effect of surfactants on the signal of chemically modified amperometric electrodes

ELSEVIER

SE@RS ACT~JA~ORS

B Sensors and Actuators B 2625 (1995) 323-327

CHEMICAL

Effect of surfactants on the signal of chemically modified amperometric electrodes

Geza Nagy ‘, Imre Kapui a, Lo Gorton b a Technical Universiry of Budapest, Inrfifule for General and Anaiyfical Chembhy, Technical Analyrical Research Group of the Hungarian

Academy of Sciences, G&W !er 4, 1111 Budapest, Hungary b Depotintent of Analytical Chemisty, University of Lund, S-221 00 Lund, Sweden

Abstract

A special electrochemical pretreatment has been employed for a finely polished glassy carbon electrode surface to increase the adsorption of Meldola blue (7-dimethylamino-1,2-benzo-phenoxazine) redox mediator. The chemically modified glassy

carbon electrode holding the adsorptive mediator layer can be used lo measure NADH concenlration amperometrically. The interfering effect of non-ionic surfactants has been studied in the case of NADH measurements using a rotating-disc electrode technique and flow-injection analysis (FIA) measurements. The glassy carbon electrode response is less affected and could be regenerated much more easily than the response of a similar spectrographic graphite-based electrode studied earlier. Surface-active agents can influence the apparent coverage of the surface by the mediator.

Keywords: Amperometric electrodes; Chemical modification; Sufactants

1. Introduction

Several widely used analytical methods are based on the catalytic action of NAD+/NADH redox coenzyme dependent different dehydrogenase enzymes. Up to now the NAD’iNADH dependent dehydrogenases rep- resent the most numerous group of known redox enzymes. Therefore it has been expected that, based on the detection of a concentration change of the coenzyme, a large group of biosensors, capable of measuring different substrates selectively, could be developed.

However, early attempts [1,2] to work out amperometric enzyme electrodes based on NADH detection revealed several major difficulties. These could be suc- cessfully overcome later by employing different elec- trocatalysts [3,4] on the electrode surface. In these cases the electrocatalyst mediates the NADH oxidation.

The influence of surfactant matrix component adsorption on the signal of biosensors is of practical importance, since surface-active additives are often used in flow analytical methods and in immunanalysis. The electrodes cannot be employed in these media as de- tectors if the additives interfere with their function.

Unfortunately, strong interference of surface-active agents was observed with the electrocatalytic oxidation of NADH on the surface of phenoxazine-modified

0925.4005/95/%09.50 0 1995 Elsevier Science S.A. All rights reserved SSDI 0925-4005(94)01368-R

spectrographic graphite electrodes. Skoog et al. [5] have found an almost complete blocking of the electrocatalytic NADH oxidation when certain surfactants were adsorbed on the surface of a phenoxazine-modified electrode.

According to their observation, the blocking was the result of the repulsive action of the adsorbed surfactant hindering the transport of the hydrophilic NADH molecules onto the electrode surface. Decrease of the amount of electroactive mediator adsorbed on the surface was not observed. The blocking appeared quite irreversible.

Benzophenoxazine-type mediator molecules do not adsorb well on a polished glassy carbon electrode, which is among the most often used electrodes in voltammetry. Reports [l&-8] appeared about different electrochemical pretreatments that could be used to modify the surface and properties of different electrodes. Elec- trochemical pretreatment can increase the adsorptivity of the glassy carbon surface.

Carrying out investigations in our laboratories with electrochemically pretreated glassy carbon electrodes, it was found that the well-known electrocatalyst mol- ecule, Meldola blue (7-dimethylamino-1,2-benzo-phenoxazine), strongly adsorbs on the treated electrode surface. The chemically modified electrode prepared

324 G. Nagy et al. I Senmrs and Achmtors B 24-25 (1995) 323-327

in this way showed good electrocatalytic properties for NADH oxidation. To study the potential applications, the well-known Meldola blue-modified spectrographic graphite electrode and the Meldola blue-modified glassy carbon electrode were compared with respect to surfactant interference with electrocatalytic NADH oxidation.

2. Experimental

Conventional, commercially available electroanalyt- ical measuring instruments and electrodes were used in the experiments. Home-made flow-injection manifold and flow-through detector cells were made for the flow- injection analysis (FIA) studies.

The working electrodes were made of glassy carbon rods with 3.0 mm diameter (Tokai Co., Japan) or of spectrographic graphite rods (KW 001; diameter 3.1 mm). In some of the experiments a glassy carbon electrode from p-Sensor was used. This electrode in- corporated a Sigradur K-type glassy carbon disc of 5 mm diameter.

Fig. 1. Cyclic voltammograms of glassy carbon electrode in pH 7

phosphate buffer. Sweep rate 100 mV s- ‘. a, freshly polished electrode;

b, electrochemically pretreated electrode; c, electrochemically pretreated electrode with adsorbed Meldola blue mediator film on the surface.

and +3.0 V peak potential with a sweep rate of 5 V s-l. After this, a constant polarizing potential of -0.5 V was applied for 60 s.

The chemicals used were of analytical grade. The Meldola blue was purchased from Aldrich, the Tween 20 from Sigma and the Triton X-100 from BDH Chem- icals.

After electrochemical pretreatment, the residual current increased substantially (Fig. 1, curve b). If the pretreated electrodes were soaked in diluted (e.g., 0.5 mgUO0 ml) Meldola blue solution, the cyclic voltammograms recorded after washing showed Meldola blue adsorption (Fig. 1, curve c). The surface coverage calculated from the current integral was in the range 2-8 nmol cm-‘.

3. Results and discussion

In our investigations the extent of adsorption of Meldola blue on a polished glassy carbon electrode surface was checked. The electrode was soaked in Meldola blue solution for different times. It was washed and cyclic voltammograms were recorded in phosphate buffer. None of the voltammograms showed noticeable electroactive material adsorption.

A completely different behaviour was observed, however, for glassy carbon electrodes after an intensive electrochemical pretreatment, as can be seen in Fig. 1. The electrochemical pretreatment changed both the electrochemical and the adsorption properties.

The formal redox potential of the Meldola blue adsorbed on a glassy carbon electrode surface measured in pH 7.0 phosphate buffer with scanning rates between 2 and 900 m V s-l was -201 mV versus SCE. This is slightly lower than the value (- 175 mV) reported [lo] for the same conditions for a spectrographic graphite electrode. The difference between the anodic and cathodic peak potentials at 5 mV s-’ scanning rate was 19 mV. Increasing the scanning rate, the difference gradually increased. (In the case of a reversible redox couple adsorbed on the electrode surface, the anodic and cathodic peaks should appear at the same potential

[91).

Electrochemical pretreatments are used in in vivo voltarnmetty to increase the sensitivity and improve the resolution of voltammetric electrodes. Many different pretreatment procedures are described in the literature. In our work, based on our previous experience with in vivo voltammetry, a few of these procedures were tried as a preliminary experiment to increase the Mel- dola blue adsorption on glassy carbon surfaces. The procedure below was found to be suitable for our purposes.

A difference was observed between the theoretically expected and the experimentally obtained log(peak height)-log(scanning rate) dependence of the voltammetric peaks measured with an adsorbed Meldola blue- modified glassy carbon electrode. For adsorbed reversible material, the dependence should show [9] a straight line with a slope of one, while for the diffusion- controlled case the Randles-Sevcik equation predicts a straight tine with 0.5 slope value. In our measurements a 0.8085 slope value was found, i.e., a value closer to the adsorptive character.

As a first step, the freshly polished electrode was The adsorbed Meldola blue acts as a mediator in continuously cycled for 120 s in 10 ml deoxygenated the electrocatalytic oxidation of NADH in pH 7.0 phosphate buffer at pH 7.0 between -0.5 V starting phosphate buffer. This could clearly be proven by cyclic

G. Nqy et al. I Sensors and Acfuafors B 24-25 (1995) 3.2-327 325

voltammetric, rotating-disc and FIA experiments. The cyclic voltammograms of the mediator adsorbed on an electrochemically pretreated glassy carbon electrode surface are shown in Fig. 2. Curve a was taken in the absence of NADH, while curve b in the presence of 1.52 mM NADH concentration. In the measurements the actual NADH concentration was determined spec- trophotometrically with 1 cm cuvettes at 340 nm (mo- lecular absorbance coefficient 6220). Plotting the amperometric current measured at 0.0 V (versus SCE) as a function of NADH concentration, a calibration curve can be drawn, on the basis of which the NADH concentration can be measured amperometrically. In the case of FIA measurements the calibration curve is drawn by plotting the amperometric peak height as a function of the NADH concentration of the solutions injected. The Meldola blue-modified electrode can serve as an amperometric detector in certain enzyme analytical measurements, or as a basic sensing element for certain chemical biosensors.

The adsorbed Meldola blue layer was quite stable. A glassy carbon-based chemically modified electrode could be used for NADH measurements for more than a week, however daily recalibration is recommended. The stability of different mediator layers, among them Meldola blue on a spectrographic graphite electrode, was studied earlier by Gorton and his research group. The reproduciblity of NADH determination with a Meldola blue-modified glassy carbon electrode was studied in FIA measurements, injecting 250 ~1 doses of 10m3 M samples. In this condition a relative standard deviation of 0.89% was found.

In our studies the glassy carbon-based (Figs. 4 and 6) and the spectrographic graphite-based (Figs. 3 and 5) Meldola blue-modified electrodes were compared concerning their amperometric NADH-measuring properties in the presence of non-ionic surfactants. The results are shown in Figs. 36

Fig. 2. Cyclic voltammograms of electrochemically pretreated glassy Fig. 5. Effect of surface-active agent on the electrocatalytic NADH carbon-basedchemicallymodified electrode in pH 7 phosphate buffer. measurement, Working electrode, spectrographic graphite electrode Sweep rate 5 mV s-‘, Concentration of NADH: a, 0; b, 1.52 mmol modified with Meldola blue. Electrode potential, 0 mV. Background I-‘. electrolyte, phosphate buffer pH 7.

Fig. 3. Amperometriccurrentvs. NADHconcentration curves obtained with chemically modified rotating-disc electrode. Working electrode, spectrographic graphite electrode modified with Meldola blue. Elec- trode potential, 0 mV, rotating speed, 152 rad s-‘. Background electrolyte, phosphate buffer pH 7: a, freshly prepared electrode, in

the absence of surfactant; b, in the presence of TWEEN 20 (c= 13.6Xcritical micelle concentration (cmc)); c, after intensive washing, and in the absence of surfactant.

2

I

Fig. 4. Amperometric current vs. NADH concentration curves obtained with chemically modified rotating-disc electrode. Working electrode, electrochemically pretreated glassy carbon electrode modified with

Meldola blue. Electrode potential, 0 mV, rotating speed, 152 rad s-‘.Background electrolyte,phosphatebufferpH7,a,freshlyprepared

electrode, in the absence of surfactant; b, in the presence of TWEEN 20 (c= 136xcmc); c, after intensive washing, and in the absence of surfactant.

326 G. Nagy et 01. I Sen.wrr and Acfuntors Ii 24-25 (1995) 323-327

Fig. 6. Effect of surface-active agent on the electrocatalytic NADH measurement. Working electrode, electrochemicallypretreated glassy

carbon electrode modified with Meld& blue. Electrode potential, 0 mV. Background electrolyte, phosphate buffer pH 7.

In the rotating-disc electrode experiments (Figs. 3 and 4) the freshly prepared electrodes were calibrated with standard NADH solution in pH 7.0 phosphate buffer at 0.0 V electrode potential (Figs. 3 and 4, curve a). The calibration was repeated in the presence of surfactants (Figs. 3 and 4, curve b). After this the cell and the electrodes were intensively washed and the calibration was repeated in surfactant-free background electrolyte (Figs. 3 and 4, curve c).

From the calibration curves it could be concluded that non-ionic surfactants (Tween 20) decrease the amperometric response of both electrodes, but to different extents. The decrease in the case of a glassy carbon-based electrode (Fig. 4) was smaller. For example, in the case of 218.8 mg I-’ (13.6 X critical micelle concentration (cmc)) Tween 20 concentration, the decrease was just slightly over 30%, while the decrease for the spectrographic graphite-based electrode was over 95%. The electrodes behave differently with respect to the regeneration of their NADH-measuring activity. After intensive washing the calibration curve of the glassy carbon electrode (Fig. 4, curve c) is almost the same as it was originally, while almost no regeneration of the original NADH-measuring activity of the spectrographic graphite electrode could be achieved by washing (Fig. 3, curve c).

Considering the interfering effect of non-ionic surfactants, the glassy carbon-based chemically modified electrode has definite advantages over the spectrographic graphite-based one. The extent of interference is less and the regeneration can be carried out easily by washing. These advantages can gain specific importance when the electrode is employed in flow measurements such as FIA.

In order to study the differences mentioned in flow conditions, the experiments described before were repeated in FIA conditions. In these measurements pH 7.0 phosphate buffer was streamed through the analysis channel at a flow rate of 1.6 ml min-‘. The previously

prepared Meldola blue-modified working electrode was introduced into the wall jet cell and the amperometric current was recorded at 0.0 V while NADH solutions of known concentration (10m3 M) were periodically injected with an injecting frequency of l/120 Hz. Be- tween two injections, 3.2 ml of clear background solution washed the electrode and the internal walls of the tubing. The height of the amperometric current peaks was evaluated. To study the interfering effect of the surfactants, after recording a few NADH peaks doses of surfactant solutions were injected using the same injection loop. Following the surfactant solution, doses of the NADH standards were injected again, and the height of the obtained peaks was evaluated.

The recorded curves (Figs. 5 and 6) showed that the injection of surfactant influences the catalytic activity at both electrodes. The peak height decreased in both cases after the non-ionic surfactant injection. However, the surface-active agent blockage is much more pro- nounced if porous spectrographic graphite is used (Fig. 5). For example, after injecting 250 yl of 8.75 g I-’ Tween 20 solution, the peak height decreased to 11.3% of the original value. For the glassy carbon-based electrode (Fig. 6) this value was 87.3% in the same conditions.

Differences appeared between the recovery properties of the two working electrodes. While the electrocatalytic activity of the glassy carbon-based electrode recovered almost completely in a relatively short time, the recovery of the spectrographic graphite electrode was a much slower process. Streaming surfactant-free background solution through the analysis channel for about an hour resulted only in the recovery of 22% of the original activity of the spectrographic graphite electrode.

The effect of Triton was similar, but the surfactant blockage of the catalytic NADH oxidation was less extensive. Experimental results with Triton are listed in Table 1.

Why do the twochemically modified electrodes behave differently with respect to the nature of surface blockage of the NADH response? It is believed that the surfactant

Table 1 Effect of Triton on the electrocatalytic NADH measurement. Values are given 8s a percentage of the peak height measured before Triton injection. Flow rate, 1.6 ml min-‘; loop volume, 250 ~1; concentration of Triton, 0.16 M; concentration of NADH, 10 -’ M

Number of NADH injections after Triton injection

1 2 15

Spectrographic graphite-based electrode

7.9 15.2 62.5

Glassy carbon- based electrode

71.6 86.3 94.3

G. Nngy et al. / Sensors and Actuators B 24-25 (1995) 323-327 327

acts by hindering the transport of the NADH towards the electrode surface. The adsorbed surfactant repels the hydrophilic NADH molecules, so their transport to the mediator decreases. This is more extensive in the case of the porous spectrographic graphite electrode, where the electrocatalysis proceeds in a relatively thick layer. The repulsive forces cannot have such a strong influence in the case of the polished glassy carbon electrode because here the electrocatalytic reaction takes place in a thin film. The regeneration of the catalytic activity in this case means the removal of the repulsive surfactant layer. It is obvious that the de- sorption of the surfactant from a polished surface is easier and faster than from a porous spectrographic graphite surface.

Skoog et al. [5] did not observe changes in the mediator surface coverage after surfactant adsorption. In our investigations the surface coverage was measured by recording cyclic voltammograms and measuring the current integral under the peaks. In the contrary to Skoog et al., we observed changes in the coverage as an effect of surface-active agents. The apparent surface coverage of Meldola blue adsorbed on the spectrographic graphite electrode decreased after soaking the electrode in Tween 20 solution. However, the decrease of the coverage was a slow process, while the blockage of the catalytic NADH oxidation was almost complete after a few seconds. It is very likely that the small decrease of coverage cannot cause the massive blockage observed in the case of Meldola blue-modified electrodes.

Interesting observations were made with a few other mediators. Sometimes the treatment with surfactants increased the apparent surface coverage. To obtain clues to explain this phenomenon, more experimental work will be needed.

The surfactant treatment, however, blocked the catalytic NADH oxidation process even in cases when the apparent surface coverage increased several times. Ac- cordingly it is believed that the change of surface

coverage has less role in the change of the electrocatalytic activity than the transport blockage.

Acknowledgements

Financial support from the Swedish Natural Science Research Council (NFR) (L.G.), the Swedish Institute (G.N.) and the Hungarian National Scientific Research Foundation (OTKA) (I.K., G.N.) for this work is highly appreciated.

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