Biosensors and Chemical Sensors - ACS Publications - American

Chapter 13

Downloaded by UNIV OF SOUTHERN CALIFORNIA on December 8, 2014 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch013

Electrochemically Prepared Polyelectrolyte Complex of Polypyrrole and a FlavinContaining Polyanion Use as a Biosensor H. F. M . Schoo and G. Challa Laboratory of Polymer Chemistry, University of Groningen, Nijenborgh 16, 9747 AG Groningen, Netherlands Pyrrole was polymerized electrochemically in an aqueous medium on a platinum electrode in the presence of a polyanion, containing covalently bound flavin units. Depending on the applied potential the polypyrrole -films thus formed were powdery and non-adherent (V < 700 mV), smooth and adherent (700 mV < V< 1000 mV) or brittle and uneven (V > 1000 mV). Besides the applied potential, medium pH and added low molar mass salt influenced the morphology and composition of the PPy -layer.The flavin-containing polyanion was incorporated in the film as a dopant. Depending on the medium pH and amount of low molar mass salt added, the film formed by this method contained various amounts of polymer-bound flavin. Cyclic voltammograms confirmed the presence of electrochemically active flavin in the layer. The oxidation of 1benzyl-1,4-dihydronicotinamide (BNAH) was used as a model reaction to test the catalytic activity of the immobilized flavin. The modified electrode showed fast current response on addition of B N A H , not only at an applied potential of 0.9 V (oxidation of H O ) but also in the absence of oxygen at 0.3 V . This might indicate that a direct transfer of electrons takes place between the flavin units and the (PPy-)electrode. Cycling of the PPy-film through its reduced and oxidized state does not lead to any loss of the flavin containing polyanion. In the case of low molar mass flavin-containing dopants most of the flavin is released from the film upon reduction of the PPy. 2

2

One of the main motivations for the development of chemically modified electrodes is the introduction of (electro-) catalytic species onto the electrode surface. These modified electrodes can be used to improve specificity and product yields in electro chemical synthesis or as the basis for a biosensor. Catalysts can be attached through (irreversible) adsorption onto a suitable substrate (i). These systems mosdy consist of an electrode covered with a monolayer of the electroactive species. In many cases this method does not lead to effective systems, due to instability of the monolayer or low loading with the catalyst. Direct modification of the electrode surface by covalent 0097-6156/92/0487-0164$06.00/0 © 1992 American Chemical Society

In Biosensors and Chemical Sensors; Edelman, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


13. SCHOO&CHALLA

Electrochemically Prepared Polyelectrolyte Complex

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binding of active moieties is also possible, but it often has drawbacks such as laborious synthesis and poor stability of the modified electrode. A more favourable approach is the incorporation of the active species in an electrically conducting polymer layer which then acts as an (electrical) intermediate between the electrode surface and the catalyst. Polypyrrole is considered to be especially suitable because it is acceptably stable under ambient conditions (2), has a high conductivity and can be easily prepared electrochemically from a great variety of solvent systems, including aqueous solutions (3-5). The catalytic species that have been applied in such polypyrrole-based systems comprise metal particles (6-9), metal chelates (10-13) (with anionic side groups) and enzymes (14-18). When redox enzymes are employed, direct electrical communication between the redox centres and the electrode is often inhibited by the insulating protein shell, surrounding the active centre of the enzyme. In this case low molar mass redox couples may be employed (1920) as mediators, but unless they are covalently bound (21) (to the enzyme) practical applications will be strongly limited due to release of the mediator into the surrounding medium, with subsequent loss of responsivity of the electrode system. In many cases the redox centre of the applied enzymes is flavin. Some efforts have been made to obtain a modified electrode, containing immobilized flavine units (22-25), but in all cases the catalyst was bound covalently to the electrode surface. Another interesting point is the possibility to incorporate polyanions as dopants in polypyrrole films (26-28), which are very effectively immobilized in the conducting polymer film. Here we describe the use of a catalytically active polyanionic dopant, containing covalently bound flavin moieties, in the formation of a flavin-containing electrode and its application as a biosensor. EXPERIMENTAL Apparatus and materials. A l l electrochemical polymerizations, amperometric measurements and cyclic voltammetry were carried out with an E G & G Princeton Applied Research Potentiostat model 273. Pyrrole was purified by vacuum distillation and by passage over neutral alumina prior to electropolymerization. The polyanions (1) used as dopants are shown in Figure 1, and were purified twice by precipitation from methanol in 0.1 M HC1. Detailed information about their synthesis and catalytic

Figure 1. Flavin-containing polyanions 1 (oc=0.10)


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properties will be published elsewhere (29). Sodium p-toluenesulphonate (NaOTs, Aldrich) was used without purification. l-Benzyl-l,4-dihydronicotinamide (BNAH) was synthesized as described elsewhere (30), and purified twice by crystallization from EtOH/H 0.


2

Procedures. Electropolymerizations were performed potentiostatically at +0.8 V (vs. Ag/AgCl) in an undivided cell with polished platinum electrodes and a saturated calomel reference electrode. For UV/VIS measurements on PPy-films Indium-Tinoxide (ITO) electrodes were used. For polymerization reactions aqueous (doubly distilled) solutions were thermostatted at 25.0°C and contained 0.1 M of pyrrole, 0.05 M COO(H)-groups (polymer 1) and 0-0.1 M sodium-p-toluenesulphonate. Solution pH was adjusted prior to the reaction, and kept constant during the reaction by addition of appropriate amounts of concentrated solutions of HC1 or NaOH. Prior to and during the polymerization nitrogen was bubbled through the stirred solution. PPy/(l)-films were rinsed with water/MeCN (5:1) mixtures and stored in doubly distilled water. Determination of the amount of polyanion 1 immobilized in the PPy-film, was performed indirectly by determining the amount of the polyanion left in the solution after the electropolymerization. The polyanion was recovered from the solution by complexation to an anion-exchanging macroporous polymer disk, containing quaternary ammonium groups (a detailed description of this procedure can be found elsewhere (57)). After washing with water/MeCN (5:1) (removal of pyrrole and soluble reaction products), the polyanion was decomplexed by flushing with acidic water/MeOH (1:2). The amount of polyanion could then be determined by means of UV/VTS-spectroscopy. Cyclic voltammetry and amperometric response experiments were performed in 0.05 M Tris buffer, Ph=8.0. RESULTS AND DISCUSSION Polymerization of pyrrole in aqueous medium. The polymerization of pyrrole was performed in aqueous medium. This imposes some restrictions upon the reaction conditions. The electiOpolymerization of pyrrole in the presence of polymer 1 in aqueous medium appeared to be very sensitive to the applied potential, especially when considering the effect upon the morphology of the PPy-film formed. Figure 2 shows typical current transient curves for the polymerization, starting with a clean, polished electrode. When potentials below 700 mV are applied, the resulting current is rather small and very brittle non-adherent films are formed, and in some cases only part of the electrode was covered with PPy. At potentials above 1000 mV transient currents initially are high, but decrease rapidly. The films formed are uneven and tend to lift off the electrode. If the potential is kept between these two values, smooth adhesive films were formed. In this case macromolecular assemblies are formed that might be described as polyelectrolyte complexes (32) (shown schematically in Figure 3). Influence of polymerization conditions upon incorporation of flavin-containing polyanion. The amount of polyanion (1) incorporated as dopant in a PPy film during electropolymerization can easily be controlled by changing the ionic strength (low molar mass salt) and/or the pH of the monomer solution. The correlation between the amount of polymer-bound flavin incorporated in the film and the concentration of the added low molar mass salt, sodium-p-toluenesulphonate (NaOTs), at pH=7 is shown


Electrochemically Prepared Polyelectrolyte Complex


13. SCHOO&CHALIA

Figure 3. Schematic representation of the macromolecular complexes of PPy and polymer (1)


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10 * [NaOTs]

(M)

Figure 4. Amount of incorporatedflavinvs. the concentration of low molar mass salt (sodium-p-toluenesulphonate) in the solution (pH=7)

in Figure 4. It can be seen that the addition of a low molar mass dopant anion has a pronounced influence upon the amount of polymer-bound flavin immobilized in the PPy film. Assuming that the degree of doping of the PPy remains about the same, this decrease in uptake of polymer 1 must be compensated by NaOTs. One might expect that the polymeric dopant would be preferred over low molar mass anions due to cooperative effects, which are known to be of great importance in polyelectrolyte interactions (33). It seems, however, that the low molar mass dopant is incorporated much easier than its polymeric antagonist, since low concentrations of NaOTs (about 0.01 M ; about one fifth of the concentration of polyanionic carboxyl groups) lead to a large decrease (factor 2) of polyanion incorporation. This might be due to kinetic restrictions during the polymerization and/or doping process, which are obviously more important for the polymeric dopant. Another important variable which determines the amount of incorporated polymeric dopant is the medium pH. As can be seen in Figure 5 the amount of polymer-bound flavin incorporated in the PPy-film decreases with higher medium pH during electropolymerization. This effect can be explained in terms of the degree of ionization of the polyanion (1). Since (1) is a weak polyacid, at low pH only few of the carboxyl groups are dissociated, whereas at high pH nearly all groups are ionized. This means that more of the polymer has to be incorporated at lower p H in order to provide a sufficient amount of dopant (-COO* groups). It is therefore not surprising that the addition of small amounts of NaOTs (0.01 M) at pH

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