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Controlled patterning of biomolecules on solid surfaces
Materials Science and Engineering C 23 (2003) 341 – 345 www.elsevier.com/locate/msec
Controlled patterning of biomolecules on solid surfaces Yves Martele´ a,1, Kristof Callewaert a, Kris Naessens b, Peter Van Daele b, Roel Baets b, Etienne Schacht a,* a
Department of Organic Chemistry-Polymer Material Research Group, Ghent University, Krijgslaan 281-S4, B-9000 Ghent, Belgium b Department of Information Technology, Ghent University, Sint-Pietersnieuwstraat 41, B-9000 Ghent, Belgium
Abstract The micropatterning of different enzymes on a solid surface to develop a multi-functional biosensor are discussed. A segmented polyurethane was used as photo-resist on a gold surface and irradiated with an ArF excimer laser (k = 193 nm) in order to obtain microarrays in the polymer structure. Alkanethiols (HS – (CH2)n – X) formed self-assembled monolayers (SAMs) on the revealed bare gold surface. Biomolecules (e.g. glucose oxidase (GOD)) were bound covalently to the end group of the SAMs. Different enzymes were attached on the same solid surface in a patterned way. To increase the sensitivity for detection of biomolecular species, multilayer films of glucose oxidase were formed on the solid surface. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Laser ablation; Patterning; Glucose oxidase; Biosensor
1. Introduction Patterning a surface impacts a number of technologies, including semiconductor circuitry [1], sensors [2], printing plates, tissue engineering and micromechanical devices [3]. Ultraviolet laser ablation is a powerful technique for patterning of very small features, which can be applied on a number of different materials. The interaction of the pulsed ultraviolet excimer laser irradiation with organic polymer surfaces leads to ablative photodecomposition, resulting in the etching of the polymer surface. This technology eliminates the need for photo-masks and allows material patterning in a ‘direct-write’ fashion. In previous work, we described the development and characterization of a series of segmented polyurethanes. These polymers were irradiated with UV excimer lasers [4,5]. In this study, a polymer is selected as photo-resist on a gold substrate and irradiated until the gold surface is reached. In a next step different biomolecules, e.g. enzymes, antibodies, can be attached on the bare gold by self-assembled monolayers (SAMs). The attachment of an enzyme to a solid surface induces a change in the 3D-conformation of the protein, which can result in a * Corresponding author. Tel.: +32-92644497; fax: +32-92644972. E-mail addresses: [email protected] (Y. Martele´), [email protected] (E. Schacht). 1 Fax: +32-9-2644972.
loss of activity. To increase the sensitivity, a multilayer is developed on the patterned surface. After the immobilization of glucose oxidase (GOD), the alternating layers are formed by consecutive adsorption of polycations and negatively charged proteins. In this way, we have a nano control over the thickness of the multilayer. The determination of the amount of glucose can be performed voltammetric by using GOD electrodes. The enzyme horseradish peroxidase (HRP) transforms H2O2, resulting in a direct electron transfer to the electrode substrate. Therefore, it would be interesting to pattern HRP next to enzymes (e.g. glucose oxidase) which produce H2O2 as side product during the enzyme reaction. In this work two different enzymes (glucose oxidase and peroxidase) are immobilized in a patterned way at the surface.
2. Materials and methods 2.1. Materials Segmented poly(carbonate –urethane) (SPU, Mw =264 000 g/mol, Aldrich), polyethylenimine (PEI, Mw = 25 000 g/mol, branched polymer, Aldrich), N-hydroxysuccinimide (Aldrich), 16-mercaptohexadecanoic acid (90%, Aldrich), 1dodecanethiol (98%, Aldrich), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulphonate (95%,
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 ( 0 2 ) 0 0 2 8 1 - 3
Y. Martele´ et al. / Materials Science and Engineering C 23 (2003) 341–345
342
tron microscopy (SEM) was used to characterize the patterned self-assembled monolayers.
3. Results and discussion 3.1. Laser ablation of segmented polyurethanes
Fig. 1. SEM picture of surface (width lines: 50 Am).
Aldrich, CMC), peroxidase (EC 1.11.1.7, Type VI-A from Horseradish, Aldrich), glucose oxidase (GOD, EC 1.1.3.4, Type VII-S from Aspergillus niger, Aldrich), sodium phosphate anhydrous (Acros), potassium phosphate monobasic (Acros), 2-(N-morpholino)-ethanesulfonic acid (MES, Acros), potassium chloride (KCl, Acros). 2.2. Methods Smooth samples were prepared by coating gallium arsenide wafers with a 100-nm-thick Au layer. The gold-substrates were functionalized with alkanethiol self-assembled monolayers (SAMs) terminated with – CH3 or – COOH. The samples were immersed for 3 h at room temperature in 1 mM solutions of the alkanethiol in ethanol and afterwards rinsed with ethanol. The functional endgroup (– COOH) of the SAMs was activated with 0.002 M CMC and 0.005 M Nhydroxysuccinimide in a 0.5 M phosphate buffer (pH = 5.5) for 1 h at room temperature. The enzyme (480 Am/ml) was covalently immobilized in a 0.1 M MES buffer for 2 h at room temperature.
In previous work [6], we have developed a series segmented polyurethanes to obtain a polymer with good ablation, in terms of no debris formation and good dimensional properties of the cavities, towards excimer lasers (ArF: k = 193 nm and KrF: 248 nm). The results reveal that segmented polyurethanes with aromatic building blocks, especially an aromatic diisocyanate (4,4V-diphenylmethane diisocyanate, MDI) showed very good properties, with a control over the depth of the cavity depending on the amount of pulses and energy density. The polymers have the highest absorption coefficient and the lowest threshold value towards the ArF excimer laser. In this work, we will use a commercially available segmented polyurethane, which is based on an aliphatic polycarbonate, namely poly(1,6-hexamethylene carbonate) diol, an aromatic diisocyanate (MDI) and 1,4-butanediol as chain extender. The polymer is spincoated from dioxane on a gold substrate and microarrays (width of the cavities = 50 Am) are ablated with an ArF excimer laser at low energy densities (E c 50 mJ/cm2). 3.2. Formation of self-assembled monolayers on gold surface It is know from literature [7] that alkanethiols (HS – (CH2)n –X) chemisorb spontaneously on a gold surface from solution forming self-assembled monolayers (SAMs). After the ablation of the polymer, an alkanethiol with a methylendgroup is attached on the patterned gold surface. After removing (stripping or dissolving) the remaining polymer
2.3. Multilayer formation The substrate with immobilized enzyme was immersed in a 0.1 M phosphate buffer and 2 M KCl solution (pH = 6.4) of polyethylenimine (1 mg/ml) for 10 min at room temperature. After rinsing with the phosphate buffer the substrate was immersed in a 0.1 M phosphate buffer (pH = 6.4) of the enzyme for 10 min at room temperature. The substrate was rinsed with the phosphate buffer. This procedure was repeated 10 times. The experiments were carried out with a Lumonics Pulse Master 848 (suitable for both KrF and ArF gas mixtures) and by means of an optical set-up. A Digital Nanoscope IIIa atomic force microscope (AFM) was used. The measurements performed in tapping mode and under air conditions were done with a scan rate of 0.1 Hz (type of probe: OTESPA-70, L = 160 Am). Scanning elec-
Fig. 2. SEM image of covalently patterned immobilized glucose oxidase (GOD).
Y. Martele´ et al. / Materials Science and Engineering C 23 (2003) 341–345
343
Fig. 3. AFM picture of the immobilised glucose oxidase.
coating, a second alkanethiol with a carboxylic acidendgroup is chemisorbed on the surface (Fig. 1). The SEM image clearly highlights the difference between the hydrophilic (light) and hydrophobic (dark) regions on the
surface, with very sharp edges between the two areas. If the alkanethiols have a reactive chemical functionality, such as a carboxylic acid, biomolecules can be bind covalently to the self-assembled monolayers.
Fig. 4. Phase image of immobilized GOD.
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Y. Martele´ et al. / Materials Science and Engineering C 23 (2003) 341–345
Fig. 5. Phase image of multilayer (GOD/PEI).
3.3. Patterning of biomolecules on gold surface
3.4. Immobilization of a co-enzyme
Enzymes (e.g. GOD, peroxidase) are bound to the selfassembled monolayer after activating carboxylic acid terminated SAMs in a phosphate buffer solution of 0.002 M CMC and 0.005 M N-hydroxysuccinimide. The results reveal that the enzyme (GOD) is covalently immobilized on the gold surface (Figs. 2 and 3). After the immobilization of the enzyme, it is possible that the conformation of the protein changes, which leads to a loss of enzyme activity. To increase the detection sensitivity of biomolecular species, multilayer films of glucose oxidase are fabricated in a patterned way on the solid surface. After the immobilization of glucose oxidase, the alternating layers are formed by consecutive adsorption of polycations (polyethylenimine, Mw = 25 000 g/mol) and negatively charged proteins (glucose oxidase) in a phosphate buffer solution (pH = 6.4). The advantage of the layer by layer (LBL) technique is the closepacked deposition of the sensing molecules in very thin layers. The morphology of the 10 bilayers of PEI/GOD LBL film is investigated with atomic force microscopy (Figs. 4 and 5). The phase image of the substrates clearly indicates the difference between the multilayer formation of the enzyme and the immobilised glucose oxidase. The root mean square (RMS) in the phase image of the multilayer formation is around 10.52j (scan size: 1 Am2, Fig. 5). The RMS of the surface with immobilised GOD is much lower, namely 0.414j (Fig. 4).
The literature [8] describes the determination of the amount of glucose by enzyme electrodes. This approach has been applied to investigate the activity of the immobilized enzymes. A mediator (e.g. benzoquinone) is used in the voltammetric determination of glucose. Some enzymes (e.g. peroxidase) allow direct electron transfer between the electrode and the enzyme [9]. The enzyme horseradish
Fig. 6. SEM picture of two patterned enzymes (left bar: peroxidase; right bar: glucose oxidase).
Y. Martele´ et al. / Materials Science and Engineering C 23 (2003) 341–345
peroxidase (HRP) transforms H2O2, leading to electron transfer to the electrode. The number of enzymes which allow direct electron transfer is very limited. Therefore, it would be interesting to pattern such enzymes next to enzymes (e.g. glucose oxidase) which produce H2O2 as side product during the enzyme reaction. In this work, two different enzymes (glucose oxidase and peroxidase) are immobilized in a patterned way at the surface. After ablating the polymer coating and immobilization of GOD on the SAM, the same procedure is repeated, leading to the immobilization of peroxidase (Fig. 6). Measuring the activity of the enzymes is still in progress.
4. Conclusions We demonstrated that laser ablation of segmented polyurethanes can be applied to pattern biomolecules on a solid surface. After irradiation of a polymer coating on a gold substrate with an ArF excimer laser, alkanethiols were chemisorbed in a patterned way on the bare gold. Different enzymes were bound covalent to self-assembled monolayers. To increase the enzyme activity multilayers were formed by using polycations.
345
Acknowledgements The authors wish to thank the Flemish Institute for the Promotion of Scientific-Technological Research in Industry (IWT), the Fund for Scientific Research-Flandern (FWO), the Research Board of Ghent University, Unite´ de Chimie des interfaces, Universite´ Catholique de Louvain (Department of Applied Physics).
References [1] T. Djenizian, L. Santinacci, P. Schmuki, Appl. Phys. Lett. 78 (19) (2001) 2940. [2] T. Von Woedtke, P. Abel, J. Kru¨ger, W. Kautek, Sens. Actuat. 42 (1997) 151. [3] T. Matsuda, Y. Nakayama, Biomed. Mater. Res. 31 (1996) 235. [4] T. Hannon, Med. Device Technol. (1999, Oct.) 34. [5] R. Srinivasan, B. Braren, Chem. Rev. 89 (1989) 1303. [6] Y. Martele´, K. Naessens, P. Vandaele, R. Baets, K. Callewaert, E. Schacht, J. Phys. Chem., B (2003) (submitted for publication). [7] G.M. Whitesides, Angew. Chem., Int. Ed. Engl. 37 (1998) 550. [8] J.J. Gooding, V.G. Praig, E.A.H. Hall, Anal. Chem. 70 (1998) 2396. [9] T. Ruzgas, E. Csoregi, J. Emneus, L. Gorton, G. Marko-Varga, Anal. Chim. Acta 330 (1996) 123.
Controlled patterning of biomolecules on solid surfaces Yves Martele´ a,1, Kristof Callewaert a, Kris Naessens b, Peter Van Daele b, Roel Baets b, Etienne Schacht a,* a
Department of Organic Chemistry-Polymer Material Research Group, Ghent University, Krijgslaan 281-S4, B-9000 Ghent, Belgium b Department of Information Technology, Ghent University, Sint-Pietersnieuwstraat 41, B-9000 Ghent, Belgium
Abstract The micropatterning of different enzymes on a solid surface to develop a multi-functional biosensor are discussed. A segmented polyurethane was used as photo-resist on a gold surface and irradiated with an ArF excimer laser (k = 193 nm) in order to obtain microarrays in the polymer structure. Alkanethiols (HS – (CH2)n – X) formed self-assembled monolayers (SAMs) on the revealed bare gold surface. Biomolecules (e.g. glucose oxidase (GOD)) were bound covalently to the end group of the SAMs. Different enzymes were attached on the same solid surface in a patterned way. To increase the sensitivity for detection of biomolecular species, multilayer films of glucose oxidase were formed on the solid surface. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Laser ablation; Patterning; Glucose oxidase; Biosensor
1. Introduction Patterning a surface impacts a number of technologies, including semiconductor circuitry [1], sensors [2], printing plates, tissue engineering and micromechanical devices [3]. Ultraviolet laser ablation is a powerful technique for patterning of very small features, which can be applied on a number of different materials. The interaction of the pulsed ultraviolet excimer laser irradiation with organic polymer surfaces leads to ablative photodecomposition, resulting in the etching of the polymer surface. This technology eliminates the need for photo-masks and allows material patterning in a ‘direct-write’ fashion. In previous work, we described the development and characterization of a series of segmented polyurethanes. These polymers were irradiated with UV excimer lasers [4,5]. In this study, a polymer is selected as photo-resist on a gold substrate and irradiated until the gold surface is reached. In a next step different biomolecules, e.g. enzymes, antibodies, can be attached on the bare gold by self-assembled monolayers (SAMs). The attachment of an enzyme to a solid surface induces a change in the 3D-conformation of the protein, which can result in a * Corresponding author. Tel.: +32-92644497; fax: +32-92644972. E-mail addresses: [email protected] (Y. Martele´), [email protected] (E. Schacht). 1 Fax: +32-9-2644972.
loss of activity. To increase the sensitivity, a multilayer is developed on the patterned surface. After the immobilization of glucose oxidase (GOD), the alternating layers are formed by consecutive adsorption of polycations and negatively charged proteins. In this way, we have a nano control over the thickness of the multilayer. The determination of the amount of glucose can be performed voltammetric by using GOD electrodes. The enzyme horseradish peroxidase (HRP) transforms H2O2, resulting in a direct electron transfer to the electrode substrate. Therefore, it would be interesting to pattern HRP next to enzymes (e.g. glucose oxidase) which produce H2O2 as side product during the enzyme reaction. In this work two different enzymes (glucose oxidase and peroxidase) are immobilized in a patterned way at the surface.
2. Materials and methods 2.1. Materials Segmented poly(carbonate –urethane) (SPU, Mw =264 000 g/mol, Aldrich), polyethylenimine (PEI, Mw = 25 000 g/mol, branched polymer, Aldrich), N-hydroxysuccinimide (Aldrich), 16-mercaptohexadecanoic acid (90%, Aldrich), 1dodecanethiol (98%, Aldrich), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulphonate (95%,
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 ( 0 2 ) 0 0 2 8 1 - 3
Y. Martele´ et al. / Materials Science and Engineering C 23 (2003) 341–345
342
tron microscopy (SEM) was used to characterize the patterned self-assembled monolayers.
3. Results and discussion 3.1. Laser ablation of segmented polyurethanes
Fig. 1. SEM picture of surface (width lines: 50 Am).
Aldrich, CMC), peroxidase (EC 1.11.1.7, Type VI-A from Horseradish, Aldrich), glucose oxidase (GOD, EC 1.1.3.4, Type VII-S from Aspergillus niger, Aldrich), sodium phosphate anhydrous (Acros), potassium phosphate monobasic (Acros), 2-(N-morpholino)-ethanesulfonic acid (MES, Acros), potassium chloride (KCl, Acros). 2.2. Methods Smooth samples were prepared by coating gallium arsenide wafers with a 100-nm-thick Au layer. The gold-substrates were functionalized with alkanethiol self-assembled monolayers (SAMs) terminated with – CH3 or – COOH. The samples were immersed for 3 h at room temperature in 1 mM solutions of the alkanethiol in ethanol and afterwards rinsed with ethanol. The functional endgroup (– COOH) of the SAMs was activated with 0.002 M CMC and 0.005 M Nhydroxysuccinimide in a 0.5 M phosphate buffer (pH = 5.5) for 1 h at room temperature. The enzyme (480 Am/ml) was covalently immobilized in a 0.1 M MES buffer for 2 h at room temperature.
In previous work [6], we have developed a series segmented polyurethanes to obtain a polymer with good ablation, in terms of no debris formation and good dimensional properties of the cavities, towards excimer lasers (ArF: k = 193 nm and KrF: 248 nm). The results reveal that segmented polyurethanes with aromatic building blocks, especially an aromatic diisocyanate (4,4V-diphenylmethane diisocyanate, MDI) showed very good properties, with a control over the depth of the cavity depending on the amount of pulses and energy density. The polymers have the highest absorption coefficient and the lowest threshold value towards the ArF excimer laser. In this work, we will use a commercially available segmented polyurethane, which is based on an aliphatic polycarbonate, namely poly(1,6-hexamethylene carbonate) diol, an aromatic diisocyanate (MDI) and 1,4-butanediol as chain extender. The polymer is spincoated from dioxane on a gold substrate and microarrays (width of the cavities = 50 Am) are ablated with an ArF excimer laser at low energy densities (E c 50 mJ/cm2). 3.2. Formation of self-assembled monolayers on gold surface It is know from literature [7] that alkanethiols (HS – (CH2)n –X) chemisorb spontaneously on a gold surface from solution forming self-assembled monolayers (SAMs). After the ablation of the polymer, an alkanethiol with a methylendgroup is attached on the patterned gold surface. After removing (stripping or dissolving) the remaining polymer
2.3. Multilayer formation The substrate with immobilized enzyme was immersed in a 0.1 M phosphate buffer and 2 M KCl solution (pH = 6.4) of polyethylenimine (1 mg/ml) for 10 min at room temperature. After rinsing with the phosphate buffer the substrate was immersed in a 0.1 M phosphate buffer (pH = 6.4) of the enzyme for 10 min at room temperature. The substrate was rinsed with the phosphate buffer. This procedure was repeated 10 times. The experiments were carried out with a Lumonics Pulse Master 848 (suitable for both KrF and ArF gas mixtures) and by means of an optical set-up. A Digital Nanoscope IIIa atomic force microscope (AFM) was used. The measurements performed in tapping mode and under air conditions were done with a scan rate of 0.1 Hz (type of probe: OTESPA-70, L = 160 Am). Scanning elec-
Fig. 2. SEM image of covalently patterned immobilized glucose oxidase (GOD).
Y. Martele´ et al. / Materials Science and Engineering C 23 (2003) 341–345
343
Fig. 3. AFM picture of the immobilised glucose oxidase.
coating, a second alkanethiol with a carboxylic acidendgroup is chemisorbed on the surface (Fig. 1). The SEM image clearly highlights the difference between the hydrophilic (light) and hydrophobic (dark) regions on the
surface, with very sharp edges between the two areas. If the alkanethiols have a reactive chemical functionality, such as a carboxylic acid, biomolecules can be bind covalently to the self-assembled monolayers.
Fig. 4. Phase image of immobilized GOD.
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Y. Martele´ et al. / Materials Science and Engineering C 23 (2003) 341–345
Fig. 5. Phase image of multilayer (GOD/PEI).
3.3. Patterning of biomolecules on gold surface
3.4. Immobilization of a co-enzyme
Enzymes (e.g. GOD, peroxidase) are bound to the selfassembled monolayer after activating carboxylic acid terminated SAMs in a phosphate buffer solution of 0.002 M CMC and 0.005 M N-hydroxysuccinimide. The results reveal that the enzyme (GOD) is covalently immobilized on the gold surface (Figs. 2 and 3). After the immobilization of the enzyme, it is possible that the conformation of the protein changes, which leads to a loss of enzyme activity. To increase the detection sensitivity of biomolecular species, multilayer films of glucose oxidase are fabricated in a patterned way on the solid surface. After the immobilization of glucose oxidase, the alternating layers are formed by consecutive adsorption of polycations (polyethylenimine, Mw = 25 000 g/mol) and negatively charged proteins (glucose oxidase) in a phosphate buffer solution (pH = 6.4). The advantage of the layer by layer (LBL) technique is the closepacked deposition of the sensing molecules in very thin layers. The morphology of the 10 bilayers of PEI/GOD LBL film is investigated with atomic force microscopy (Figs. 4 and 5). The phase image of the substrates clearly indicates the difference between the multilayer formation of the enzyme and the immobilised glucose oxidase. The root mean square (RMS) in the phase image of the multilayer formation is around 10.52j (scan size: 1 Am2, Fig. 5). The RMS of the surface with immobilised GOD is much lower, namely 0.414j (Fig. 4).
The literature [8] describes the determination of the amount of glucose by enzyme electrodes. This approach has been applied to investigate the activity of the immobilized enzymes. A mediator (e.g. benzoquinone) is used in the voltammetric determination of glucose. Some enzymes (e.g. peroxidase) allow direct electron transfer between the electrode and the enzyme [9]. The enzyme horseradish
Fig. 6. SEM picture of two patterned enzymes (left bar: peroxidase; right bar: glucose oxidase).
Y. Martele´ et al. / Materials Science and Engineering C 23 (2003) 341–345
peroxidase (HRP) transforms H2O2, leading to electron transfer to the electrode. The number of enzymes which allow direct electron transfer is very limited. Therefore, it would be interesting to pattern such enzymes next to enzymes (e.g. glucose oxidase) which produce H2O2 as side product during the enzyme reaction. In this work, two different enzymes (glucose oxidase and peroxidase) are immobilized in a patterned way at the surface. After ablating the polymer coating and immobilization of GOD on the SAM, the same procedure is repeated, leading to the immobilization of peroxidase (Fig. 6). Measuring the activity of the enzymes is still in progress.
4. Conclusions We demonstrated that laser ablation of segmented polyurethanes can be applied to pattern biomolecules on a solid surface. After irradiation of a polymer coating on a gold substrate with an ArF excimer laser, alkanethiols were chemisorbed in a patterned way on the bare gold. Different enzymes were bound covalent to self-assembled monolayers. To increase the enzyme activity multilayers were formed by using polycations.
345
Acknowledgements The authors wish to thank the Flemish Institute for the Promotion of Scientific-Technological Research in Industry (IWT), the Fund for Scientific Research-Flandern (FWO), the Research Board of Ghent University, Unite´ de Chimie des interfaces, Universite´ Catholique de Louvain (Department of Applied Physics).
References [1] T. Djenizian, L. Santinacci, P. Schmuki, Appl. Phys. Lett. 78 (19) (2001) 2940. [2] T. Von Woedtke, P. Abel, J. Kru¨ger, W. Kautek, Sens. Actuat. 42 (1997) 151. [3] T. Matsuda, Y. Nakayama, Biomed. Mater. Res. 31 (1996) 235. [4] T. Hannon, Med. Device Technol. (1999, Oct.) 34. [5] R. Srinivasan, B. Braren, Chem. Rev. 89 (1989) 1303. [6] Y. Martele´, K. Naessens, P. Vandaele, R. Baets, K. Callewaert, E. Schacht, J. Phys. Chem., B (2003) (submitted for publication). [7] G.M. Whitesides, Angew. Chem., Int. Ed. Engl. 37 (1998) 550. [8] J.J. Gooding, V.G. Praig, E.A.H. Hall, Anal. Chem. 70 (1998) 2396. [9] T. Ruzgas, E. Csoregi, J. Emneus, L. Gorton, G. Marko-Varga, Anal. Chim. Acta 330 (1996) 123.