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AMPEROMETRIC ENZYME-BASED BIOSENSORS FOR APPLICATION IN FOOD AND BEVERAGE INDUSTRY

ELISABETH CSÖREGI1*, SZILVESZTER GÁSPÁR1, MIHAELA NICULESCU1, BO MATTIASSON1, WOLFGANG SCHUHMANN2

1Lund University, Centre for Chemistry and Chemical Engineering, Department ofBiotechnology, P.O. Box 124, 221 00 Lund, Sweden 2Ruhr-Universität Bochum, Analytische Chemie - Elektroanalytik and Sensorik, Universitätsstr. 150; 0-44780 Bochum, Germany

Summary

Continuous, sensitive, selective, and reliable monitoring of a large variety of different compounds in various food and beverage samples is of increasing importance to assure a high-quality and tracing of any possible source of contamination of food and beverages. Most of the presently used classical analytical methods are often requiring expensive instrumentation, long analysis times and well-trained staff. Amperometric enzyme-based biosensors on the other hand have emerged in the last decade from basic science to useful tools with very promising application possibilities in food and beverage industry. Amperometric biosensors are in general highly selective, sensitive, relatively cheap, and easy to integrate into continuous analysis systems. A successful application of such sensors for industrial purposes, however, requires a sensor design, which satisfies the specific needs of monitoring the targeted analyte in the particular application, Since each individual application needs different operational conditions and sensor characteristics, it is obvious that biosensors have to be tailored for the particular case. The characteristics of the biosensors are depending on the used biorecognition element (enzyme), nature of signal transducer (electrode material) and the communication between these two elements (electron-transfer pathway).

Therefore, the present chapter presents the different existing biosensor designs describing the possible electron-transfer pathways, discusses their advantages and disadvantages, and shows their possible application in food and beverage industry. Three practical examples are given describing biosensor designs developed in our laboratory, demonstrating their usefulness for industrial applications.

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1. Biosensors - Fundamentals

Biosensors are selective devices that involve a communication between a biorecognition element (enzymes, microorganisms, cells, antibodies, tissues etc.) and a physical transducer, which is able to transform the chemical information i. e. the concentration of the target analyte, into a measurable signal (electrical, optical, mass, thermal, etc.) as shown in figure 1. Most biosensors use enzymes in the complementary recognition process, since their catalytic action implies a self-regeneration of the binding pocket. In addition, many enzymes are readily available and easy to couple with a large variety of transducers.

Figure 1. Schematic presentation ofa biosensor.

Amperometric biosensors based on enzymes are the most studied branch, since they combine the selectivity and specificity of the enzymatic reactions with the simplicity of the electrochemical detection method. In general, the analyte is catalytically converted (oxidised or reduced) by a specific enzyme, which is usually immobilised on an electrode surface. Mostly, the redox equivalents are intermediately stored in the cofactor of the enzyme, from/to which an electron-transfer process has to occur, whereby the biocatalytic process is linked to the electrode. Thus, the chemical energy of the enzyme-catalysed reaction is transduced to an electrochemical reaction, which occurs at a certain potential determined by the nature of the compound used in the final step of the electron-transfer process. Due to a stoichiometric relation between the number of transferred electrons and the analyte, a current proportional to the concentration of the target analyte is yielded and used for quantification.

Enzyme-based biosensors attracted much attention for their possible use in food and beverage industry, representing a very promising alternative to the traditional timeconsuming and often expensive analysis techniques. Due to their simplicity, low cost, and possibility of integration into on-line measurement systems needed in automated industrial technologies, enzyme-based biosensors have been widely used in this area, resulting in a large number of review articles published in the last 10 years [1-7]. As demonstrated, biosensors can be useful tools for monitoring the conversion of raw materials, the presence and concentration of possible contaminants, the product content, and product freshness [3].

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Amperometric enzyme-based biosensors for application in food and beverage industry

2. Prerequisites for application of biosensors in food industry

A successful industrial application of biosensors in food industry, requires a sensor design assuring besides low fabrication costs the following:

•selectivity versus all possible enzymatic and electrochemical interferences

•adequate sensitivity

•appropriate response time

•stability (operational and/or storage stability)

•reliability

•simplicity of monitoring

Since the concentration of the target analytes is significantly different and each individual application requires particular operational conditions, it is obvious that biosensors have to be tailored for the considered application. E.g., long term monitoring applications, such as monitoring in fermentation processes, require biosensors, which guarantee a good mechanical and thermal stability. In addition, any source of contamination by e.g. component leakage has to be strictly avoided using an appropriate sensor design, while fast response time (<s) and high selectivity issues are often not crucial. The choice of enzyme(s), immobilisation procedure, electrode configuration, use of additional sensor elements (e.g. additional membranes for improved stability and/or interference elimination) will therefore always be determined by the considered application.

However, every enzyme-based biosensor has to take into account that its performance is determined simultaneously by the biorecognition element (the enzyme), the signal transducer (polarised electrode), and by the communication between these two elements (electron-transfer pathway). Therefore, the electrode configuration determined by the above mentioned two elements and the immobilisation procedure of the enzyme, has to be designed in such a manner, that the biochemical information is translated into a measurable electrical current with the highest efficiency (optimal electron-transfer pathway). Below, a short overview is given on the various existing biosensor designs, the electron transfer possibilities, and the advantages and disadvantages of various sensor architectures.

3. Existing biosensor configurations and related electron-transfer pathways

The simplest electron-transfer mechanism would be represented by the direct electrochemical regeneration of the active site (prosthetic group) of the enzyme at the electrode surface (see figure 2).

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Figure 2. Direct electron transfer pathway (via electron tunnelling) between the active site ofan enzyme and the surface ofa transducer.

However, this approach can be applied only for a very few enzymes (e.g. peroxidases), which have their active site situated close to their surface, and thus allow its direct regeneration on the transducer. To insulate the enzyme-integrated active site, most of the enzymes have their prosthetic group deeply buried within the protein shell and thereby, the distance for a direct electron transfer is - according to the Marcus theory [S, 9] - too long. Therefore, in general the electron-transfer pathway has to be artificially designed, either by (i) using “electron shuttles” (e.g. redox mediators, see figure 3) or by (ii) shortening the electron-transfer distance (e.g. orientation of enzymes on electrodes, modification of the active site of an enzyme, etc).

Figure 3. Mediated electron transfer pathway using a redox mediator (Mox) as the “electron shuttle between the active site of the enzyme and the transducer exemplifiedfor an oxidation reaction.

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3.1. BIOSENSORS BASED ON O2 OR H2O2 DETECTION

The first developed enzymatic biosensors were based on the fact that in nature the active site of many enzymes is regenerated by their co-substrate (e.g. O2 or NAD+). Since these co-substrates can be directly oxidised or reduced on the surface of a polarised electrode, they could be successfully used as “electron shuttles”. The quantification of the analyte was based in these cases on measuring either the (i) decrease of the co-substrate concentration or (ii) the increase of the co-product (e.g. hydrogen peroxide), (see scheme 1).

Scheme 1. Substrate detection possibilities exemplifiedfor an oxidation reaction.

Unfortunately, both above mentioned electrochemical reactions occur at high overpotentials and thus, the signal transduction suffers of electrochemical interferences when the biosensor is used in real applications implying complex matrices. Additional problems may occur at high substrate concentrations, due to the lack of molecular oxygen. Therefore, next generation biosensors were often based on a mediated electron transfer principle, the earlier types using freely diffusing redox mediators (see section 3 2).

Despite the mentioned drawbacks, biosensors based on the direct detection of either O2 or H2O2 were used to measure different analytes in food stuff and beverages, mainly due to their relative simplicity (see Table 1).

Table 1. Biosensors using direct H2O2 or O2 detection with potential use in food and/or beverage industry

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Table I. Biosensors using direct H2O2 or O2 detection with potential use in food and/or beverage industry

* LR denotes the linear range and DL the detection limit of the biosensors

3.2. BIOSENSORS BASED ON FREE-DIFFUSING REDOX MEDIATORS

Considering any real application, it is of great importance that the biosensors are operated within an optimal potential window (approximately between -0.10 to +0.05 V vs. SCE), where electrochemical interference is minimal [10]. This can be practically realised in two different sensor architectures.

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The first sensor types use artificial, free-diffusing, electron-transfer mediators (redox couples with a formal potential lying within the optimal potential range), replacing the natural electron acceptor the hydrogen peroxide/oxygen, couple (see scheme 2).

Scheme 2. Detection ofa substrate using an artificial redox mediator.

Despite assuring detection at low potentials, these biosensor configurations often cause problems by contaminating the sample. Thus, the application of these biosensors in food and/or beverage industry is almost impossible, unless extra protection membranes are applied in the particular sensor design. Moreover, a competition between the natural cofactor of the enzyme (e.g. O2) and the artificial mediator occurs. Therefore, the search for and use of, oxygen independent enzymes was and is obvious (e.g. measurement of D-fructose in food samples based on PQQ-dependent D-fructose dehydrogenase [11-13]).

The second type of sensors is based on coupled enzymes (oxidase-peroxidase). These bi-enzyme electrodes are mostly applied for the detection of the substrates of H2O2- producing oxidases and make use of the selectivity of peroxidases (POD) towards H2O2. Since peroxidases are able to directly exchange electrons with the electrode (via electron tunnelling), many of the reported electrode designs make use of a direct electron-transfer pathway [14] as shown in scheme 3a. The most often used peroxidase in this context is horseradish peroxidase (HRP). Since only 48 % of the randomly immobilised HRP molecules were reported to be able to undergo a direct electron transfer [ 15], often an orientation of the peroxidase molecule is required to improve the rate of the electron transfer. Besides the accessibility of the enzyme's redox centre, the glycosylation degree of the enzymes is also playing an important role. However, even when using an orientated binding of peroxidases, (improved electron-transfer reaction rate [ 16]), the major drawback of this sensor design is a small current response.

Scheme 3a. Electron-transfer pathway in coupled enzyme electrodes, the final electron transfer step is via direct electron tunnelling.

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Since the direct electron transfer between the commonly used horseradish peroxidase (HRP) and the electrode was shown to be sluggish, often the use of an additional mediator is required, as shown in scheme 3b.

Scheme 3b. Electron-transfer pathway in coupled enzyme electrodes, the final electron transfer step is via mediated electron transfer.

Moreover, HRP is characterised by a low selectivity vs. reducing substrates, thus, highly motivating search for new, more selective, and/or stable peroxidases. Use of lactate peroxidase [17-19], tobacco peroxidase [20, 21], microperoxidase [17, 18, 22, 23], peroxidase from Arthromyces ramosus [19, 24, 25], soybean peroxidase [19, 26, 27] or sweet potato peroxidase [21] in biosensor designs was already reported.

Examples of biosensor architectures based on electron transfer principles mentioned in this section with possible application in food and/or beverage industry are given in Table 2.

Table 2. Biosensors using artificial mediators with potential use in food and/or beverage industry

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* LR denotes the linear range and DL the detection limit ofthe biosensors

3.3. INTEGRATED SENSOR DESIGNS (REAGENTLESS BIOSENSORS)

An improved communication between enzyme(s) and the electrode, meeting the requirements for real sample applications is respected in the greatest extent by using integrated ("reagentless")biosensors. These electrodes contain all needed components integrated in a sensing layer without any leakage possibility, since there is no need of

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