Enzyme Entrapment in Electrically Conducting Polymers

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J. Chem. SOC.,Faraday Trans.I, 1986,82, 1259-1264

Enzyme Entrapment in Electrically Conducting Polymers Immobilisation of Glucose Oxidase in Polypyrrole and its Application in Amperometric Glucose Sensors

Nicola C. Foulds and Christopher R. Lowe The Biotechnology Centre, University of Cambridge, Downing Street, Cambridge CB2 3EF A technique is described for the entrapment of glucose oxidase in a polypyrrole matrix electrochemically deposited on a printed platinum electrode. The enzyme activity incorporated into the polymer was quantified by spectrophotometric assay using a flow-through cell and found to be proportional to the activity in the electropolymerisationmedium. Polymerentrapped glucose oxidase electrodes can be operated as amperometric glucose sensors in the presence or absence of soluble mediators. These electrodes respond rapidly, reaching a steady state within 20-40 s, with the enzymatic response being current limiting. The potential of this new immobilisation technique to the development of biosensors is discussed.

Amperometric biosensors that combine the specificity and selectivity of biological molecules with the analytical powers of electrochemical techniques are beginning to be commercialised. Much effort has been directed at devising an amperometric sensor for the clinically significant substrate glucose using the archetypal and inexpensive redox enzyme glucose oxidase. In order to achieve this objective it is important that electron transfer between the enzyme and electrode material be facilitated. Thus, in our work we have concentrated on techniques to immobilise the enzyme that will promote proximity between the enzyme active site and the conducting surface of the electrode. In addition, the entrapment of enzymes in conducting polymer films provides a controlled method of localisation of biologically active molecules to defined areas on electrodes.Polypyrrole was chosen because it is one of the few conducting polymers that is acceptably stable in ambient conditions’ and can be easily prepared electrochemically from a variety of electrolytes, including aqueous solutions.2 The polymerisation occursduring the electrochemicaloxidationof a solutioncontaining pyrrole monomers. The generation of an extremely reactive n radical cation that reacts with neighbouring pyrrole speciesproduces a polymer that is predominantly a,a’-c~upled,~ although some branching of the polymer chains is thought to take place by jkoupling. The resulting polymer has a net positive charge (ca. one positive charge per four pyrrole units)4 and incorporates anions from solution during the film growth process. The chemical and physical properties of the film are dependent upon factors such as pH, charge, potential and the anion incorporated. In this report we describe a method for incorporating glucose oxidase into polypyrrole films in a manner which is capable of producing glucose-sensing electrodes in a one-step process.

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Enzyme Entrapment in Conducting Polymers

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Preparation of Enzyme Electrodes The base electrode used for the preparation of the polypyrrole/glucose oxidase (GOD) electrodes consisted of a thin film of Pt ink printed on a ceramic backing. All but two small areas of the ink had been masked: one was used as an electrical contact, the other, an area of 0.16 cm2, was used as the working electrode. Polypyrrole films were electrochemically grown on these electrodes from an aqueous solution containing up to 0.2 mol dm-3 pyrrole (Aldrich Chemical Co., redistilled once). The films were grown using a three-electrode cell at a constant potential of 0.8 V (us. Ag I AgCl) supplied by an EG & G model 273 potentiostat/galvanostat. The average time of synthesis was ca. 2.5 h. Enzyme immobilisation was performed by the addition of 0-0.13 pmol dm-3 GOD (type VII from Aspergillus niger; Sigma Chemical Co.) to the aqueous solution prior to polymerisation. All solutions were bubbled with nitrogen for 25 min prior to use and maintained at 25 "C by means of a Julabo FlO water bath and circulator. The polypyrrole/GOD electrodes were stored in 0.1 mol dm-3 potassium phosphate buffer pH 7.0 at 4 "C when not in use.

+

Estimation of the Amount of Enzyme Entrapped in the Electrodes Estimation of the amount of glucose oxidase present on each enzyme electrode was performed using a Perkin-Elmer A5 u.v.-visible spectrophotometer. A flow-through cell was established and linked to a small vessel containing the bulk of the assay medium located outside the spectrophotometer. The rate of increase of absorbance at 510 nm with the 4-aminoantipyrine assay5 was used as a measure of enzyme activity.? A standard curve was constructed by adding known amounts of enzyme to the external vessel and starting the reaction by the addition of the 4-aminoantipyrinecolour reagent. A magnetic stirrer bar ensured homogeneity of the solution in the external vessel, and a constant, fast flow rate to and from the spectrophotometer cell was provided by a Gilson HP4 peristaltic pump. The amount of enzyme activity present on each electrode was estimated by securing the electrode in the external vessel in place of the soluble enzyme. The enzyme electrodes were aged for two days after synthesis in order to stabilise their activity. Operation of the Enzyme Electrodes as Amperometric Glucose Sensors Steady-state current measurements were made in a three-electrode cell with a working volume of 10 cm3 and supporting electrolyte of 0.1 mol dm-3 potassium phosphate pH 7.0. The reference and counter-electrodes were sintered Ag I AgCl and Pt wire, respectively. Constant potential to the cell was supplied by an EG & G model 273 potentiostat/galvanostat and current was monitored as a function of time by a Gould series 60000 XYt recorder or an Apple IIe microcomputer. For current measurements in which oxygen was the electron acceptor to GOD the enzyme electrodes were poised at a potential of +0.7 V (vs. Ag I AgCl). This potential had been predetermined by linear-sweep voltammetry as being in the diffusion-controlled plateau region for hydrogen peroxide oxidation on the Pt ink electrodes. When alternative electron acceptors or mediators were included in the supporting electrolyte a less anodic potential could be selected, and all solutions were bubbled with nitrogen for 25 min prior to use. After application of the appropriate potential to the enzyme electrode the background current was allowed to decay to a steady state before aliquots of stock glucose were added. After brief stirring (2 s) by means of a magnetic stirrer, the current in quiescent solution was recorded, the steady state being reached within 20-40 s.

t Enzyme activity is expressed in units (U); 1 U is that amount of enzyme which transforms 1 pmol min-l of substrate at pH 7.0 and 25 "C.

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N . C . Foulds and C . R. Lowe

I

0:3

0:s

0:9

1:2

1;5

1261

1:8

211

GOD/mg cmd3 (during polymerisation)

Fig. 1. Relationship between the amount of glucose oxidase present during electropolymerisation of pyrrole and the enzymatic activity of the resulting electrode.

Results and Discussion Enzyme Immobilisation Glucose oxidase can be incorporated into polypyrrole films by electrochemical codeposition of the enzyme and conducting polymer on the Pt ink electrode surface. Fig. 1 shows the correlation between the amount of enzyme present in the electropolymerisation medium and the resultant enzymatic activity incorporated in the electrode. Under the conditions specified up to 125 mU of oxidase activity could be incorporated per cm2 of electrode surface. However, the mechanism by which the enzyme is entrapped in the polypyrrole matrix is uncertain. The immobilisation procedure described here for glucose oxidase results in a dark brownish-black film of oxidised polypyrrole being deposited on the previously silvery-grey electrode surface. Scanning electron micrographs of both the Pt ink surface and of the polypyrrole covered Pt ink surface show an extremely porous structure with typical pore sizes up to a few pm. However, when the polypyrrole film is grown in the presence of glucose oxidase the gross morphology of the film changes to a more fibrillar structure. Glucose oxidase from Aspergillus niger has an isoelectric point of 4.2s and therefore at the pH usually used for the immobilisation procedure (pH 7.0) is negatively charged. Since the topology of the growing surface of polypyrrole films is known to be influenced by the anion present in the electrolyte,’ it is possible that incorporation of glucose oxidase as the anion may play some part in the immobilisation. Despite uncertainties as to the mechanism of entrapment, electrochemical deposition offers advantages over the more traditional methods of enzyme immobilisation, particularly in the simplicity and reproducibility of one-step electrode construction.

Electrode Characteristics The reaction between glucose oxidase and its substrate glucose is illustrated by the following scheme :

GOD,,*, GOD,,,,H,

+glucose tGOD,,,,,,

+glUCOniC acid

+ ‘electron’ acceptor(ox) f GOD2FAD+ ‘electron’ acceptor(red)

with the natural electron acceptor being oxygen.

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Enzyme Entrapment in Conducting Polymers

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4-65

3-88

2 291

2 M

4 4

+

g

z

1.94

0.97

0

20

40

60

80

100

[ glucose]/mmol dm-3

Fig. 2. Response curve for glucose. Steady-state currents measured at +0.7 V (us. Ag I AgCl) in 0.1 mol dmm5phosphate buffer, pH 7.0.

,

0 2 0

04

0.8

1.2

1.6

2-0

( l/[glucose])/dm3 mmol"

Fig. 3. Determinationof the apparent Michaelis-Menten constant (Km) for the polypyrrole/GOD electrode. Km= 30.7 mmol dm-3. Linear correlation coefficient = 0.9997. Maximum response = 6.37 FA. (Calculated using constant absolute error in current.)

In principle, a glucose sensor could be constructed with reference to the above reaction scheme by exploiting several approaches. The most direct parameter to monitor would be that of reduced glucose oxidase. Although flavin enzymes have been reduced on mercury electrodes without overtension,8direct (as opposed to mediated) redox reactions of these proteins on solid electrodes have yet to be reported.

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N . C. Foulds and C. R.Lowe

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6

5

3

5

15

10

20

25 I

time/days

Fig. 4. Stability of the polypyrrole/GOD electrode on storage in 0.1 cm3phosphate buffer, pH 7.0, at 4 "C.

An alternative approach to glucose monitoring has been to measure the oxidation current of the hydrogen peroxide produced by glucose ~ x i d a s ewhich ,~ under constant oxygen conditions can be related to the amount of glucose in the initial sample. However, the potential needed to oxidise hydrogen peroxide is sufficiently anodic that several other interfering compounds could contribute to this current if the background solution were not sufficiently defined. Finally, glucose can be measured with the glucose oxidase system at a less anodic potential if a suitable mediator between the reduced enzyme and the electrode is found. Ferrocene and its derivatives have a range of redox potentials between 100 and 400 mV vs. SCE and have been reported as mediating this electron transfer.1° Both the measurement of hydrogen peroxide and the use of mediators between reduced enzyme and the electrode have been investigated for glucose monitoring using the polypyrrole/glucose oxidase electrode. The steady-state responses at +0.7 V (vs. Ag I AgCl) in the polypyrrole-immobilised enzyme electrode to the addition of varying amounts of substrate were employed to construct response curves for glucose. The steady-state current appeared to be virtually unaffected by turbulence in the solution and it was therefore concluded that the enzymatic reaction was current limiting. If the limiting factor had been mass transfer to the electrode then a large difference in current between quiescent and turbulent solutions would be observed. Wilson and Shull have suggested that for an enzymatic reaction that is catalytically controlled, an apparent Michaelis-Menten constant (Km) can be calculated for the immobilised enzyme by amperometric methods. A LineweaverBurke-type plot can thus be constructed according to the following equation : 1 - 1

- --+-x-

iss

Zmax

1 K k S imax

where iss is the steady-state current, imaxis the maximum current and S is the substrate concentration. Thus a double reciprocal plot of issagainst S should be linear with a y-axis intercept of l/imaxand an x-axis intercept of - l/Km. Fig. 2 and 3 show these response characteristics for a polypyrrole-immobilised glucose oxidase electrodewith an estimated units (100 mU cm-2) maximum current response for this electrode activity of 1.6 x was 6.4 PA, with KL = 3 1 mmol dm-3. The latter value is of the same order of magnitude as that for solubleglucose oxidase from Aspergillusniger."? l2The responsecharacteristics of the polypyrrole/glucose oxidase electrode to glucose with the addition of various mediators to the supporting buffer have also been observed. For instance, with phenazine


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Enzyme Entrapment in Conducting Polymers methosulphate and benzoquinone the electrode can be poised at +0.35 and +0.4 V (US. Ag I AgCl), respectively, and oxidation currents due to reoxidation of the mediator can be measured on addition of glucose. The stability of the polypyrrole/glucose oxidase electrode under defined storage conditions is illustrated by fig. 4. Responses were measured as the oxidation of resultant hydrogen peroxide caused by the addition of 50 mmol dm-3 glucose. The useful lifetime of the electrode was >21 days. This paper describes an extremely simple and easily controlled enzyme-immobilisation procedure that has application in the development of glucose sensors. The inevitable ' shelf-life' problem of devices using a biological component can be circumvented by the use of ' one-step ' electrode construction techniques that are amenable to mass production and hence end-product disposability. Extensions to this work aimed at the production of fast and disposable glucose sensors have focussed on the synthesis of pyrrole monomers having pendant functionalities that are good oxidants for reduced glucose oxidase. Electropolymerisation of these functionalised monomers by the method described should result in the entrapment of glucose oxidase in a conducting matrix to which electron-transfer from the enzyme to the electrode can occur.

References 1 A. F. Diaz and B. Hall, ZBM J1 Res. Dev., 1983, 27(4), 342. 2 S. Asavapiriyanont, G. K. Chandler, G. A. Gunawardena and D. Pletcher, Electroanal. Chem., 1984, 177, 229. 3 A. Diaz, Chem. Scripta, 1981, 17, 145. 4 A. F. Diaz, J. I. Castillo, J. A. Logan and Wen-Yaung Lee, Electroanal. Chem., 1981, 129, 115. 5 G. I. Ling, M. Ramstorp and B. Mattiasson, Anal. Biochem., 1982, 122, 26. 6 R. Bentley, in P. D. Boyer, H. A. Lardy and H. Myrback, The Enzymes (Academic Press, New York, 1973), vol. 7, 567. 7 M. Salmon, A. F. Diaz, A. J. Logan, M. Krounbi and J. Bargon, Mol. Cryst. Liq.Cryst., 1982,83,265. 8 F. R. Duke, R. N. Kust and L. A. King, Electrochem. Soc., 1969, 116, 32. 9 R. M. Ianniello and A. M. Yacynych, Anal. Chem., 1981,53,2090. 10 A. E. G. Cass, G. Davis, G. D. Francis, H. Allen, 0. Hill, W. J. Aston, I. J. Higgins, E. V. Plotkin, L. D. L. Scott and A. P. F. Turner, Anal. Chem., 1984, 56, 667. 1 1 F. R. Shu and G. S. Wilson, Anal. Chem., 1976,48, 1679. 12 B. E. P. Swoboda and V. Massey, J. Biol. Chem., 1965, 240, 2209. 13 Q. H. Gibson, B. E. P. Swoboda and V. Massey, J. Biol. Chem., 1960,91,230. Paper 511955; Received 6th November, 1985