Novel approaches for the use of mediators in enzyme electrodes

Biosensors & Bioelectronics 8 (1993) 315-323

Novel approaches for the use of mediators in enzyme electrodes llana Rosen-Margalit Department

of Molecular

Microbiology

& Judith Rishpon

and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel

(Received 6 July 1992; revised version received and accepted 11 January

1993)

Abstract: This work describes the preparation

of glucose electrodes consisting of an enzyme (glucose oxidase) and a mediator embedded in a colloidal graphite emulsion matrix. These components are homogeneously mixed in an organic medium that evaporates rapidly while the enzyme activity remains intact. The appropriate conditions for preparation and measurement, as well as electrochemical characterization of these electrodes, are discussed. The problem of mediator leaching in electrodes using vinyl ferrocene was overcome by the use of a special membrane that prevented leaching of positive ferricinium ions by electrostatic repulsion under the appropriate measurement conditions, including minimal exposure of the electrode to anodic potentials, thus increasing the long-term stability. The resulting electrodes are easily prepared, have fast and high response independent of oxygen tension and can be stored in dry form. The linear range can be extended by the introduction of a dialysis membrane and can thus be adjusted to the measurement of glucose concentration in the range of medical interest. Keywords: glucose oxidase, vinyl ferrocene,

1. INTRODUCTION In the last two decades the development

of cheap and reliable enzyme electrodes for industrial and medical applications has attracted major efforts. In the case of enzymes involved in oxidation/ reduction, the redox centre of active site is usually built as a ‘pocket’ within the molecule. Such steric constraints do not allow direct exchange of electrons between an enzyme and an electrode surface; e.g. glucose oxidase, where the distance between the redox centers (FAD/ FADH2) and the enzyme surface is too large for direct electron transfer with simple electrodes such as gold, platinum or carbon. This, together with the difficulties encountered in efficient 09565663/93/$06.00

mediated enzyme electrode.

electrochemical regeneration of naturally reduced cofactors (such as NAD(P)H), had led to the construction of enzyme electrodes employing a variety of techniques based mainly on the use of mediators (Cass ef al., 1984; Brooks ef al., 1987; Claremont ef al., 19&r, b; Frew et al., 1986; Green & Hill, 1986; Schuhmann et al., 1990; Ikeda et al., 1985,1986) or of modified properties of electrodes (Narashimhan & Wingard, 1985, 1986u, b, c; Ianniello et al., 1982; Benneto et al., 1988u, b, c). Other electrodes were based on conducting salts (Kulys, 1986; Albery ef al., 1985; Cardosi & Turner, 1991), electropolymerization, e.g. polypyrrols (Foulds & Lowe, 1986; Umana & Waller, 1986; Bartlett & Whitaker, 1987u, b;

@ 1993 Elsevier Science Publishers Ltd.

315

I, Rosen-Margalit & J. Rishpon

Dicks et al., 1989; Schuhmann et al., 1991) or redox polymers (Hale et al., 1989, 1991a, b; Ianagaki et al., 1989; Gorton et al., 1990; Degani & Heller, 1989; Gregg & Heller, 1990, 1991a, b; Foulds & Lowe, 1988; Heller, 1990; Pishko et al., 1991). A major obstacle in the case of the combination electrode-enzyme-mediator, despite several advantages, is the lack of long-term operational stability, low electron transfer efficiency and oxygen tension dependence, which is especially relevant in in vivo measurements as well as in anaerobic systems. The aim of this study was the construction of a mediator-based enzyme electrode capable of more efficient electron transfer between enzyme and electrode. Such electrodes should be simple to use, rapid, sensitive and, most importantly, stable.

2. MATERIALS

AND METHODS

2.1. Materials Glucose oxidase EC.1.1.3.4 (Cat. No. G-2133) from Aspergillus niger, type VII lyophilized powder (150000 units/g solid) was purchased from Sigma, St Louis, MO, USA; D-glucose (Cat. No. 10117) from BDH Ltd, Poole, England; vinyl ferrocene (Cat. No. 4503) from Polyscience, Warrington, PA, USA; colloidal graphite emulsion (dag 568~ Acheson) and ethanol absolute (Cat. No. 983) from Merck, Darmstadt, Germany. All other chemicals were of analytical grade. 2.2. Preparation

of electrodes

100 ~1 of colloidal graphite emulsion was mixed with an equal volume of vinyl ferrocene solution in ethanol (20 mg.ml-l). After mixing, 20 ~1 of glucose oxidase (100 mg.ml-l in phosphate buffer, 0.1 M, pH 6) was added and the solution was mixed again vigorously. 10 ~1 of this solution was applied onto glassy carbon (gc) electrodes (built in a Teflon housing, O-3 or 0.5 cm diameter). After evaporation of the solvents (within a few minutes) the electrodes were ready for use. The electrodes were stored at 4°C in buffer phosphate, pH 6, or dry. In some cases the electrodes were covered with a special cationic membrane (see below). 316

Biosensors & Bioelectronics

2.3. Preparation

of cationic membrane

Membranes were prepared by modification of a procedure employed for the synthesis of butylamine-substituted polyacrylamids (Reuveny et al., using 1983) polyacrylamide hydrazidemetacrylamide hydrazide 30% (Tor & Freeman, 1986) and 2-diethyl amino ethyl amine (26235-8 Aldrich). The highly hygroscropic polymer was stored at -20°C over CaC12 in a closed vessel. The polymer was dissolved in O-1 M pH 6 phosphate buffer (5% w/v). 5 ~1 of that solution was then applied onto the prepared electrode and, after drying (1 h), 5 ~1 of glyoxal 1% was added for crosslinking. 2.4. Electrochemical

measurements

A conventional three-electrode cell was used with a Pt gauze as counter electrode and a KClsaturated calomel electrode (Radiometer K-401, Copenhagen, Denmark). The cell was thermostatted (25°C) and argon was bubbled before and during the experiments. The experiments were performed with an IIT model 303-C potentiostat including a multiplexer and programmer (The Technion, Haifa, Israel), an EG&G Princeton Applied Research model 273 potentiostat, an x-y recorder (model 3086, Yokogawa, Japan), a rotating disc electrode system from Pine Instruments (model AFMSRX), an Ims S-100 64K microcomputer equipped with Tecmar A/D and D/A units, an 8253 PIT timer unit and an 8253 PPI parallel port, IBM PC compatible, 256K, equipped with Tecmar A/D and D/A units with a 9513 timer unit and a 3300 A Hewlett-Packard function generator. The method of measurement, explained elsewhere (Rosen & Rishpon, 1989), is based on connecting and disconnecting the electrode in a repetitive mode, measuring the current during the connection period and integrating it so that the resulting response is expressed in charge units. Integration increases the sensitivity and improves the signal-to-noise ratio. Electrochemical techniques such as cyclic voltammetry, rotating disc electrode and AC measurements (Bard & Faulkner, 1980) were used for characterization of the electrodes. Activity of the electrodes was determined by dipping the electrode in buffer solution containing all the essential components for the enzymatic reaction except the substrate. The electrode

Biosensors & Bioelectronics

Use of mediators in enzyme electrodes

current at the appropriate potential was recorded until a steady value was obtained (a few minutes). The substrate was then added and the difference in electrode current recorded. The response time in this mode is defined as the time needed to obtain a steady reading after addition of glucose.

3. RESULTS

of low stability, particularly during the application of anodic potential. Repetition of measurements resulted in decrease of peak current with time. This effect was attributed to leaching of relatively water-soluble ferricinium ion, produced when ferrocene is oxidized at anodic potential. Similar phenomena have also been reported by other groups (Schuhmann ef al., 1990).

AND DISCUSSION 3.2. Improvement

of electrodes

3.1. Vinyl ferrocene electrode A voltammogram of a colloidal graphite emulsionbased vinyl ferrocene-glucose oxidase electrode, with and without glucose, is shown in Fig. 1. Reversible electrochemical behaviour and high current at a potential typical of vinyl ferrocene (O-36 V anodic peak, 0.30 V cathodic peak vs SCE) were observed, with a typical catalytic current in the presence of glucose. Measurements carried out at a constant potential showed high response (high currents) to glucose addition in less than 10 s. These electrodes were, however,

220zoo180

-

160

-

140

-

To improve the stability of the electrode, tests were made on reducing the anodic potential applied and shortening the time that the electrode was held at this potential. The optimal conditions found were a measurement potential of 0.25 V vs SCE (the potential tested ranged from 0.25 to 0.5 V in steps of 0.05 V), applied for only 0.5 s, and then disconnection of the electrode for 10 s repetitively. This method enabled the sensitivity to be amplified. During the disconnection period the enzyme continued to generate the product, which accumulated, but leaching of ferricinium ion was diminished. However, the stability of the electrode was still poor: complete loss of activity after ten measurements, as shown in Fig. 2 (for the uncovered electrode). To overcome this problem, the electrode was covered with a cationic membrane (see Section 2.3) to prevent leaching of the positively charged ferricinium ions by electrostatic repulsion. Fig. 2 shows the effect of this membrane on the

120100

160~

80-

140. 2

60-

120.

s .-

40

loo80.

zo-

60. o-

4020-

-2o-

07 0

-4o-

:

10

20

30

40

50

DAYS FROM PREPARATION -0.4

-a2

0.4

EO(“) .,.“,

0.6

ce

Fig. 1. Cyclic voltammograms of gc electrode coated with colloidal graphite emulsion containing glucose oxidase and vinyl ferrocene with cationic membrane. Measurement conditions: deaerated O-1M phosphate buffer, pH 6, 37°C 5 mV.s-‘. -, buffer only; ----, after addition of 23.5 mM glucose.

Fig. 2. Storage stability of gc electrodes coated with colloidal graphite emulsion containing glucose oxidase and vinyl ferrocene (percentage from initial activity). Measurement conditions: deaerated 0-I M phosphate buffer, pH 6, 0.25 V vs SCE (0.5 s connection, 10 s disconnection), 23.5 m.+tglucose. W, with 5% cationic membrane coating; +, with 20% cationic membrane coating; *, without any membrane. 317

I. Rosen-Margalit & J. Rishpon

long-term stability of the electrodes. The chosen polymer concentration was 5% because of the higher activity as well as the lower viscosity. It was found that the membrane slowed down leaching of the ferricinium but did not completely prevent it. This membrane had no effect on the response time or the linear range of glucose concentrations. The optimal electrode stability required the combination of short (O-5 s) applications of low anodic potential (0.25 V) and the use of the cationic membrane. 3.3. Other ferrocene derivatives Other ferrocene derivatives. tested by the same method were ferrocene and ferrocene mono- and dicarboxylic acid. The best results, in terms of stability and sensitivity, were obtained with vinyl ferrocene, which is the most hydrophobic among the derivatives tested. 3.4. Optimization and characterization Other ratios of graphite emulsion to ethanol were tested (3:1, 2:1, 1:2, 1:3). The optimal ratio was found to be one volume of graphite emulsion:one volume of ethanol solution of vinyl ferrocene. Under these conditions the weight of coating of graphite emulsion/mediator/enzyme thickness was 0.67 mg ? 12% and the 11.5 pm 2 15%. The effective surface area, as calculated from the capacitive current in cyclic voltammograms with the same gc electrode with and without the coating, was larger by a factor of 166 (32 cm* whereas the geometric area was 0.196 cm*). Vinyl ferrocene concentrations in the range 0.5, 1, 5 and 10 mg.ml-’ were tested. Electrodes prepared with the two lowest concentrations responded very poorly to glucose. The response with 10 mg.ml-’ (the highest concentration used, due to solubility limitation) was slightly higher and both showed high than with 5 mg.ml-l, response to glucose addition. Attempts to increase the amount of ferrocene by building sequential layers had a small effect on the response but caused mechanical instability. The effect of increasing the enzyme concentration was as follows. When the amount of enzyme per electrode was increased from 0.08 to 2 mg the response to 23.5 mM glucose increased from 30 to 116 j&/O.5 s. The middle range enzyme 318

Biosensors & Bioelectronics

amounts (0.4, 1 mg) resulted in middle range responses. This shows that electrode activity is best with the higher enzyme loading. The highest molar ratio of ferrocene to enzyme employed (2 mg enzyme:O.l mg vinyl ferrocene) was 38 molecules of ferrocene for each enzyme molecule. Table 1 shows the effect of pH, temperature, buffer type and concentration, and oxygen on the electrode response to 23.5 mM glucose. The results are expressed as &/0*5 s. The data show that the optimal pH is between 7 and 7.5. At lower pH the response is lower, by a factor of 1.6 between pH 5 and pH 7. At higher pH the response also decreases. At 37°C the response increased by a factor of 2 compared to 25°C (at the optimal pH), as expected for an enzyme system. The buffer concentration affects the response: 0.05 M is too low, probably due to the need for minimal supporting electrolyte or because of poor buffer capacity. A concentration of O-2 M does not improve the response in comparison to 0.1 M. Buffer types hepes and phosphate were found to have no effect on electrode response, which suggests that large buffer ions do not interact with or penetrate the cationic membrane covering

TABLE 1 Effect of different factors on electrode response. pH

Temp.

Buffer

W)

type

Buffer cont. (M)

5 5

6 6 6 6.5 6.5 7 7 7 7 7 7.5 7.5 8 8

25 25 25 25 25 25 25 37 25 37 37 25 25 25 25 25

phosphate hepes-NaCl phosphate phosphate hepes-NaCl hepes-NaCl phosphate phosphate phosphate phosphate phosphate hepes-NaCl phosphate hepes-NaCl hepes-NaCl phosphate

0.1 0.05,O.l 0.1 0.1 0.05,0-1 0.05,O.l O-1 0.05 O-1 0.1 0.2 0.05,O.l 0.1 0.05,O.l 0.05,O.l 0.1

Oxygen Response to 23.5 mM glucose (pCiO.5 s).

-

+ _ -

153 159 157 147 168 200 200 256 255 440 469 208 261 236 249 237

Use of mediators in enzyme electrodes

Biosensors & Bioelectronics

the electrode. Oxygen did not affect the response to glucose. This could probably be related to the high excess of accessible mediator (in conditions under which the oxygen does not compete (Dicks ef al., 1989)). Fig. 3 shows a calibration curve for glucose under optimal conditions. The linear range with this type of electrode was too low for medical interest (O-0.5 mM, whereas blood glucose concentration is around 5 mM). This problem was solved by the introduction of diffusional constraint; i.e., the electrode was covered by a dialysis membrane (MW cutoff 3500). By this method the range of linearity was extended by a factor of 10 (up to 5 mM) and glucose concentrations up to 50 mM could be determined (Fig. 4). The response time was increased only by a factor of 2. Usually the electrodes were stored in 0.1 M phosphate buffer solution, pH 6, at 4°C. Dry storage of the electrodes at 4°C showed that the

0

03

;

15 IGLUCOSE

2 25 .3 CONCENTRATION

J 35 (mlM)

45

5

Fig. 4. The same as Fig. 3 but with electrode covered with dialysis membrane, pH 6, 25°C. See text for details.

activity, stability affected.

and response

time were not

3.5. Electrochemical study

Ij!!l

GLUCOSE

30 CONCENTRATION

35 40 (mM)

45

50

3.5.1. Rotating disc electrode Experiments with a rotating disc electrode (RDE) were performed with an electrode covered with a cationic membrane under optimal pH and temperature conditions. With glucose (23.5 mM, the saturation range of the electrode), a linear dependence was found between the square root of rotation speed and current, with a slight deviation from linearity at high rotation speed (Fig. 5), probably due to turbulent streaming at high rotation speed or to the fact that the measurement potential (O-25 V) is lower than the diffusion-limited current range (which starts

iq GLUCOSE

CONCENTRATION

(mM)

Fig. 3. Glucose calibration curve of gc electrode coated with colloidal graphite em&ion containing glucose oxidase and vinyl ferrocene. Measurement conditions: deaerated 0.1 Mphosphate buffer, pH 7.5, 37”C, O-25 V vs SCE (0.5 s connection, 10 s disconnection). (a) full range; (b) linear range.

Ib

*~’

j 30 40 ROTATION

/ 50 SPEED

/

:

;

r

60 70 A 5 (rpm)

80

90

1 100

Fig. 5. Dependence of electrode response on rotation speeds of gc electrode coated with colloidal graphite emulsion containing glucose oxidase and vinyl ferrocene. Measurement conditions: deaerated 0-I M phosphate buffer, pH 7.5, 37°C O-25 V vs SCE. n, with 23.5 mM glucose; +, buffer only. 319

I. Rosen-Margalit & J. Rishpon

Biosensors & Bioelectronics

at O-4 V). At high rotation speeds, the ratedetermining step is not the mass transport but the electrode reaction rate. An attempt to measure at higher anodic potential (at the diffusion-limited current range) was not possible because of loss of ferricinium ions at higher anodic potentials. For this reason, dependence of the current on rotation speed without the cationic membrane could not be measured, even at low potential. The result suggests that the reaction rate at the electrode is very fast compared to mass transport through the cationic membrane (up to a certain limit, starting at 4900 rev/min, where the graph starts to deviate from linearity, beyond which the reaction rate becomes rate determining). 3.5.2. Cyclic voltammograms Cyclic voltammograms (CVs) at different scan rates were performed in the potential range -0.3-O-6 V vs SCE with and without glucose. Fig. 6 shows the dependence of anodic peak current (with glucose) on scan rate, on a logarithmic scale. The slope of this graph, O-6, is typical for diffusion-controlled reactions. Glucose transport to the electrode is rate limiting and the enzymatic and electrochemical reactions are relatively fast. Measurement of pH effect on peak positions at different scan rates without glucose showed that at pH 5 the anodic peak potential was 40-60 mV higher than at pH 8, and the difference between the position of the peaks at those two pHs decreases with decreasing scan rate. Since protons do not participate in the ferrocene oxidation reaction, pH change does not affect the position of the peaks. A possible explanation to this phenomenon is film charging

3.5 3

3-

s

2.5.

E %

2-

y

1.5.

2

l-

% 2

0.5. 01 -2.4

-------m---------

-2.2

-2

-1.8

-1.6 -1.4 LOGV (vmlc)

-1 2

-1

-0.8

-0.6

Fig. 6. Anodic peak currents as a function of scan rates (log. scale) for gc electrode coated with colloidal graphite emulsion containing glucose oxidase and vinyl ferrocene. Measurement conditions: deaerated 0.1 M phosphate buffer, pH 7.5, 37T, no stirring, 23.5 rniu glucose oxidase. 320

during oxidation (neutral ferrocene becomes positively charged ferricinium), which creates a problem of charge balancing. Consequently, protons are getting out to the solution or anions are coming in near the electrode. Thus, at lower pH, i.e. at higher proton concentration, the reaction will take place at higher anodic potential. This effect is emphasized when electrodes covered with the cationic membrane are compared with electrodes without the membrane. The anodic peak potential is higher with the membranecovered electrode than with the non-covered electrode at both pH values investigated (5 and 8). The cationic membrane prevents removal of protons, and so the local pH (due to proton accumulation) is lower than the bulk pH and the reaction will occur at a higher anodic potential. This phenomenon also explains the higher response obtained with higher pH, as mentioned earlier. Another possible explanation could be that the pH value near the electrode surface (behind the cationic membrane) is lowered by the hydrolysis of the product gluconic acid. Due to this effect, the pH within the membrane might become lower. 3.5.3. Experiments with AC AC (alternating current) experiments encountered some difficulties as they require (especially in the low frequency range) that the electrode be biased at anodic potential for relatively long times while losing a substantial amount of ferrocene (even with the cationic membrane). Hence, comparison of two measurements with the same electrode was impossible and the data could be treated only qualitatively. Fig. 7 shows an example of an AC measurement at 0.25 V v.s SCE without and with glucose sequentially. The electrode behaviour resembles that of a transmission line (Rishpon et al., 1985) of resistors and capacitors. Addition of glucose decreases the capacity and adds a diffusional impedance. The AC measurements also confirm that instability problems of ferrocene electrodes are due to leaching of soluble ferricinium ions produced during anodic potentials.

4. CONCLUSIONS A novel method for preparation of mediated enzyme electrodes in a matrix of colloidal graphite emulsion has been described. The method consists

Use of mediators in enzyme electrodes

Biosensors & Bioelectronics

I

0

,001

I

I

.002

c

,003

.004

real

Fig. 7, Complex representation of the AC capacitance of gc electrode coated with colloidal graphite emuhion containing glucose oxidase and vinyl ferrocene. Measurement conditions: 0.1 Mphosphate buffer, pH 6, DC bias 0.25 V vs SCE. 0, buffer only; +, with 23.5 mM glucose.

of drying a mixture containing colloidal graphite emulsion, mediator and an enzyme on the electrode surface, and leads to the formation of a uniform layer of high surface area, which has great adsorption capacity for proteins. The layer functions as an immobilization matrix as well as an electrode surface, so that close contact between all components is possible. The enzyme activity remains intact in spite of the organic medium. Stability problems due to ferricinium leakage under anodic potential were solved by the use of low anodic potentials (O-25 V), short connection times (O-5 s), and by covering the electrode with a positively charged membrane that prevented the leakage of positively charged ferricinium ions. In this way the lifetime of the electrode was substantially improved: 100% activity after two months of intensive work, followed by two more months with a decrease of 50% of the initial activity. The electrode lost only 30% of its initial activity after 6 months in storage, during which only a few measurements were performed. Such lifetimes are longer than those reported for similar electrodes described in the literature (Schuhmann et al., 1990,50% decrease in 8 days; Chen et al., 1992, maximum stability after 15 measurements; Hale er al., 1989, 1991a, b, 50%

loss of activity after two months, 100% in another system). Other advantages of the electrodes are: simplicity of preparation, independence of oxygen tension (in contrast with other works: Hale et al., 1989, 1991u, b; Gregg & Heller, 1990; Dicks ef al., 1989) very fast response (5-10 s), possibility of dry storage and high response for glucose. The linear range of these electrodes could be extended by the introduction of a dialysis membrane, and thus adjusted to the concentration range of medical interest. The electrodes are operated at low potential (0.25 V vs SCE), which is advantageous for testing biological fluids. These optimized electrodes could form the basis for the construction of devices used for routine determination of analytes in biological fluids.

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Ikeda, T., Hamada, H. & Senda, M. (1986). Electrocatalytic oxidation of glucose at glucose oxidase immobilized benzoquinone mixed carbon paste electrode. Agric. Biol. Chem., 50(4), 883-90. Kulys, J.J. (1986). Enzyme electrodes based on organic metals. Biosensors, 2, 3-13. Narashimhan, K. & Wingard, L.B. (1985). Direct electron transfer with glucose oxidase immobilized on aminophenyl boronic acid modified glassy carbon. Ann. N. Y. Acad. Sci. 501, Enzyme Engineering, 8, 298301. Narashimhan, K. & Wingard, L.B. (1986a). Immobilization of flavins on electrode surfaces. Part I: Attachment of riboflavin through 8 methyl to glassy carbon. J. Mol. Catal., 34, 253-62. Narashimhan, K. & Wingard, L.B. (19866). Immobilization of flavins on electrode surfaces. Part II:

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