whole microbial cells have many advantages in terms of convenience, cost and durability, a major limitation of these sensors is often their inability to distinguish between different substrates of interest. This paper demonstrates that it is possible to use sensors entirely based upon whole microbial cells to selectively measure ethanol and glucose in mixtures. Amperometric sensors were constructed using immobilized cells of either Gluconobacter oxydans or Pichia methanolica. The bacterial cells of G. oxydans were sensitive to both substrates, while the yeast cells of P. methanolica oxidized only ethanol. Using chemometric principles of polynomial approximation, data from both of these sensors were processed to provide accurate estimates of glucose and ethanol over a concentration range of 1.0 8.0 mM (coefcients of determination, R 2 = 0.99 for ethanol and 0.98 for glucose). When data were processed using an articial neural network, glucose and ethanol were accurately estimated over a range of 1.010.0 mM (R 2 =0.99 for both substrates). The described methodology extends the sphere of utility for microbial sensors. Published by Elsevier Science B.V.
Keywords: Amperometric microbial sensor; Articial neural network; Chemometrics; Ethanol; Glucose; Selectivity

1. Introduction

1.1. Selecti6ity of biosensors

Soon after biosensors appeared in the 1960s, efforts began to improve their characteristics. One of the major remaining problems is to provide highly selective analyses (Riedel et al., 1989). As a general rule, one sensor is used to determine the concentration of one substrate
Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. * Corresponding author. Tel.: + 1-309-681-6377; fax: + 1-309-6816689. E-mail address: leathetd@mail.ncaur.usda.gov (T.D. Leathers). 0956-5663/01/$ - see front matter Published by Elsevier Science B.V. PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 2 4 6 - 9


contained within a sample (the scheme one sensor one substrate). In this approach, wide substrate specicity, or low selectivity, appreciably restricts practical applications (Turner et al., 1987). The problem of low selectivity is particularly prevalent in sensors based upon whole cells, organelles, or tissue cuts and signicantly compromises the advantages of these sensors. However, low selectivity can also be a problem for enzyme electrodes and, in some cases, for immunosensors as well (Smolander et al., 1992, 1993; Weller et al., 1998). Numerous attempts have been made to increase the specicity of a single sensor. Approaches have included substrate adaptation, the use of selective membranes, screening for superior organisms and mutant isolation (Racek, 1995). In some cases, these methods have led to a satisfactory solution of the problem.

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However, a new approach pertaining to the problem of low selectivity has recently emerged, in which a set of low-selectivity sensors are used in concert to provide a highly selective analysis (the scheme N sensorsM substances). A prominent example of this strategy is articial tongue/nose systems (Vlasov et al., 1997; Ziegler et al., 1998; Bessant and Saini, 1999). Such analyzers consist of low-selectivity biological and/or chemical detectors and a signal processing system. In this scheme, it is possible to estimate the ratio of mixture components and/or their actual concentrations by using principles of chemometrics or articial neural networks. In practice, multidimensional calibration dependencies are rst obtained for mixtures of the analytes in different ratios; then the ratios of components, or the concentrations of selected components, are estimated from a complex of signals using chemometric principles. These theoretical principles were developed for low-selectivity chemical sensors (Schierbaum et al., 1990; Weimar et al., 1990, 1991; Slama et al., 1996).

a way that the vector of output signals (O%) maximally corresponds to the vector O. The action of the neural network is determined not only by neuron properties and weights of connections between them, but also by net topology, i.e. the relative positions of neurons. The development of a particular training algorithm, called the delta rule of error back propagation (Werbos, 1974; McClelland and Rumelhart, 1988), has made multilayer feed forward networks the most popular type (Fig. 1b).

1.3. Aim of the study

We previously showed that a low-selectivity wholecell biosensor could be used in conjunction with an enzyme electrode to effectively estimate ethanol content in ethanolglucose mixtures (Reshetilov et al., 1998). The parameters of these sensors were characterized and optimal measuring conditions were reported (Reshetilov et al., 1998). The current work was designed to further develop this approach. Specically, the potential for differential analysis of ethanol glucose mixtures using two whole-cell microbial sensors was investigated. Sensors based upon bacterial cells of Gluconobacter oxydans, known to be highly sensitive to both ethanol and glucose, were constructed. Similarly, sensors based on yeast cells of Pichia methanolica, known to be highly sensitive to ethanol and not to glucose, were also constructed. Chemometric principles and an articial neural network were then used to process signals from these biosensors. The reliability and accuracy of the methodology are described in this paper.

1.2. Biosensors and the concept of articial neural networks

Articial neural networks represent a comparatively new trend in the study of articial intelligence. They are, in effect, mathematical models of biological neural systems. Computation strategies based on neural models facilitate the solution of complex problems that require a great deal of time and calculation when solved by traditional methods. In this regard, biosensor response functions are often characterized by signicant deviations from linearity, requiring complex mathematical descriptions. Accordingly, coupling biosensors with articial neural networks is growing in importance as a tool for multicomponent analyses (Hanaki et al., 1996; Ping and Jun, 1996; Vlasov et al., 1997; Ziegler et al., 1998). While an articial neural network provides a non-linear approach that needs no a priori knowledge of functional dependencies, it does require training. Training or learning is based upon cumulative experimental data. Neural networks consist of simple processing elements or neurons linked with each other in a particular conguration (Fig. 1a). Each neuron is a non-linear transducer of input signals. Input signals (Xi ) are given weight coefcients (Wi ), summed and transferred to a non-linear function of activation (transfer function, F) that forms an output signal (Y). Training of the network then consists of the adjustment of the weight coefcients of input neuron signals. Values of the vector of input signals (I) and the vector of desired output signals (O) are presented to the network. Weight coefcients are chosen in such

Fig. 1. Conceptual design of an articial neural network. (a) Structure of a single neuron. (b) Structure of a three-layer feed forward network.

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2. Materials and methods

2.1. Microorganism strains and their culti6ation

G. oxydans strain B-1280 and P. methanolica strain Y-2621 were obtained from the All-Russian Collection of Microorganisms, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences. G. oxydans was cultured on a medium containing 100 g/l sorbitol and 10 g/l yeast extract (Difco, Detroit, MI) at pH 6.0. Growth was monitored by optical density. Cells from early stationary phase cultures (16 18 h) were harvested by centrifugation (3000 g, 15 min), washed twice with a sterile physiological solution and immediately immobilized in receptors. P. methanolica cells were initially grown in a medium containing 10 g/l glucose, 1.0% (v/v) methanol, 5.0 g/l yeast extract (Difco, Detroit, MI), 1.0 g/l KH2PO4, 3.5 g/l (NH4)2SO4, 0.5 g/l MgSO47H2O, 0.1 g/l CaCl2, 40 mg/l adenine, 40 mg/l arginine and 20 mg/l methionine. When cultures reached 0.5 1.0 mg/ml (wet wt.), cells were harvested by centrifugation, washed and then placed in fresh medium containing 1.0% ethanol in place of glucose. After an additional 8 10 h of incubation at 30 C, cells were harvested and immediately immobilized in receptors.

rate of change of electrode current was used in further calculations as sensor response. Simultaneously, analog signals were registered on a two-coordinate recorder (H-307/1, ZIP, Russia). All measurements were made at 20 C in a continuously stirred open cuvette with a working volume of 5.0 ml. The working solution was 20 mM potassium-phosphate buffer, pH 7.0. Assays were initiated by the introduction of the sample (50 ml) into the cuvette. The measuring time for a single sample was no more than 2 min. After each measurement, the electrode was washed with buffer for 1015 min until oxygen concentrations were restored to initial levels.

2.4. Chemometric approach 2.4.1. Calibration surfaces An unambiguous description of a sample consisting of m components requires either direct knowledge of the concentrations of all m components, or an indirect expression of these values, such as the actual concentration value of one substrate and the ratio of concentrations of all substrates. Objects characterized by several values can be mathematically described as vectors. Thus, each sample will correspond to a vector with coordinates equal to the concentration of components comprising the sample. The vector is graphically depicted as a line segment in m-dimensional space connecting the origin of coordinates with a point whose coordinates are equal to the vector components. For a mixture of two components, the locus of all possible concentrations forms a plane (i.e. a two-dimensional space), called the concentration plane. A point on this plane represents each particular sample. The calibration dependence is a surface in three-dimensional space, with the concentration values of constituent components as abscissa and ordinate and the sensor response value as applicate. Since the overall sensor response to a sample represents the sum total of individual responses to each constituent substrate, there may be equivalent overall responses to samples containing substrates in different ratios. A curved line that connects all points corresponding to different samples inducing an identical sensor response value is called an isocline. An isocline is thus the projection to the plane of substrate concentrations of the curved line formed by a sensor response plane intersecting the calibration surface. Individual component concentrations can be determined using the isoclines derived from two different sensors. The point at which these isoclines intersect on the concentration plane describes the concentration values for each component. 2.4.2. Building of calibration surfaces Amperometric sensors based upon whole cells of G. oxydans were previously described as being sensitive to

2.2. Cell immobilization

Receptor elements for both types of biosensors were formed by immobilizing cells by adsorption onto chromatographic paper (Whatman GF/A, UK). A 5 ml portion of a cell suspension (80 mg/ml dry wt.) were applied to the paper surface with a micropipet and dried for 20 min. The receptor element was then placed onto the measuring surface of a Clark-type amperometric electrode and was xed utilizing nylon netting.

2.3. Measurements
An industrially manufactured amperometric transducer (Ingold 531-04, Ingold Mettler-Toledo, Wilmington, MA) was coupled to the microbial biosensor. Sensor signals were then amplied in conjunction with a built-in lter for noise suppression (U7-1, ZIP, Russia) and analog signals were directly converted to digital form by an ADD device (ADDA-12, Flytech Technology, Taipei, Taiwan). Digital signals were processed on a personal computer using the program Sensor for Windows developed by the authors. The program recorded sensor responses, performed signal preprocessing (smoothing, removal of signal peak outbursts and zero drift), calculated signal parameters (amplitude and rate of change), built calibration dependencies and calculated substrate concentrations. The

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3. To simplify the mathematical processing, calibration surfaces were approximated by piecewise or other dependencies (Table 1). 4. The responses of both sensors to the unknown sample were measured. 5. The possible ratios of substrate concentrations in the sample were determined by the calibration dependence and sensor response value (described in more detail in Reshetilov et al., 1998). 6. The nal estimated substrate values were determined as those congruent for both sensors. Sensors suffered from slight losses in response values over time. This was overcome by normalization to a control sample (10 mM glucose and 10 mM ethanol).

2.5. Articial neural networks

There are several variations on the back propagation algorithm. In batch back propagation, the weights are changed after presentation of all patterns of the training set. However, in the case of large training samples, the network may be adjusted more rapidly by incorporating a weight change upon presentation of every forward and backward pass of the network. Other frequently used training methods include Quickprop (Fahlman, 1988) and the method of resilient back propagation (Rprop) (Riedmiller and Braun, 1993). Rprop has a number of advantages over other training methods. Rprop institutes a change in weight at every change of the sign of a partial derivative of the error function of the corresponding weight of connection (Wij ) between neurons i and j. The value of the partial

Fig. 2. Calibration surface for the sensor based on Gluconobacter oxydans cells, showing the normalized response (nA/s) to mixtures of glucose and ethanol over the range of 0.0 10.0 mM. Interpolation was performed using a polynomial of the second degree.

both glucose and ethanol, resulting from highly active aldose- and alcohol dehydrogenases in the cytoplasmic membrane (Kitagawa et al., 1987; Smolander et al., 1993). Sensors based upon whole cells of P. methanolica are sensitive to ethanol but not to glucose (Morozova et al., 1996). Although it would have been sufcient to calibrate the P. methanolica sensor using only ethanol standards, for convenience, a single set of glucose ethanol mixtures was used to calibrate both the P. methanolica and G. oxydans sensors. The sensors were calibrated over a range of substrate concentrations from 0.0 to 10.0 mM, for both glucose and ethanol. Preliminary studies showed that saturating substrate levels did not impede the subsequent analysis of data. The calibration surface for the response of the G. oxydans sensor to glucose and ethanol is shown in Fig. 2. The response of the P. methanolica sensor to ethanol is shown in Fig. 3. As noted, the latter sensor exhibits no response to glucose.

2.4.3. Chemometric determination of mixture component concentrations Chemometric principles were used to estimate the concentrations of substrates in samples by the following scheme: 1. Both sensors were calibrated as described in Section 2.4.2. 2. A calibration surface reecting the dependence of the obtained response value on the concentrations of substrates in a sample was built for each sensor (Figs. 2 and 3).

Fig. 3. Calibration surface for the sensor based on Pichia methanolica cells, showing the normalized response (nA/s) to ethanol over the range of 0.0 10.0 mM, in the presence or absence of glucose.

A.V. Lobano6 et al. / Biosensors & Bioelectronics 16 () 10011007 Table 1 Approximation function coefcients Parameter A gl oxydans G. A et oxydans G.
et B gl, oxydans G.

1005

K gl oxydans G.

K et oxydans G.

R 0 oxydans G.

K et methanolica P.

R P. methanolic a0 0.016

Value

0.003

0.008

0.001

0.082

0.128

0.096

0.025

derivative itself is not taken into account, avoiding the problem of blurred adaptivity. Compared with the gradient descent algorithm, weight coefcients are changed evenly throughout the network, independent of the distance to the output layer. Another important advantage of this method is the algorithm stability in relation to the choice of training parameters. The choice of training rate and momentum parameter is often of critical importance when using standard back propagation. In practice, Rprop generally provides better solutions for most problems, with fewer training cycles. The Stuttgart Neural Network Simulator ver. 4.1 (SNNS Group, IPVR, University of Stuttgart) was used to create an articial neural network. Calculations were performed on a personal computer based on a Pentium 233MHz processor. Different learning rules (Rprop, Quickprop, Backprop) and transfer functions of activation (sigmoid, binary) were tested. A neural network with one internal layer was used. 3. Results and discussion

RG. oxydans = A gl oxydans[gl]2 + A et oxydans[et]2 G. G.

et + B gl, oxydans[gl][et]+ K gl oxydans[gl] G. G.

+ K et oxydans[et]+R 0 oxydans G. G. where RG. oxydans is sensor response value; K gl oxydans and G. K et oxydans are sensor sensitivities to glucose and ethaG. nol, respectively; [gl] and [et] are glucose and ethanol concentrations in the analyzed sample; A gl oxydans and G. A et oxydans are coefcients reecting the second order G. non-linearity of the sensor response dependence on substrate concentration; B gl,et G. oxydans is a parameter showing the degree of interaction of glucose and ethanol effects on the sensor response value; R 0 oxydans is the G. sensor response value in the absence of both substrates. The solution of the rst degree equation is substituted into the second degree equation, which is then solved by nding the root of the second degree polynomial. It should be noted that the polynomial of the second degree restricts the useful range of this analysis. For example, due to a nonzero value of the parameter R 0 oxydans, the approximation gives large errors with G. low concentrations (B 0.5 mM) of either substrate. Furthermore, the useful range must be restricted to substrate concentrations that do not result in two possible values of component concentrations. In this case, this restriction means that glucose concentrations must be B 8 mM. The values of parameters obtained are given in Table 1.

3.1. Approximation of calibration surfaces and determination of mixture component concentrations

Calibration surfaces were approximated with polynomials of different degrees in order to simplify the determination of mixture component concentrations. The simplest subprograms for nding the roots of polynomials were used for building isoclines, in place of complex mathematical programs. The calibration dependence for the sensor based upon the strain P. methanolica (Fig. 3) was adequately described by the following equation: RP. methanolica = K et methanolica[et]+ R 0 methanolica, P. P. where RP. methanolica is the sensor response value, K et methanolica is the sensor sensitivity to ethanol, [et] is P. the ethanol concentration in the analyzed sample, and R 0 methanolica is the sensor response value in the absence P. of ethanol. Effects of the second order (dependence of the sensor response value on the square of the ethanol concentration) can be ignored in describing the calibration surface. The calibration surface for the sensor based upon the strain G. oxydans (Fig. 2) was described by the following polynomial of the second degree:

3.2. Determination of mixture component concentrations using articial neural networks

A neural network with one internal layer and sigmoid transfer functions of activation was used in this study. Analytical signals of the two biosensors, RG. oxydans and RP. methanolica, arrived at the input layer. The third input signal T, characterizing the time from the start of measurement, was used to increase the accuracy of determination. Network outputs were values of glucose and ethanol concentrations normalized by the formula Cnorm = C/Cmax. The best results were achieved with Rprop training (Riedmiller and Braun, 1993). Fig. 4 shows examples of dependencies of the sum of square errors (SSE) at output neurons on the number of training cycles at different distributions of the weights of connections between neurons. Expectedly, since the weights were initialized at random, the

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training proceeded in different ways. The training process was often stopped after reaching one of the local minima of the error function (Fig. 4). To solve the problem of local minima in some modications of the back propagation algorithm, a dynamic change of the training rate was used. Local minima could also be avoided (after the values of weight coefcients are stabilized) by the addition of a random constituent to start the gradient descent from a new point. Fig. 4 shows that 8000 cycles provided sufcient training. No signicant decrease of the error was observed upon further training. Fig. 5 shows a plot of SSE dependence at output neurons of the network on the number of neurons in the internal layer. Due to the above described peculiarities of weights initialization, the errors in each point were measured three times to choose the least value. The dependence analysis showed that 12 neurons in the internal layer were sufcient for optimal training. Further increases in the number of neurons did not result in a signicant decrease of SSE and required more training time. Under the optimal conditions established above, the minimal SSE was 0.039. R 2 = % wi (Yobsi Yobs)2 % wi (Yobsi Ycali )2
i=1 i=1

Fig. 5. Dependence of the sum of squared errors (SSE) on the number of neurons (n) in the internal layer.

,

chemometric approach and articial neural networks was estimated by the coefcient of determination (R 2) described by the formula:

i=1

% wi (Yobsi Yobs)2, where

3.3. Error of determination of concentrations

The accuracy of determining concentrations using the

Yobs = % wi Yi
i=1

% wi

i=1

is the weighed mean value of substrate concentration (Yobsi ), wi is weight coefcient for each measurement, Ycal are concentration values obtained from data processing and n is number of measurements. The coefcient of determination describes the degree of data deviation from standard values. Thus, when R 2 =1 the model provides a precise determination of the substrate concentration.

3.4. Comparison of the efciency of mixture analysis using chemometric methods and articial neural networks
The analyses of experimental data processed by chemometric and articial neural network methods are summarized in Table 2. These results testify to the potential of using a system consisting of two microbial
Table 2 Coefcient of determination Analyte Fig. 4. Examples of dependence of the logarithm of the sum of squared errors (log(SSE)) on the number of training cycles (N). Curves a d correspond to different initial distributions of weights on the number of connections between neurons. Value of R 2 for Value of R 2 for articial polynomial approximation neural networks 0.976 0.993 0.995 0.992

Glucose Ethanol

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1007

sensors to analyze a two-component mixture. Data processing was successful using either traditional chemometric methods or an articial neural network. Indeed, differences in the accuracy of determinations were not found to be signicant. Since polynomial approximations restricted the range of substrate concentrations that could be analyzed, however, processing of data using the neural network was preferable. The choice of a method for experimental data processing should consider such factors as the availability of sufcient data for neural network training and the difculty of designing a portable device using the polynomial approximation dependence, as well.

4. Conclusions Results demonstrated that a system consisting of only two microbial sensors can be used in the quantitative analysis of ethanolglucose mixtures. An articial neural network was shown to be highly efcient for the analysis of data from this system. The accuracy of the articial neural network was compared with that of a traditional chemometric method. The coefcients of determination (R 2) were 0.99 for ethanol and 0.98 for glucose in data processed by polynomial approximation and 0.99 for each substrate in data processed by the articial neural network.

Acknowledgements This work was conducted under Specic Cooperative Agreement 58-3620-8-F005 between the Institute of Biochemistry and Physiology of Microorganisms, Pushchino, Moscow Region, Russia and the Agricultural Research Service of the US Department of Agriculture. The authors thank N.O. Morozova for providing data on measurements with the sensor on the basis of P. methanolica.

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