APPLICATION OF VOLTAMMETRIC AND AMPEROMETRIC TECHNIQUES TO DESIGN ENZYMATIC BIOSENSORS FOR FOOD PROCESSING INDUSTRY

Jolanta Wawrzyniak¹, Antoni Ryniecki¹, Małgorzata Przybyt^2
1 Institute of Food Technology of Plant Origin, Agricultural University of Poznan, Poland
² Institute of General Food Chemistry, Technical University of Łodz, Poland

ABSTRACT

The paper has been devoted to a review of problems associated with designing voltammetric biosensors dedicated for the food processing industry. Such biosensors are based on techniques of the cyclic voltammetry and amperometry. Voltammetric biosensors can be obtained by the immobilisation of the biological agents (e.g. enzyms) specific for a given substance on the working electrode of the traditional measuring system of the voltammograph. Short characteristics of the voltammetric methods, the salient features of redox mediators and conducting polymers and their applications in construction of voltammetric biosensors are described. Moreover, the mechanism of action and some examples of the manufacture of voltammetric biosensors are presented. The observed development of research on voltammetric biosensors allows assuming that in future they will find application alongside conventional analytic methods in qualitative and quantitative studies of food products.

Key words: biosensor; voltammetry, enzym, redox mediator, conducting polymer, food analysis.

INTRODUCTION

There is a continuing demand in the food processing industry for new analytical methods, which would provide a rapid and easy way not only to control final products but also to monitor the correctness of the course of the technological processes. The above-mentioned needs arise from the modernisation and automation of food-processing plants, increase of production efficiency as well as from the necessity to have access to information about the qualitative and quantitative conditions of the processed food products.

There are many methods allowing the determination of qualitative and quantitative composition of chemical substances such as, for example, gas and liquid chromatography, refractometry or spectrophotometry. However, the above-mentioned methods require complicated and, frequently, expensive specialised equipment, qualified personnel and are usually labour- and time-consuming.

The demand on the part of the food processing industry encouraged many researchers to seek new analytical methods and, therefore, in recent years, many investigations have been devoted, among others, to issues connected with broadening the area of application of amperometric and voltammetric methods. Numerous papers present attempts to combine the above-mentioned techniques with biologically active agents, e.g. enzymes.

This paper has been devoted to a review of problems associated with designing voltammetric biosensors dedicated for the food processing industry. The above-mentioned biosensors are based on techniques of the cyclic voltammetry and amperometry [2, 4, 5, 6, 24, 31, 36, 45]. In order to elucidate the mechanism of action of the biosensors, short characteristics of the voltammetric methods themselves will be presented in the first part of the paper.

VOLTAMMETRY AND ITS APPLICATION IN THE ANALYSIS OF FOOD PRODUCTS

Voltammetry belongs to the group of polarographic methods developed by Jaroslav Heyrovsky [9, 16, 31]. A typical set of equipment for voltammetric analyses consists of a measuring chamber containing three electrodes, namely the working electrode (microelectrode), reference electrode and auxiliary electrode as well as the voltage source (of the set characteristics) and devices for measuring current and voltage – voltmeters and ammeters (as a rule, both devices allow registration of the measured values in time). The method employs measurements of changes in time (τ) in the current (I) flowing through the system of electrodes in relation to potential (E) applied to the working electrode. The registered changes in the current allow drawing the I(τ) = f[E(τ)] relationship, which is called the voltammogram. An example of a voltammetric system is presented in Figure 1. If measurements of the current are carried out at constant potential, then we obtain a classical amperometric system. Such measurements must be done in stirred solution or with rotating electrode (hydrodynamic amperometry).

The voltammetric methods, because of the character of the applied potential, can be divided into:

• Stripping voltammetry (SV) – this is a technique in which two phases can be distinguished. In the first phase, which precedes the proper voltammetric process, concentration of the examined substance takes place as the result of electrolysis or adsorption on the working electrode. The concentration occurs by way of convection at constant potential. During the second phase, the polarisation of electrodes changes and the proper electrochemical process begins. Its course is registered in the form of the voltammogram, which is used for analytic determinations [9, 23]. Stripping voltammetry is characterised by high sensitivity and allows determining electroactive compounds even at the range of 10^-9–10^-11 mol·dm^-3. The technique is applied in the trace analysis of metal cations and to determine a large group of organic compounds [40].

• Linear sweep voltammetry (LSV) – this technique investigates the dependence of the current flowing through the measuring cell in which the examined substance is on the linear increase of the potential. The scan rate of the electrode potential usually alters in the range from 20 to 100 mV·s^-1, [40]. In this method, the registered relationship I = f(E) has the shape of a peak (Figure 2). The electrode potential (E_p) at which the peak is observed is characteristic for a given substance and its height is proportional to the concentration of this substance. The dependence allows the application of the LSV method in the quantitative analysis [9].

• Cyclic voltammetry (CV) – this is a variant of the LSV technique in which changes in time of the current between the working and counter electrodes in relation to the applied potential (cyclically varying at a constant scan rate within definite boundaries from E₁ to E₂ and back from E₂ to E₁) are registered. Changes in the electrode potential in time for the cyclic voltammetry and the shape of the voltammogram are presented in Figure 3. The cyclic voltammetry finds wide application in the quantitative and qualitative analysis. On the basis of the course of the voltammogram, it is possible to determine not only the type and quantity of the examined substance but also to establish if the electrode process is reversible [9, 21]. The cyclic voltammetry is also applied in studies of mechanisms of electrode processes [9, 40].

From among numerous research studies, possibilities of the application of voltammetry to the determination of vitamin C concentrations in the result of the electrochemical oxidation of ascorbic acid arise considerable interest [1]. Ijeri et al. [18] found that voltammetric techniques employing chemically modified (by a redox mediator) working electrodes can be applied successfully to determine concentrations of ascorbic acid in multivitamin pharmaceutical preparations, fruit juices, jams and wine. Esteve et al. [11] comparing concentrations of vitamin C in infant formulas obtained using the voltammetric method and employing high performance liquid chromatography, obtained comparable results. They concluded that, since voltammetric techniques are characterised by measurement simplicity, low costs and low time expenditure, the method could be used for routine determination of vitamin C. Moreover, voltammetric techniques can also be applied to determine simultaneously several substances found in the same solution. Jaiswal et al. [20] employing voltammetric techniques carried out a simultaneous determination of the ascorbic acid and α-tocopherol in multivitamin-multimineral pharmaceutical preparations. Similar experiments were carried out by Pournaghi-Azar et al. [32], who succeeded to determine simultaneously ascorbic acid and dopamine, while Ernst and Knoll [10]monitored the mechanism of a simultaneous electrochemical oxidation of ascorbic and uric acids. In their investigations, Saurina et al. [38] made an attempt to determine simultaneously concentrations of three oxidizable amino acids (cysteine, tyrosine and tryptophan) using voltammetric methods. However, due to the poor accuracy of the method, about 10% for Cys and Trp and 18% for Tyr, they concluded that the method developed by them required further improvement and, in the form described by them, could be used in food processing industry only in the situation when the accuracy of the analysis is not as critical as the speed in obtaining the approximate concentration of these amino acids. The voltammetric technique was also employed by Navarátil et al. [27] to determine As (III) (the obtained limit of detection was 3.2·10^-7 g·dm^-3) who found that this method could be applied for the analysis of samples of various origins of natural waters [27].

Examples of voltammogram obtained in the course of determination of vitamins B₂ and C, are presented in Figures 4 and 5, respectively [37, 43]. It is evident from the examples given above that voltammetric methods provide an alternative for other analytical methods allowing quantitative and qualitative determination of many chemical substances. However, they can only be used to investigate electroactive substances. The combination of voltammetric methods with enzymes make it possible to build voltammetric biosensors that allow expanding voltammetry to a new area of analysis of substances which are not electroactive by themselves, but which, in an enzymatic reaction, form electroactive products.

STRUCTURE AND MECHANISM OF VOLTAMMETRIC BIOSENSORS

Voltammetric biosensors can be obtained by the immobilisation of the enzyme specific for a given substance on the working electrode of the traditional measuring system of the voltammograph. It should be mentioned here that, basically, the construction of voltammetric biosensors does not differ from biosensors applied in amperometric systems. The difference consists in the kind of techniques employed to read signals generated by these biosensors.

Principles used in measurements carried out with the assistance of voltammetric biosensors
The measurement of the concentration of the analyte with the assistance of voltammetric biosensors usually consists of two stages. During the first stage (calibration), the voltammetric curves for the known concentrations of the examined substance are drawn. The obtained voltammograms are used to read potential value at which the peak occurred and the corresponding values of the current and, next, the calibration curve is developed which expresses the relationship between the product concentration and the current. In the course of the second stage (determination of the unknown concentration), measurements can be carried out in two ways; either by an amperometric measurement (at the potential for which the peak occurs) and the comparison of the obtained result with the calibration curve (amperometric biosensor) or by carrying out a voltammetric measurement whose result is compared with voltammograms obtained earlier. Voltammetric curves obtained for the biosensor designed to determine glucose at its various concentrations as well as the correlation between the current values (at the potential of peaks of voltammetric curves) and glucose concentrations are shown in Figure 6, [45].

A specific interaction between the substance to be determined and the enzyme (biomolecule) takes place in the course of measurements employing voltammetric biosensors resulting in an electrochemical signal. The recorded signal may develop as the result of the exchange of electrons between the electrode and the substrate, the product or the enzyme active centre. The measurement is characterised by high selectivity resulting from the fundamental property of enzymes, namely their ability to respond to one specific compound [35]. In additions, the substrate specificity of the enzyme action simplifies operations needed to prepare samples [17, 23].

Mechanism of voltammetric biosensors and the role of redox mediators and conducting polymers
Before proceeding to discuss the mechanism of voltammetric biosensors, it is important to devote some attention to factors improving the work of these biosensors, namely redox mediators and conducting polymers.

Redox mediators
Bearing in mind the placement of the enzyme active centre applied in biosensors which usually is found inside the polypeptide structure of the protein enzymatic molecule (in the so called deep pocket inside the apoenzyme), the electron transport can be significantly slowed down and, therefore, the recorded voltammetric signals can be too weak and indistinguishable to determine the concentration of the examined substance. In some groups of enzymes, this process can be improved by the introduction of redox mediators, which act as electron transmitters between the enzyme active centre and the electrode surface [6, 30, 35]. In the case of oxidases, which are flavoproteins, the natural electron mediator – oxygen (1) can be substituted by artificial mediators, i.e. ferrocene (the Fe³⁺ complex with a cyclopentadienyl anion), quinones, indophenols, viologens [35] which can be used either in dissolved form or co-immobilised with the enzyme.

reaction with natural mediator:

(1)

reaction with synthetic redox mediator:

(2)

electrode reaction:

(3)

The redox mediator is reduced in the course of enzyme reaction (2), then is reoxidized at the electrode (3) generating current which is proportional to the concentration of enzyme substrate. Thanks to the application of mediators of electron passages, the measurement range of the biosensor can be expanded because it does not depend on the oxygen concentration in the examined solution [34, 35].

In the case of oxidases containing prosthetic groups other than FAD, oxygen cannot be replaced by synthetic mediators [34]. The application of enzymes belonging to the group of NAD⁺ dependent dehydrogenases is also limited because high overpotential of NADH oxidation and not full reversibility of its oxidation. In this case direct application of synthetic mediators is not possible. Dehydrogenase substrates can be determined by the electrodes on which dehydrogenase is immobilised together with another enzyme – diaphorase, oxidizing NADH with redox mediator which is reoxidized in reaction (3).

electrode reaction:

(4)

(5)

This allows employing synthetic mediators, i.e. methylviologen, ferrocene, Fe(CN)₆^-3, [34]. Dehydrogenases, which contain quinone derivatives as a co-factor (PQQ), constitute a separate class of dehydrogenases where it is possible to introduce synthetic mediators [28]. It should be added that redox mediators cannot only mediate the transport of electrons between the enzyme active centre and the electrode but they can also transfer electrons between the product of the enzymatic reaction and the electrode.

The application of mediators allows, on the one hand, (as in the case of traditional voltammetry) reducing the operational potential (the potential at which the peak of the voltammetric curve is observed) and on the other, increasing and improving the quality (amplification) of the signal resulting from the reaction, which reduces the influence of factors interfering with the voltammetric signal [7, 8, 18]. Examples of mediators applied to manufacture biosensors with selected enzymes were complied in Table 1.

Current conducting polymers
Much attention has been devoted, in recent years, to polymers capable of conducting electric current – especially those, which could be utilised in electrochemical biosensors [14, 39]. Polymers most frequently applied to design biosensors include: polyaniline, polypyrrole, polyindole and polytiophen [5, 6, 7, 29, 41, 42]. One of the examples can be poly-(N-methylpyrrole) used as an electron transmitter between the electrode and FAD, which is a component of the active centre of the glucose oxidase [6] and poly-(1,3-phenylenediamine) used by Yang et al. to design a biosensor for the determination of lactic acid [44].

In order to increase the stability and cut the response time of biosensors, mediators are frequently combined with conducting polymers [12, 39]. The choice of the mediator, conducting polymer and the method of enzyme immobilisation all exert a strong influence on properties of the manufactured biosensor. Garcia investigated the impact of the method of fructose dehydrogenase immobilisation on the activity of the biosensor used to determine fructose [12]. In their investigations, they employed two methods of enzyme immobilisation. In the first one, the enzyme was cross-linked in a polymer (polypyrrole) immobilised on the platinum electrode, whereas in the second, they used the method of covalent enzyme binding (with the aid of glutaraldehyde) on the polymer surface (also polypyrrole) immobilised on the platinum electrode. The biosensor manufactured using the first method was characterised by a shorter response time (8 s) than that for the biosensor obtained with the assistance of the second method (15 s) but the stability of the first one was about 1 week, while that of the second – 2 weeks [12].

Mechanism of voltammetric biosensors
The mechanism of action of the voltammetric biosensor can be illustrated taking as the example the biosensor manufactured by Niculescu et al. [28] to determine ethanol, glucose and glycerol. In the presented example (Figure 7), as the biological agent, they used appropriate dehydrogenases (of similar structure) in which quinones derivatives (PQQ) play the role of co-factors (alcohol dehydrogenase PQQ-ADH, glucose dehydrogenase PQQ-GDH and glycerol dehydrogenase PQQ-GlyDH), whereas the redox polymer, which is a complex of the poly(vinylimidazole) with Os(4,4’-dimethylbipyridine)₂Cl (PVl₁₃dmeOs), mediates in the transport of electrons. Substrate molecules (ethanol, glucose and glycerol) diffuse into the layer with the immobilised enzymes where they are oxidised in the result of the enzymatic reaction into, respectively: acetaldehyde, gluconic acid and dihydroxyacetone. At the same time, enzymatic co-factors undergo reduction and the active form of enzymes is regenerated following PQQ oxidation on the carbon electrode through the polymer (PVl₁₃dmeOs). This process generates signals in the form of peaks whose heights are proportional to the concentration of ethanol, glucose and glycerol in the examined sample.

Examples of the manufacture of voltammetric biosensors
Numerous literature publications dealing with biosensor development emphasise considerable multiplicity of methods that can be employed to design them. Work continues in many laboratories with the aim to manufacture biosensors, which could be marketed. However, this requires analysing many different factors that might influence the reproducibility and repeatability of the designed biosensor. Below we present a review of designs, which have been adopted so far in research work on voltammetric biosensors.

The essential step during biosensor fabrication is choice of the immobilization technique for an enzyme and other factors (redox mediators and conducting polymers). One of these methods is screen-printing. This technique was employed by Nagata et al. to fabricate a glucose biosensor [25]. Special ink prepared with glucose oxidase (modified with ferrocene derivatives as the electron – transfer mediator), soluble and insoluble polymer resins and organic solvent was printed on a thin gold film electrode on a plastic film. In the next stage, employing the method of cyclic voltammetry, they determined the potential at which the peak corresponding to the redox reaction occurred and then they measured the value of the current in the concentration function of the determined substance. However, the designed biosensor was characterised by poor stability lasting barely several hours. The range of glucose concentrations within which the observed signal was proportional to its content varied from 2.8 ÷ 11 mM. Avramescu et al. employing the screen-printing technique designed a simple biosensor for D-lactate in wine [2]. In the process of manufacturing the above-mentioned biosensor, D-lactate dehydrogenase and NAD⁺ were spread on the surface of the carbon electrode modified with an insoluble salt of Medolda’s Blue. The range of D-lactate concentrations obtained by them for which it was possible to perform measurements with the manufactured biosensor varied from 0.1 ÷ 1.0 mM and the biosensor response time was equal to 150 s. After two weeks, the biosensor showed 75% of its initial activity.

Better results were achieved by Qian et al. [36] who developed a biosensor for glucose using a porous and conductive carbonaceous material – wood ceramics onto which, by way of physical adsorption, glucose oxidase was immobilised. The best results were obtained when the enzyme was stabilised by polyethyleneimine and using Toluidine Blue O (TBO) as a mediator. The voltammetric analysis of the biosensor activity revealed that measurements of glucose concentrations should be carried out at the potential of +500 mV. The linear correlation between the current and the glucose concentration was obtained within the range of 0.5 ÷ 7.0 mM, whereas the biosensor response time was about 1 min. After 50 days, the designed biosensor retained 72.4% of its initial activity (response). Also Chaubey et al. [6] used the technique of physical adsorption of two bio-catalysers – lactate oxidase and dehydrogenase on the polyaniline membrane when designing a biosensor for L-lactate. In the result of immobilising two enzymes, they manufactured a biosensor sensitive to low lactate concentrations (5 · 10^-5 M) due to the substrate recycling. Their biosensor showed stability for about three weeks.

Attempts have also been made to miniaturise the designed biosensors. A miniaturised electrode for glucose determination, in which glucose oxidase alongside ferrocene perchloride was cross-linked in a Nafion film on the surface of a platinum electrode, was designed by Zhou et al. [45]. The developed electrode was characterised by good stability and reproducibility. The operational potential determined by the voltammetric method was +0.25 V. The electrode lost about 20% of its activity after one month.

Another method employed to design biosensors is the technique of covalent binding of the enzyme to the electrode or membrane surface by means of low-molecular compounds containing two functional groups, the so called activating-binding factors, e.g. glutaraldehyde (cross-linking). Chaubey et al. employed the above-described technique to immobilise L-lactate dehydrogenase (LDH) via glutaraldehyde on the surface of conducting polymers covering the electrode and developed a biosensor for L-lactate [5]. The performed investigations showed that the polypyrrole–polyvinyl sulphonate (PPY-PVS) membrane on whose surface the lactate dehydrogenase was immobilised acted like an electron acceptor and enhanced the stability of the microenvironment during the biochemical reaction. The PPY-PVS-LDS electrode obtained in this way could be used to determine L-lactate at 0.5 ÷ 6.0 mM concentrations. The response time of the examined biosensor was 40 s and a shelf-life of about 2 weeks.

Some publications describing the designing of biosensors needed for the determination of more complex substances recommend the application of two or more enzymes [15]. In such systems, products of some enzymatic reactions constitute substrates of the consecutive ones. Liu et al. [24] manufactured a glucose and lactose specific biosensor designed on the basis of three enzymes: β-galactosidase, mutarotase and glucose oxidase immobilised in the β-cyclodextrin polymer together with a ferrocene mediator on the carbon electrode. In the course of the lactose determination with the obtained biosensor, the β-galactosidase hydrolyses lactose to monosaccharides, and then α-D-glucose, which is one of the products of this hydrolysis, is transformed by the mutarotase into β-D-glucose, which undergoes oxidation in the presence of glucose oxidase. The determined content of glucose is proportional to the lactose concentration. The potential determined with the cyclic voltammetric method at which lactose and glucose concentrations were determined equals +0.35 V. The designed biosensor was characterised by a short response time and high sensitivity to lactose and glucose and its activity dropped only by about 15% after two months.

CONCLUSION

Much attention has been paid in recent years to the improvement of the applied analytical methods aiming at:

• shortening the time required for analyses,




• improvement of the sensitivity and selectivity of the applied methods,




• miniaturisation,




• multi-functionality and




• computerisation of measuring equipment.

The combination of voltammetric methods with properties of biological agents opens up wide possibilities to employ them for the determination of both ions and substances taking part in the redox reactions as well as for compounds inactive electrochemically. The observed development of research on voltammetric biosensors allows assuming that in future they will find application alongside conventional analytic methods in qualitative and quantitative studies of food products.

REFERENCES

1. Arya S.P., Mahajan M., Jain P., 2000. Non-spectrophotometric methods for the determination of Vitamin C. Anal. Chim. Acta 417, 1–14.

2. Avramescu A., Noguer T., Magearu V., Marty J. L., 2001. Chronoamperometric determination of D-lactate using screen-printed enzyme electrodes. Anal. Chim. Acta 433, 81-88.

3. Bassi S., Lee E., Zhu J. X., 1998. Carbon paste mediated, amperometric, thin film biosensors for fructose monitoring in honey. Food Res. Int. 31(2), 119-127.

4. Boujtita M., Hart J.P., Pittson R., 2000. Development of a disposable ethanol biosensor based on a chemically modified screen-printed electrode coated with alcohol oxidase for the analysis of beer. Biosensors Bioelectronics 15, 257-263.

5. Chaubey A., Gerard M., Singhal R., Singh V.S., Malhotra B.D., 2000. Immobilization of lactate dehydrogenase on electrochemically prepared polypyrrole-polyvinylsulphonate composite films for application to lactate biosensors. Electrochim. Acta 46, 723-729.

6. Chaubey A., Pande K.K., Singh V.S., Malhotra B.D., 2000. Co-immobilization of lactate dehydrogenase on conducting polyaniline films. Anal. Chim. Acta 407, 97-103.

7. Chaubey A., Malhotra B.D., 2002. Mediated biosensors. Biosensors Bioelectronics 17, 441-456.

8. Chrusciński L., 1991. Amperometryczne i enzymatyczne elektrody do oznaczania mleczanów [Amperometric and enzymatic electrode for the lactate determination]. Przem Spoż. 4, 106-108 [in Polish].

9. Cygański A., 2004. Podstawy metod elektroanalitycznych [Basis of the electroanalytical methods]. WNT, Warsaw [in Polish].

10. Ernst H., Knoll M., 2001. Electrochemical characterisation of uric acid and ascorbic acid at platinum electrode. Anal. Chim. Acta 449, 129-134.

11. Esteve M. J., Farré R., Frígola A., López J. C., Romera J. M., Ramírez M., Gil A., 1995. Comparison of voltammetric and high performance liquid chromatographic method for ascorbic acid determination in infant formulas. Food Chem. 52, 99-102.

12. Garcia C.A.B., Netob G. de Oliveira, Kubotaa L. T., 1998. New fructose biosensors utilizing a polypyrrole film and D-fructose 5-dehydrogenase immobilized by different processes. Anal. Chim. Acta 374, 201-208.

13. Garjonyte R., Yigzaw Y., Meskys R., Malinauskas A., Gorton L., 2001. Prussian Blue- and lactate oxidase-based amperometric biosensor for lactic acid. Sensors Actuators B 79, 33-38.

14. Gerard M., Chaubey A., Malhotra B.D., 2002. Application of conducting polymers to biosensors. Biosensors Bioelectronics 17, 345-359.

15. Hagget B., Dembczyński R., Jankowski T., 2001. Biosensory – procedury oznaczeń [Biosensors – procedures of determinations] in: Metody pomiarów i kontroli jakosci w przemysle spożywczym i biotechnologii [Measurement and quality control method in food industry and biotechnology]. M. Jankiewicz, Z. Kędzior (eds.), Wyd. AR, Poznań [in Polish].

16. Hulanicki A., 2004. Jaroslav Heyrovsky (1890-1967). Chem. Anal. (Warsaw) 49, 763.

17. Horubała A., 1988. Biosensory i ich zastosowanie [Biosensors and their application in the food technology]. Przem. Spoż. 10, 292-293 [in Polish].

18. Ijeri V. S., Jaiswal P. V., Srivastava A.K., 2001. Chemically modified electrodes based on macrocyclic compounds for determination of Vitamin C by electrocatalytic oxidation. Anal. Chim. Acta 439, 291-297.

19. Imamura M., Haruyama T., Kobatake E, Ikariyama Y., Aizawa M., 1995. Self-assembly of mediator-modified enzyme in porous gold-black electrode for biosensing. Sensors Actuators B 24-25, 113-116.

20. Jaiswal P.V., Ijeri V.S., Srivastava A.K., 2001. Voltammetric behavior of α-tocopherol and its determination using surfactant + ethanol + water and surfactant + acetonitrile + water mixed solvent systems. Anal. Chim. Acta 441, 201-206.

21. Kisza A., 2001. Elektrochemia [Elektrochemistry]. WNT, Warsaw [in Polish].

22. Kress-Rogers E., 1997. Handbook of biosensors and electronic noses in medicine, food, and the environment. CRC Press, Boxa Roton.

23. Kuncewicz A., Panfil-Kuncewicz H., 1997. Enzymatyczne i biosensoryczne układy w analizie żywnosci [Measurement systems in food analysis using enzyms and biosensors]. Przem. Spoż. 7, 15-18 [in Polish].

24. Liu H., Li H., Ying T., Sun K., Qin Y., Qi D., 1998. Amperometric biosensor sensitive to glucose and lactose based on co-immobilization of ferrocene, glucose oxidase, β-galactosidase and mutarotase in β-cyclodextrin polymer. Anal. Chim. Acta 358, 137-144.

25. Nagata R., Clark S.A., Yokoyama K., Tamiya E., Karube I., 1995. Amperometric glucose biosensor manufactured by a printing technique. Anal. Chim. Acta 304, 157-164.

26. Nalini B., Narayanan S.S., 2000. Amperometric determination of ascorbic acid based on electrocatalytic oxidation using a ruthenium (III) diphenyldithiocarbamate-modified carbon paste electrode. Anal. Chim. Acta 405, 93-97.

27. Navarátil T., Kopanica M., Krista J., 2003. Anodic Stripping voltammetry for arsenic determination on composite gold electrode. Chem. Anal. (Warsaw), 48, 265.

28. Niculescu M., Mieliauskiene R., Laurinavicius V., Csöregi E., 2003. Simultaneous detection of ethanol, glucose and glycerol in wines using pyrroloquinoline quinone-dependent dehydrogenases based biosensors. Food Chem. 82, 481-489.

29. Pandey P.C., Aston Robert W., Weetall Howard H., 1995. Tetracyanoquinodimethane mediated glucose sensor based on a self-assembling alkanethiol/phospholipid bilayer. Biosensors Bioelectronics 10, 669-674.

30. Pandey P.C., Upadhyay S., Upadhyay B., 1997. Peroxide biosensors and mediated electrochemical regeneration of redox enzymes. Anal. Biochem. 252, 136-142.

31. Pawełkiewicz J. 1954. Fizykochemiczne metody w analizie żywnosci [Physical chemistry in food analysis]. PWN, Poznań [in Polish]

32. Pournaghi-Azar M.H., Ojani R., 1995. Catalytic oxidation of ascorbic acid by some ferrocene derivative mediators at the glassy carbon electrode. Application to the voltammetric resolution of ascorbic acid and dopamine in the same sample. Talanta 42, 1839-1848.

33. Pournaghi-Azar M.H., Ojani R., 1997. A selective catalyticvoltammetric determination of vitamin C in pharmaceutical preparations and complex matrices of fresh fruit juices. Talanta 44, 297-303.

34. Przybyt M., 1999. Zastosowanie biosensorów wnalizie srodków spożywczych [Application of biosensors in analysis of food products]. Przem. Spoż. 6, 32-35 [in Polish].

35. Przybyt M., 2001. Biosensory – podstawy teoretyczne [Biosensors – theoretical background] in: Metody pomiarów i kontroli jakosci w przemysle spożywczym i biotechnologii [Measurement and quality control method in food industry and biotechnology]. Jankiewicz M., Kędzior Z. (eds.), Wyd. AR, Poznań.

36. Qian J.M., Suo A.L., Yao Y., Jin Z.H., 2004. Polyelectrolyte-stabilized glucose biosensor based on woodceramics as electrode. Clin. Biochem. 37, 155-161.

37. Qijin W., Nianjun Y., Haili Z., Xinpin Z., Bin X., 2001. Voltammetric behavior of vitamin B2 on the gold electrode modified with a self-assembled monolayer of L-cysteine and its application for the determination of vitamin B2 using linear sweep stripping voltammetry. Talanta 55, 459-467, Talanta 56, 1167.

38. Saurina J., Hernández-Cassou S., Fàbregas E., Alegret S., 2000. Cyclic voltammetric simultaneous determination of oxidizable amino acids using multivariate calibration methods. Anal. Chim. Acta 405, 153-160.

39. Schuhmann W., 1995. Electron-transfer pathways in amperometric biosensors. Ferrocene-modified enzymes entrapped in conducting-polymer layers. Biosensors Bioelectronics 10, 181-193.

40. Szczepaniak W., 2002. Metody instrumentalne w nalizie chemicznej [Instrumental methods in chemical analysis]. PWN, Warsaw [in Polish].

41. Tká J., Vostiar I., Sturd’ık E., Gemeiner P., Mastihuba V., Annusd J., 2001. Fructose biosensor based on d-fructose dehydrogenase immobilised on a ferrocene-embedded cellulose acetate membrane. Anal. Chim. Acta 439, 39-46.

42. Warriner K., Higson S., Christie I., Ashworth D., Vadgama P., 1996. Electrochemical characteristics of two model electropolymerised films for enzyme electrodes. Biosensors Bioelectronics 11 (6/7), 615-623.

43. Wawrzyniak J., Ryniecki A., Zembrzuski W., 2005. Application of voltammetry to determine vitamin C in apple juices. Acta Sci. Pol., Technol. Aliment. 4 (2), 5-16.

44. Yang Q., Atanasov P., Wilkins E., 1999. Needle-type lactate biosensor. Biosensors Bioelectronics 14, 203-210.

45. Zhou D. M., Ju H. X., Chen H.Y., 1997. A miniaturized glucose biosensor based on the coimmobilization of glucose oxidase and ferrocene perchlorate in nafion at a microdisk platinum electrode. Sensors Actuators B 40, 89-94.

Accepted for print: 11.12.2006

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