Development of a thin-film glucose biosensor using semiconducting amorphous carbons

Biosensors & Bioelectronics 10 (1995) 237-241

Development of a thin-film glucose biosensor using semiconducting amorphous carbons A.D. Clark, M.F. Chaplin, S.A. Roulston & L.J. Dunne School of Applied Sciences, South Bank University, Borough Road, London, SE1 0AA, UK Tel: [44] (0)171 815 7900 Fax: [44] (0)171 815 7999 (Received 13 June 1994; revised 23 September 1994; accepted 3 October 1994)

Abstract: Carbonaceous materials formed by the thermal decomposition of

sulphanilic acid in the temperature range 700 to 1400°K have considerable potential as enzyme supports and electron mediators in biosensors. These carbons have a variety of functional groups for enzyme binding and their electric and magnetic properties have been studied extensively. This paper describes a thin-film biosensor for glucose, formed by immobilizing glucose oxidase on pellets of the carbon prepared at 1400°K. We show the current response of this glucose electrode at 1100 mV (versus SCE) in aerated solutions; we also show its temperature and pH dependence, and report results using a ferrocyanide redox system. Keywords: amorphous carbons, biosensor, glucose oxidase, surface functional groups, ferrocyanide, redox

INTRODUCTION There continues to be much interest in amperometric enzyme biosensors for the analysis of glucose. Semiconducting carbonaceous materials have demonstrated great potential for the formation of the electrodes central to these sensors. Such materials possess good electrical conductivity and an abundance of available functional groups for coupling to enzymes. We have reported previously our investigations concerning the electrical and magnetic properties of a number of amorphous carbons that are prepared by the thermal decomposition of sulphanilic acid in an inert environment at heat treatment temperatures in the range 700 to 1400°K (Roulston et al., 1990). Our investigations included a study of the functional groups present 0956-5663/95/$07.00 © 1995 Elsevier Science Ltd

in these carbons, a description of the immobilization of glucose oxidase to amorphous carbon and suggestions as to the potential of such carbons for binding immobilized enzymes for use in biosensors (Roulston et al., 1991). We have also discussed recently a model for the electrical and magnetic properties of these amorphous carbons (Dunne et al., 1994). Here we report results obtained from a biosensor for glucose, which was prepared by immobilizing glucose oxidase on these carbons.

EXPERIMENTAL The preparation and characterization of amorphous carbon pellets and the immobilization of glucose oxidase were performed according to the 237

A.D. Clark et al.

method described previously (Roulston et al., 1990; Roulston et al., 1991). 1400°K Sulphanilic acid carbon (SAC; 60% w/w) and 40% (w/w) paraffin wax were mixed thoroughly at 50°C and pressed into a disc 1-0 mm thick using a 10 ton hydraulic press. The pellets were mounted on the end of a cylindrical polyacetate tube with an internal electrical connection formed by means of a mercury pool connected to a platinum wire. The surface of each disc was shown to be fiat by scanning electron microscopy, each having a geometric surface area of 1.42 cm2. Mounted pellets were immersed in 12 ml of sodium acetate buffer, pH 5.5, containing 10 mM ethyl-dimethylaminopropyl-carbodiimide, and allowed to react for 24 h. The pellets were washed to remove unbound carbodiimide and then coupled to glucose oxidase (12 ml, 9 U/ml) in sodium acetate buffer, pH 5-5, for 48 h. Finally, the pellets were washed repeatedly until no enzyme activity was present in the supernatant, as detected colorimetrically after the addition of glucose, the chromogen azino-bisethyl-benzothiazoline-sulphonic acid (ABTS) and horseradish peroxidase. The resultant electrode consisted of a thin film of the enzyme attached covalently to the surface of the carbon electrode. Voltammetry of these electrodes was undertaken using a saturated calomel reference electrode and a platinum foil counter electrode. A Princeton Model 362 scanning potentiostat was used for potential generation; the potentiostat was interfaced with a microcomputer equipped with a Condecon 300 interface and software supplied by EG&G Instruments. Chemical assay of apparent immobilized enzyme activity was achieved via a two-step colorimetric assay in acetate buffer, and was based on the method of Childs and Bardsley (1975). In the first step, the immobilized glucose oxidase pellet was immersed in an aerated solution of glucose; the supernatant, which did not contain any glucose oxidase activity, was removed and assayed at 420 nm for hydrogen peroxide using the enzyme peroxidase and ABTS. The pH dependence of the immobilized enzyme was examined in the following buffers: 50 mM citric acid for pH 5 to 7-6; 50mM tris (hydroxymethyl) aminomethane for pH 7.1 to 8.9, and 50 mM glycine/NaOH for pH 8.6 to 10. The glucose and temperature dependence of the electrochemical reaction were determined in 50 mM citrate buffer, pH 7.0. 238

Biosensors & Bioelectronics

RESULTS AND DISCUSSION When the chemical activity of immobilized glucose oxidase is monitored, it is usual to detect the hydrogen peroxide produced because this is more sensitive than determining the rate of oxygen depletion. The use of standard hydrogen peroxide solutions showed that no chemical reaction occurred between the hydrogen peroxide and the amorphous carbon pellets. It was found, however that the chromogen (ABTS) used in the assay of the peroxide was reduced chemically by the amorphous carbon. This proved to be a noncatalytic chemical reaction that correlated with the phenolic content of the carbon (Fig. 1), as found by base neutralization studies (Roulston et al., 1991), suggesting the involvement of a quinol/quinone type redox centre in the carbon. To avoid this destructive side-reaction, the carbons were removed from the assay system before the peroxidase/ABTS reagent was added. Figure 2 shows the electrode response due to glucose and hydrogen peroxide. A buffer solution containing peroxide at 59 mM and no glucose showed a similar plateau to the glucose response. This does not necessarily imply that peroxide is being detected in the analysis of glucose. However, the magnitude of the potentials at which these current plateaus occur is consistent with peroxide decomposition, and a glucose solution outgassed with nitrogen for one hour gave a response of similar magnitude to the buffer

mmol phenol per 100 g carbon I ~/~eolour loss 25 100

20 80

15 613

10 40

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700

800

900

1000 1100 1200 1300 1400 Heat treatment temperature (K)

Fig. 1. Interaction between particulate S A C and the electrochemically oxidized form of A B T S in 0.1 M acetate buffer, p H 5.5. Phenolic content of particulate heat treatment temperature carbon.

Biosensors & Bioelectronics

Development of a Thin-film Glucose Biosensor

1.6

C u r r e n t difference(taA) 100

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Fig. 3. Current response at 1100 m V (versus SCE) of the glucose electrode in aerated solution over a wide range of glucose concentrations at p H 7.0. Triplicate determinations were made.

)0 800 1000 1200 Potential (mV) versus SCE -10

Fig. 2. Current response of the glucose oxidase electrode in solution at steady state potential: - - × - - × - - 0.1 M acetate buffer (pH 5.5); - - ~ - - ~ - - - 0.1 M acetate buffer (pH 5.5) containing 59 m M H202; 0.1 M acetate buffer (pH 5.5) containing 1.0 M glucose.

solution alone. This demonstrated the need for oxygen in the overall reaction. The glucose concentration dependence is shown in Fig. 3. Three distinct regions are observed in this Figure. A central linear portion to the calibration curve spans the useful range of glucose concentrations of between approximately 1 and 30 mM, which constitutes the practical range. Above and below these values, quantitative estimation of concentration is made difficult through reaction rate limitation and background current phenomena, respectively. The kinetic parameters for the immobilized enzyme were determined by both chemical and electrochemical means (Fig. 4). The two weighted double reciprocal plots of inverse substrate concentration against inverse reaction rate showed linearity. In both cases the apparent Michaelis constant for glucose was about 20 mM. The maximum rate of enzymatic reaction was 0.95 U

whereas the limiting current was 40 p,A. The enzyme activity may be calculated to be equivalent to about 300 IxA of electron movement (two electrons per molecule of oxygen reduced). The difference between these values can be accounted for in terms of the increased agitation and aeration conditions in the chemical reaction, which cannot be approached for a static electrode, and the loss of the peroxide from the electrode by diffusion. The temperature dependence of the electrode function is shown in Fig. 5. A shift in the temperature peak is seen from 35°C for the soluble form to 25°C for the immobilized form. Such a shift is expected because, as stated by Ianniello and Yacynych (1981), the act of immobilization of an enzyme confers conformational differences on the subsequent structure that may be reflected in activity-temperature behaviour. Temperatures over 45°C were unsuitable for this electrode system as the paraffin wax, used in the fabrication of the pellets, melted at 50°C leading to the physical detachment of the pellet from the electrode assembly. The pH dependence of the electrode performance is shown in Fig. 6. There is a shift in pH optimum towards more alkaline pH for the biosensor. This may have been due to the 239

A.D. Clark et al.

Biosensors & Bioelectronics m a x i m u m activity

I/V (Abs'l.min) 1/iss (laA-1)

100

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60 80

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Fig. 6. p H dependence o f apparent glucose oxidase activity. Triplicate determinations were made: immobilized enzyme; - soluble enzyme.

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0.2

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0.4

0.5

0.6

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Fig. 4. Double reciprocal plots o f the rate o f reaction against glucose concentration: chemical reaction; - electrochemical reaction; iss is the steady state current. Triplicate determinations were made.

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240

microenvironment of the immobilized enzyme: the SAC surface contained many acidic groups which would have attracted hydrogen ions into the microenvironment and changed the pH optimum to a more alkaline pH. There are no basic positively charged groups present on the SAC surface but the positive charge on the electrode might be expected to reverse this. We should also note that there was a difference in the assay method between the immobilized and free enzyme for both the temperature and pH dependence studies. The soluble enzyme was assayed chemically using the peroxidase/ABTS system, whereas the immobilized enzyme was determined electrochemically from the rate of peroxide decomposition. The electrochemical decomposition of hydrogen peroxide is more favourable at higher pH (Kinoshita, 1988), and such a shift as found here has been reported by other workers using oxygen as the mediator (Ang et al., 1987; Jonsson & Gorton, 1985). The detection of glucose by means of the electrochemical decomposition of peroxide is not ideal: the approach renders the electrode reliant on a coreactant (oxygen), results possibly in the poisoning of the electrode with peroxide, and is close to the potential limits of the electrode. Substitution of the natural redox system with an artificial system avoids this problem and results in well behaved and characterized redox behaviour.

Biosensors & Bioelectronics

Development of a Thin-film Glucose Biosensor

The natural redox couple (oxygen/peroxide) competed with the ferrocyanide/ferricyanide redox couple under aerated conditions. This reduced the oxidation current below that of the unaerated system (Fig. 7).

C u r r e n t (pA) 601-

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CONCLUSIONS

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200

300

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/ l

The formation, characterization and use of a glucose biosensor based on a semiconducting amorphous carbon has been described. The enzyme electrode is simple to manufacture and the carbons involved have a variety of surface functional groups available for enzyme binding, and so the method has considerable potential for further development.

i

-20

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-30 ¢

Fig. 7. The effect of ferrocyanide and solution aeration on the potential-dependent current output of the glucose electrode (zero ramp rate): - - l l m l l - - (a) 1 m M ferrocyanide in 0.1 M aerated buffer at p H 7.0 with a 1400°K SAC~GOD electrode; ~ (b) as in (a) with the addition of 0.5 M glucose; - - x - - × - - (c) as in (b) with an outgassed solution; --0- (d) as in (a) in the presence of 440 tzM H202.

Figure 7 shows the response of the electrode to the ferrocyanide/ferricyanide redox couple at zero ramp rate. At a working electrode containing no glucose, a background current arose due to the oxidation of ferrocyanide at the electrode, and this current reached a plateau at around 200 mV. In a buffer solution containing 0.5 M glucose, that has undergone outgassing with nitrogen for one hour, this oxidation current increased by 3-5 times. This increase in current was consistent with the value of imax observed for the detection of enzyme activity via peroxide decomposition under aerated conditions (Fig. 3). As no oxygen was present, and no peroxide could therefore be produced, the ferrocyanide/ferricyanide redox system must have been acting as an artificial electron transfer mediator. Furthermore, from the similarity between the currents obtained from these two approaches, their efficiencies seem to be comparable under these extremes of aeration.

REFERENCES Ang, K.P., Gunasingham, H., Tay, B.T., Herath, V.S., Teo, P.Y.T., Thiak, P.C., Kush, B. & Tan, K.L. (1987). Determination of glucose using flow injection with a fibre based enzyme reactor. Analyst, 112, 1433-1435. Childs, R.E. & Bardsley, W.G. (1975). The steady state kinetics of peroxidase with 2,2'-azino-di-(3ethyl-benzthiazoline-6-sulphonic acid) as chromogen. Biochem. J., 145, 93-103. Dunne, L.J., Clark, A.D., Topping J. & Bryant A. (1994). A model for the electrical and magnetic properties of amorphous carbons based on Fullerene-type structures. Int. J. Electron., in press. Ianniello, R.M. & Yacynych, A.M. (1981). Immobilised enzyme chemically modified electrode as an amperometric sensor. Anal. Chem., 53, 2090-2095. Jonsson, G. & Gorton, L. (1985). An amperometric glucose sensor made by modification of a graphite electrode surface with immobilised glucose oxidase and adsorbed mediator. Biosensors., 1, 355-368. Kinoshita, K. (1988). In: Electrochemical Oxygen Technology. John Wiley, New York. Roulston, S.A., Dunne, L.J., Clark, A.D. & Chaplin, M.F. (1990). Electronic and structural properties of amorphous semiconducting carbons prepared by the thermal decomposition of sulphanilic acid in the solid state. Phil. Mag., 62, 243-260. Roulston, S.A., Chaplin, M.F., Dunne, L.J. & Clark, A.D. (1991). Development of an enzyme substrate incorporating semiconducting amorphous carbons for use in biosensors. Biosensors & Bioelectronics, 6, 325-332. 241