Flow injection system for potentiometric determination of alkaline phosphatase inhibitors

Analytica Chimica Acta 577 (2006) 134–139

Flow injection system for potentiometric determination of alkaline phosphatase inhibitors Robert Koncki ∗ , Katarzyna Rudnicka, Łukasz Tymecki University of Warsaw, Department of Chemistry, Pasteura 1, PL-02-093 Warsaw, Poland Received 3 February 2006; received in revised form 10 May 2006; accepted 14 May 2006 Available online 17 June 2006

Abstract A simple flow injection system for potentiometric detection of alkaline phosphatase (ALP) activity has been developed and adapted for determination of selected inhibitors of this enzyme. In this system monofluorophosphate (MFP) has been applied as a specific ALP substrate. The use of this substrate enables application of fluoride ion selective electrode (FISE) as a detector of the product of the enzyme catalyzed reaction. Moreover, chemical stability and low cost of MFP enables the use of the substrate as a component of the carrier. This way, fluoride ions contained in this substrate define and stabilize baseline signal generated by the detector. Effects from several potential ALP inhibitors and interfering species were studied and discussed. The system allows inhibitive detection of beryllium and vanadate ions at ppb levels with relatively high selectivity, short time of analysis and high throughput of the system (near 8 samples h−1 ). © 2006 Elsevier B.V. All rights reserved. Keywords: Flow injection analysis; Fluoride ion selective electrode; Alkaline phosphatase; Inhibitors; Beryllium; Vanadate

1. Introduction Alkaline phosphatase (EC 3.1.3.1, ALP) is commonly assayed enzyme in the clinical practice, because its blood activity significantly rises in case of many skeletal and liver diseases. Moreover, ALP is often applied as biocatalytic active label in immunohistology, immunoblotting and various immunoassays, including immunosensing devices. The popularity of ALP is based on its low cost, high stability, high turnover-rate, relatively small size and the large number of commercially available ALP conjugated immunoreagents. Owing to the same reasons this enzyme is often used as a marker in genosensing devices. Detection of ALP activity also has applications in bioanalytical methods and biosensors dedicated to determine the enzyme inhibitors and (re)activators [1–14]. Free and immobilized ALP was applied for fluorimetric inhibitive determination of cyanide anions [1]. Reversible inhibition of ALP by vanadate and arsenate anions was investigated using capillary electrophoresis with laser-induced fluorescence detection [2]. In the course of this study it was found that EDTA influences ALP as irreversible

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inhibitor or as an activator depending on its concentration. Similar electrophoretic investigations demonstrated high inhibitive effect from theophiline on the ALP activity [2,3]. Using fluorigenic substrate dedicated for ALP activity assay this drug was determined at therapeutic levels [4]. ALP activity is influenced by many metal ions. Fluorimetric inhibitive detection of silver ions at ppm levels was reported [1]. Simple visual determination of lead ions by inhibition of ALP immobilized on polyurethane foam was possible using p-nitrophenylphosphate (as ALP substrate), malachite green and molybdate as colorforming system [5]. ALP-catalyzed chemiluminescence was used for inhibitive detection of Zn, Bi and Be ions [6,7]. The same bioanalytical system was also used for detection of Zn ions as a reactivator of ALP apoenzyme. Amperometric ALPapoenzyme electrode was applied as a biosensor for Zn and Co ions [8]. Determinations of cadmium and lead cations with tissue optical algal biosensor were based on inhibition of ALP present on the external membrane of Chlorella vulgaris [9]. The same microalgae were applied for inhibitive detection of heavy metal ions in connection with conductometric device [10]. On the other hand, potentiometric ALP-based biosensor exhibited no sensitivity towards heavy metals ions (effects from Hg and Cu ions were tested), but was affected by some insecticides (carbofuran, aldicarb and heptachlor) [11]. Several voltam-

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metric and amperometric ALP-based biosensors were applied for inhibitive determination of a series of pesticides including carbamate, malathion, dichlorvos and 2,4-D [1,12,13]. Also optic fiber chemiluminescence-based biosensor with immobilized ALP was applied for inhibitive determination of paraoxon [14]. A variety of electrochemical and optical methods for ALP activity and consequently for ALP inhibitor detections is possible because of low biocatalytic selectivity of this enzyme. Unfortunately, in the cited investigations [1–14], ALP substrates are rather expensive (if commercially available) and often chemically unstable phosphates of electroactive, chromogenic, fluorigenic or chemiluminescent compounds. In the present study a potentiometric system for determination of ALP inhibitors based on monofluorophospate (MFP) as the enzyme substrate and fluoride ion selective electrode (FISE) as the detector has been investigated. This substrate is much cheaper than other commercially available ALP substrates and allows the application of inexpensive potentiometric equipment for the bioanalytical investigations. In this paper analytical characteristics of the developed FIA system based on this ALP/MFP/FISE (bio)sensing scheme will be presented and discussed. 2. Experimental 2.1. Reagents ALP isolated from bovine intestinal mucosa (powder, 15 U mg−1 ) was purchased from Sigma (USA). Activities of the enzyme solutions are given using enzyme activity units established with the use of p-nitrophenylphosphate—a common substrate recommended for spectrophotometric determination of ALP activity in the clinical settings. Disodium monofluorophosphate salt (MFP) was obtained from Aldrich (USA). TRIS (base form), theophilline and caffeine were obtained from Sigma (USA). Pesticide standards were purchased from IChO (Poland). Other reagents of analytical grade were obtained from POCh (Poland). Human serum

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standards were obtained from Cormay (Poland). Enzyme and substrate solutions were prepared immediately before use. All solutions were prepared with doubly distilled water. 2.2. Measurements The FIA setup used in this work is schematically shown in Fig. 1. To pump solutions the Minipuls 3 peristaltic pump (Gilson, France) was used. Line 1 carries inhibitor segment (total volume 0.5 mL) injected by rotary valve (VI). Line 2 carries ALP segment (total volume 0.1 mL) injected by rotary valve (VE). Both injection rotary valves were from Rheodyne (product no. 5020, USA). Distances between these valves and the lines connector are matched so that after mixing, when the valves are switched simultaneously, the enzyme/inhibitor segment is surrounded by the excess of inhibitor. Line 3 carries MFP solution in TRIS buffer. After mixing the obtained solution flows through reaction coil (RC). After RC and before detector (D) the solution is acidified by acetic acid carried by Line 4. Simple, laboratory-made, wall-jet type detector cell consisting of fluoride ion selective electrode (FISE, type 9409BN, Orion, USA) and a part of syringe is shown in the inset of Fig. 1. The electrode and a double junction saturated calomel reference electrode (Moller Glasblaserei, model RH-44/2-SD1) were coupled to digital pH-meter (model OP208/1, Radelkis, Hungary) connected with a data-collecting PC. 3. Results and discussion 3.1. Determination of ALP activity ALP is able to catalyze hydrolysis of MFP leading to formation of free fluoride ions, which could be detected using FISE [15]. Contrary to many other ALP substrates MFP is extremely cheap (less than 0.1 D g−1 ) and relatively stable in neutral solutions. Long-time (5 h) measurements performed under FIA conditions in several buffers (in pH range from 4 to 11) show that non-enzymatic hydrolysis of MFP is negligible. In the course of

Fig. 1. FIA setup (VI—inhibitor injection port, VE—enzyme injection port, RC—reaction coil, D—detector). Flow cell (with FISE) made of part of disposable plastic syringe is depicted in the inset.

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the stability tests baseline potential of FISE was constant. These observations stay in line with results obtained in the course of stationary measurements [15]. Effective hydrolysis of MFP only occurs under strongly acidic or alkaline conditions. This high stability and low cost cause that MPF could be used as a component of the carrier applied in the developed FIA system. On the other hand, the presence of fluoride ions in MFP improves quality of the FIA measurements by stabilizing the potential of the FISE, as these ions determine the baseline signal generated by the electrode before injection of enzyme/sample. However, the presence of free fluoride ions reduces the maximal signal, which could be measured with the FISE in the course of monitoring of MFP hydrolysis. In case of total conversion of MFP into fluoride ions maximal decrease of the potential is limited to approximately 120 mV, because the content of free fluorides in the MFP reagent is around 1%. This limitation was observed and discussed in detail previously [15]. Following the name of the enzyme, optimal pH for its maximal biocatalytical activity ranges from pH 8 to 10. Such pH values of reaction solution are well defined by TRIS buffer. However, optimal pH for FISE ranges from pH 4 to 6. In more alkaline solutions corrosive processes of LaF3 membrane (formation of lanthanum hydroxide) take place. In well-buffered alkaline solutions the electrodes are still useful for determination of fluoride ions and monitoring of the enzymatic process, but with evidently higher detection limits and slightly smaller sensitivity [15]. To improve working conditions of FISE in the developed FIA system an additional channel (Line 4 in Fig. 1) was installed. 0.2 M acetic acid flowing through this channel reacts with TRIS forming acetate buffer that provides optimal condition for FISE operation. Primary experiments show that for enzymatic hydrolysis of MFP the value of pH near 9.0 is optimal. This result is consistent with earlier findings [15], but this study clearly confirms that a decrease of signal observed for higher pH values of TRIS buffer is caused by lowering of ALP activity, rather than changes of FISE characteristics. In the present investigations and in all cases FISE works at the same optimal pH defined by acetate buffer formed before the cell detector (Fig. 1). Concentration of the substrate determines sensitivity of the developed FIA system. Effect of MFP concentration on the response of the system is shown in Fig. 2. For 0.1 U mL−1 ALP activity under given FIA conditions optimal concentration of MFP is 0.10 mM. An increase in MFP concentration in the carrier solution causes an increase of level of background free fluoride ions (and therefore a decrease of the baseline potential shown in Fig. 2) that overlap fluorides generated in the course of enzymatic hydrolysis of MFP. This means that the same amounts enzymatically generated fluorides cause smaller changes of the FISE potential. On the other hand, the decrease of signal (peaks height) for lower MFP levels is connected with sub-nernstian sensitivity of FISE observed at such low concentrations. Moreover, for very low MFP concentrations, effects connected with depletion of the substrate in the carrier zone of the enzyme reaction could be observed. Similar effects from MFP concentration were observed in the course of measurements performed under stationary conditions [15].

Fig. 2. Effect of MFP concentration in the carrier on the response of the system for ALP (0.1 U mL−1 ). In this experiment VI was not used.

Taking into account that enzyme activity measurements have kinetic character, the effects from flow rate and length of the reaction coil are not surprising. A decrease of the flow rate as well as an increase of the RC length causes an increase of time of the biocatalytic reaction. In consequence, more MFP is enzymatically converted into fluoride ions. On the other hand, at slow flows and long flow distances the resulting sensitivity does not increase significantly because of the sample dispersion. Additionally, a sample throughput obviously decreases and stability of the system is worse due to electric noises. The elongation of the enzyme reaction time without undesired dispersion effects is possible in the course of stop-flow mode of measurements; however, such operations are convenient rather in SIA than FIA systems. Reassuming, the lowering of the detection limit of ALP activity (and in consequence further higher sensitivity of the bioanalytical system towards ALP inhibitors) is possible, however at the expense of time of the analysis. Evaluation of optimal conditions is a result of compromise among the needs of high sensitivity and short time, acceptable reproducibility and accuracy of the measurements. Such optimization could be easily performed for specific analyte and sample. The main goal of this study is a general characterization of the bioanalytical system in respect of the ALP inhibitors determination and evaluation of its advantages and disadvantages. Further experiments were carried out at 3.8 mL min−1 final flow rate, using 7.2 m long reaction coil and 0.1 mM concentration of MFP in 0.1 M TRIS carrier in Line 3. As a carrier in Lines 1 and 2 water was applied. 0.2 M acetic acid, pumping through the channel 4, caused that the final pH at FISE was 4.8. Response of the system for ALP under these conditions is illustrated by Fig. 3a. Possible frequency of injections enables analysis around 8 samples h−1 . The baseline is stable, drift- and noise-free. It is worth noticing that under given conditions serum ALP activities at physiological and pathological levels could be easily determined (Fig. 3b). Application of the FIA system for clinical analysis will be presented in the future in the form of separate contribution.

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Fig. 3. Response of the developed FIA system on ALP activity (a), and physiological (NP) and pathological (HP) human serum standards (b). In this experiment valve VI was not used.

3.2. Determination of ALP inhibitors Analytical applications of ALP inhibition reported in Section 1 of this paper were demonstrated in experiments performed under various conditions, using different ALP substrates and determination methods of the respective products. Owing to differences in nature of substrates (P–F versus P–O bonds), and conditions of assays (differences in kind, pH and concentration of the used buffers, in the temperature as well as in the presence/absence of activating/stabilizing compounds) reported in the literature [1–14] and in this paper, the utility of the developed FIA system is difficult for comparison. Moreover, some effects especially from cations could be changed by some side reactions, i.e. complexation with MFP or fluoride ions. Finally, the inhibitive detections have kinetic character in two additional aspects. Both, the time of inhibition and the time of enzyme activity detection as well as conditions of these experimental steps decide about ranges of determination of any inhibitor. Taking into account all these aspects we decided to investigate response of our bioanalytical system on the effects from a majority of ALP inhibitors reported until now in the analytical papers [1–14]. Firstly, effects from selected metal cations were investigated. In the primary experiments inhibition processes were performed off-line. After defined incubation time samples of enzyme (0.1 U mL−1 ) and inhibitor (0.02 mM) in 0.11 M TRIS buffer (pH 9.3, without MFP) were injected into FIA system through the VE injection valve. In these experiments VI valve was not used. This way, the FIA system was used only for determination of fluoride ions generated in the course of reaction catalyzed by residual ALP activity. The enzyme reaction takes place in RC and its time was defined by the length of this coil. Results of these experiments for a series of metal ions tested as potential ALP inhibitors are shown in Fig. 4 as percentage of the signal obtained in the course of injection of the enzyme solution without any inhibitor. In general, all tested cations can be divided into four groups according to the kinetics of the observed effects. Large number of ions including Na, K, Ca, Mg as well as Ag, Pb, Cu, Cr, Mn

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and Zn did not influence the signal generated by the bioanalytical system. Bismuth and mercury ions, as well as solutions of nitric acids, used for preparation of these metal ions standards (but without them), suppressed peaks from ALP and these effects increased with the time of the off-line incubation. Al(III) and Be(II) ions decreased peaks height and this effect was practically independent of the time of the off-line incubation. Analysis of data shown in Fig. 4 leads to conclusion that mercury ions are stronger inhibitors than Be(II) ions. However, Be(II) causes very fast ALP inhibition. After 6 min (first points in Fig. 4) the process is complete, whereas Hg ions still react with the enzyme. Finally, uncommon effects were observed for Ni(II) and Co(II) ions. The corresponding peaks were higher than those obtained in the absence of the metal ions. Main investigations were performed using both valves of the FIA system schematically depicted in Fig. 1. This way, the whole procedure of ALP inhibitor determination is automated. Moreover, measurement procedure is shorter and more reproducible than in the previous experiments. In these experiments enzyme (0.1 U mL−1 ) and inhibitor standards were injected simultaneously by the valves VE and VI, respectively. In such system inhibition and enzyme activity detection steps were not separated. Both of them take place in the same time. The time of the whole process (defined by the flow rate and RC length) was shorter (around 4 min) than in the course of the previous experiments. Obviously this shortening of the time causes a decrease of the sensitivity. On the other hand, such methodology of measurements could result in an improvement of the selectivity of the inhibitive determinations because slower processes should be discriminated. In other words, slowly interacting species cannot be detected. For example, influences from acidic solutions used for preparation of mercury and bismuth ions standards were not detected as slower than inhibition processes caused by the metal ions (see Fig. 4). This way, the system allows determina-

Fig. 4. Effect of enzyme-inhibitor incubation time on the response of the system. Signal is defined as percentage ratio of peak heights obtained in the presence and absence of an inhibitor. Off-line inhibition performed in solutions containing 20 ␮M metal ion and 0.1 U mL−1 ALP in the absence of MFP. In this experiment VI was not used. After inhibition process samples were injected using VE.

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Fig. 5. Response of the FIA system on beryllium ions. Peaks for control injections without the inhibitor are shaded. Concentrations of the metal ion in the solutions injected through the VI valve are given in the figure. 0.1 U mL−1 ALP solution was injected through the VE valve. Corresponding calibration graph is shown in Fig. 8.

Fig. 7. Effect of selected cations (at 0.1 mM concentrations) on the baseline signal of the FIA system. These metal ion solutions were without (A) or with 0.01 mM KF (B). In these experiments ALP solution was not injected (VE was not used).

tion of mercury and bismuth ions not influenced by acidity of the used solutions. Typical calibration of the system is shown in Fig. 5. Between every two injections of inhibitor standard a single injection of water were done. This way, every third peak represents the response of the system for ALP without inhibitor. Such data illustrate well the stability and reproducibility of the signal (and baseline) generated by the developed system as well as are useful for the evaluation of the percentage of the inhibition. These are data for beryllium ions which exhibited the strongest effect in comparison to all other tested cations. Corresponding calibration graph is shown in Fig. 6. The calibrations were performed for 100 ␮M and smaller concentrations of the tested inhibitors. As can be seen from Fig. 6 noticeable effects were observed only for mercury(II), bismuth, cadmium, aluminum and nickel ions. The highest sensitivity was found for beryllium ions with

detection limit below 10 ppb (where signal change is smaller that 3%). These results confirm previous findings [6,7] that Be(II) is a potent ALP inhibitor. It is worth noticing that the determination of Be(II) is rather selective. Some heavy metal ions, sometimes reported as ALP inhibitors or activators, such as silver, copper, lead, cobalt, zinc and chromium, as well as alkaline cations (Na, K, Mg and Ca) do not interfere at relatively high levels (0.1 mM). Effects for higher concentrations were not investigated. Effects from the cations influencing the bioanalytical system (shown in Fig. 6) are significantly smaller than for Be(II). It seems to be possible to enhance the selectivity of the bioanalytical system towards beryllium ions by the use of appropriate reagents masking these interfering ions, however it requires further detailed investigations. The origin of the responses shown in Fig. 6 requires more detailed consideration. Relatively low sensitivity of the system towards some metal ions (namely Hg(I), Hg(II) and Bi(III)) is rather obvious taking into account slow kinetics of the inhibition (Fig. 4). Owing to the same reason the system was insensitive for much slower inhibition caused by acidity of some metal ion standards. On the other hand, the inhibition of ALP by Be(II) ions is quite fast and therefore well detected by the developed system. The obtained results clearly confirm high thermodynamic and kinetic bioaffinity of ALP to this inhibitor. The results for Al(III) and Ni(II) ions shown in Figs. 4 and 6 are surprising. At first glance, aluminum ions act as strong ALP inhibitor and nickel ions seem to be a potent ALP activator. Both these statements are wrong. These statements were excluded by two simple experiments. In the first of them 0.1 mM metal ion standards were injected into system whereas enzyme solution was not injected (VE was not used). In the course of such experiment only for Ni(II) and Al(III) ions peaks were recorded (Fig. 7A). Moreover, peaks for Al(III) ions were inverted. For other tested cations (including Be(II), Hg(II) and Cr(III) ions) no change of the baseline potential was recorded. These results clearly confirm that changes for Be(II) ions shown in Fig. 4 are

Fig. 6. Calibration graphs for selected metal cations. Signal is defined as percentage ratio of peak heights obtained in the presence and absence of an inhibitor.

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caffeine, formaldehyde and glycine were found. Only EDTA was found as weak ALP inhibitor. Its detection at 10 ␮M level was possible. This inhibiting effect is explained by extraction of Zn ions by EDTA from active center of ALP. Activation effects caused by lower concentrations of EDTA were not confirmed. Contrary to previous reports [1,11–14] the system was insensitive for pesticides. At 10−4 M concentrations no effects from selected chlorophenyl (2,4-D, 2,4,5-T, MCPA), carbamate (carbofuran, aldiarb) and phosphoorganic (dichlorvos, malathion, parathion) compounds were observed. These findings could be explained as previously by slow kinetics of ALP inhibition by these compounds or by protecting effect of MFP on ALP in case of these compounds. It could be concluded that the developed bioanalytical system enables detection of selected metal ions in the presence of organic toxic substances. 4. Conclusion Fig. 8. Calibration graphs for selected ALP-inhibiting anions. Signal is defined as percentage ratio of peak heights obtained in the presence and absence of an inhibitor.

not side-effects, but originated from ALP inhibition. In the second experiment (Fig. 7B), the same samples beforehand spiked with fluoride ions at 0.01 mM concentration were injected. This level of fluoride mimics reaction mixture after the hydrolysis of ca. 10% MFP presented in the carrier. The peaks recorded for all investigated cations were the same as for fluoride ion solution without any metal ions. Only in the presence of Al(III) ions the peaks were smaller and slightly deformed. For Ni(II) samples the recorded peaks were still higher than for reference samples. The results for the both considered cations indicate that the observed effects are not connected with the enzyme reaction. The effects observed for Al(III) ions are caused by the formation of fluoride complexes. Surprisingly, such side-effects are not observed for other ions (including Be(II) and Cr(III) ions) probably due to slower kinetics of the complexes formation process, although thermodynamic constants for complex formation by Be(II) and Al(III) ions with fluorides have comparable values. The mechanism of influences by Ni(II) ions is completely different. Results shown in Fig. 7 for Ni(II) ions clearly indicate that under given conditions these cations catalyzes hydrolysis of MFP independently of the ALP presence. This phenomenon was confirmed experimentally by monitoring the changes of fluoride ions concentration in the mixture of MFP and Ni(II) ions in TRIS buffer (without ALP). Similar but significantly weaker influences were observed from Co(II) ions. The developed biosensing system was also tested with several inorganic anions and organic species reported in Section 1 as potential ALP inhibitors. Procedures of these investigations were the same as in case of studies with metal cations. Typical anions commonly existed in real samples (nitrate, sulphate, chloride—all used as sodium salts) did not change the signal generated by the FIA system. The system was highly sensitive for arsenate, tungstate and vanadate anions (Fig. 8). Strong and fast inhibition caused by vanadate ions enables their determination at ppb levels. Surprisingly, no effects from theophilline,

Analytical characteristics of the ALP–MFP–FISE–FIA biosensing system and its application for ALP inhibitors determination has been reported in this paper. This bioanalytical system based on simple potentiometric equipment and easily available substrate and enzyme is especially attractive from economic point of view. Although the system has not been optimized, it allows relatively sensitive, selective and fast determination of beryllium and vanadate ions in the micromolar range of concentration. It was pointed that lower detections limits could be achieved; however, this kind of optimization has sense only in the context of specific, selected kind of samples and analytes. Acknowledgement This work was supported by the Polish Committee for Scientific Research (KBN-3-T09-034-28). References [1] F. Garcıa-S´anchez, A. Navas-Dıaz, M.C. Ramos-Peinado, C. Belledone, Anal. Chim. Acta 484 (2003) 45. [2] A.R. Whisnant, S.D. Gilman, Anal. Biochem. 307 (2002) 226. [3] A.R. Whisnant, S.E. Johnston, S.D. Gilman, Electrophoresis 21 (2000) 1341. [4] G.H. Sarpara, S.J. Hu, D.A. Palmer, M.T. French, M. Evans, J.N. Miller, Anal. Commun. 36 (1999) 19. [5] I.A. Veselova, T.N. Shekhovtsova, Anal. Chim. Acta 413 (2000) 95. [6] S.D. Kamtekar, R. Pande, M.S. Ayyagari, K.A. Marx, D.L. Kaplan, J. Kumar, S.T. Tripathy, Mater. Sci. Eng. C 3 (1995) 79. [7] S.D. Kamtekar, R. Pande, M.S. Ayyagari, K.A. Marx, D.L. Kaplan, J. Kumar, S.T. Tripathy, Anal. Chem. 68 (1996) 216. [8] I. Satoh, Y. Iijima, Sens. Actuators B 24–25 (1995) 103. [9] C. Durrieu, C. Tran-Minh, Ecotoxicol. Environ. Saf. 51 (2002) 206. [10] C. Chouteau, S. Dzyadevych, C. Durrieu, J.-M. Chovelon, Biosens. Bioelectron. 21 (2005) 273. [11] T. Danzer, G. Schwedt, Anal. Chim. Acta 318 (1996) 275. [12] Y. Su, A. Cagnini, M. Mascini, Chem. Anal. 40 (1995) 579. [13] F. Mazzei, F. Botre, S. Montilla, R. Pilloton, E. Podesta, C. Botre, J. Electroanal. Chem. 574 (2004) 95. [14] Z.P. Chen, D.L. Kaplan, H. Gao, J. Kumar, K.A. Marx, S.K. Tripathy, Mater. Sci. Eng. C 4 (1996) 155. [15] R. Koncki, D. Ogo´nczyk, S. Gł˛ab, Anal. Chim. Acta 538 (2005) 257.