Amperometric enzyme electrode for the determination of phenols in chloroform

Geoffrey F. Hall, David J. Best and Anthony P. F. Turner

Biotechnology Centre, Cranfield Institute o f Technology, Cranfield, Bedfordshire, MK43 OAL, U K

(Received 5 November 1987; revised 14 January 1988)

An enzyme electrode has been developed that operates in chloroform. The enzyme polyphenol oxidase (E.C.1.14.18.1) was used to detect p-cresol via the electrochemical reduction of the product of the enzyme reaction at a graphite foil electrode. The response was linear for p-cresol concentrations of O- 100 iXM. After an initial rise from 1.9 to 4.0 tzA in the first three assays, the response of the electrode to p-cresol (100/zM) remained stable for 13 consecutive assays. The enzyme electrode lost no activity over 7 weeks' storage in chloroform at 5°C and is sensitive to a broad range of phenols.

Keywords: Enzyme electrode, organic solvents; p-cresol, polyphenol oxidase

Introduction

The ability of enzymes to catalyse reactions in organic solvents has been recognized for over 20 years/ and the potential for using enzymes in organic synthesis has recently generated intense research activity, z-6 The enzyme electrode was first reported in 1962, 7 and there is now a wide range of devices undergoing devel- opment. 8-H The combination of both technologies could create enzyme electrode systems that operate in an organic, nonpolar environment. In chloroform, polyphenol oxidase oxidizes p-cresol to 4-methyl-l,2- benzoquinone, ~2 and this reaction is utilized for the enzyme electrode described below. Operating an en- zyme electrode in an organic solvent enables a more simple and rapid enzyme immobilization procedure to be employed. Since the enzyme is insoluble in the or- ganic phase, it remains in the aqueous phase, retained by a hydrophilic support. Enzyme loadings can be achieved that are higher than those with the covalent immobilization techniques required for aqueous sys- tems. ~3 Other advantages of operating an enzyme elec- trode in organic solvents include higher concentrations of nonpolar reactants, extended substrate range, re- duction of side reactions occurring in the aqueous phase (for example the o-quinone product of poly- phenol oxidase undergoes rapid chemical polymeriza- tion in an aqueous environment, inactivating the en- zymO 2) and better integration with chemical steps in a synthetic sequence. The nonaqueous enzyme elec- trode could permit analyses to be carried out on previ- ously inaccessible clinical and industrial samples by

partitioning of a very low concentration analyte from a large aqueous volume into a small organic volume. The phenol electrode described could be used in this way for the detection of phenolic contamination of waste water.

Materials and methods

E l e c t r o d e c o n s t r u c t i o n

Polyphenol oxidase (1.7 mg tyrosinase from Sigma, Poole, Dorset) was dissolved in sodium phosphate buffer (15/~1, 50 mM, pH 7.0). This solution was al- lowed to soak into a rectangle (5 × 14 ram) of Hybond- N nylon membrane (Amersham International plc, Little Chalfont, Bucks.) and left to dry for 1 h at room temperature. A length of bare nickel-chromium wire (60 mm, 24 swg, Griffin and George Ltd., Lough- borough, Leicestershire) was folded as in Figure la. One end of the dry nylon membrane was clamped into fold 1 of the length of wire. The membrane was then folded around a block of graphite foil (5 x 6 × 1 ram, Le Carbone, Portslade, Sussex), which had been soak- ing in a solution of tetrabutylammonium toluene-4-sul- fonate (TBATS) (0.I M, Fluka, Fluorochem Ltd., Glossop, Derbyshire) in chloroform (HPLC grade, Al- drich Chemical Co., Gillingham, Dorset) for at least 1 h. All chloroform used in this work had been previ- ously saturated with sodium phosphate buffer (50 raM, pH 7.0). The shorter edge of the graphite foil block and the unclamped end of the nylon membrane were clamped into fold 2 of the wire. A length of nickel-

© 1988 Butterworth Publishers Enzyme Microb. Technol., 1988, vol. 10, September 543

Pap ers

0,, b,

1 1 - - ] - - 2

2

1

c, 6 ~ ~ 5

Figure 1 (a) The shape of nickel-chromium wire required for electrode construction. Fold 1 clamps one end of the nylon membrane and fold 2 clamps the graphite block and the mem- brane. (b) The enzyme electrode. 1. Nickel-chromium wire clamp to hold the nylon membrane supporting the enzyme in close contact with the graphite block. 2. Nickel-chromium wire shown in la. 3. Nylon membrane support for enzyme immobili- zation, cut away (dotted line) to show 4, graphite foil block. (c) The electrochemical cell used for all assays. 1. Saturated Calo- mel reference electrode. 2. Enzyme electrode. 3. Platinum wire counter electrode. 4. Hole for addition of p-cresol solution. 5. Truncated boiling tube. 6. Circular magnetic flea

chromium wire (10 mm) was clamped around the graphite block and membrane to hold the membrane in close contact with the graphite. The enzyme electrode is shown in Figure 1 b.

Cyclic voltammetry

The electrochemical properties of p-cresol and the product from the enzyme reaction were studied in chloroform (0.1 M TBATS) using the technique of cy- clic voltammetry. ~4 The working electrode was a glassy carbon electrode. Voltammograms between +600 mV and -500 mV versus SCE in chloroform were recorded using a BAS 100 Electrochemical Ana- lyzer (BAS, West Lafayette, IN 47906, USA) at a sweep rate of 50 mV s -~.

Apparatus

A three-electrode system was employed for all work with the enzyme electrode. The potential was main- tained by a precision potentiostat (Ministat, Thomp- son and Associates, Newcastle upon Tyne) and the current was recorded on an x-t chart recorder (Gal- lenkamp, Loughborourgh, Leicestershire) via a resis- tance board (J.J. Junior, J.J. Instruments, Southamp- ton, Herts.). A capacitor (47 /~F) was connected across the input terminals of the chart recorder to

smooth any background noise. A saturated Calomel electrode (Russell pH Ltd., Auchtermuchty, Fife, Scotland) was used as a reference and the auxiliary electrode was a platinum wire (0.4 mm diameter). The electrodes were immersed in chloroform (5 ml, 0.1 M TBATS) contained in a truncated boiling tube. The enzyme electrode was poised at -275 mV versus satu- rated calomel electrode in chloroform, and additions of small volumes of stock p-cresol (90 mM, Sigma, Poole, Dorset) in chloroform (0.1 M TBATS) were made via a small hole in the lid of the electrochemical cell (Figure lc).

Calibration of the enzyme electrode

Nine discrete assays were performed over a range of p-cresol concentrations (0 to 267/~M) on five different electrodes. Before each assay, sodium phosphate buffer (2/~1,50 mM, pH 7.0) was placed onto each side of the electrode to rehydrate the polyphenol oxidase. The electrode was placed into the cell described above and poised at -275 mV versus SCE in chloroform. After 25 min, the current became constant and an addi- tion of p-cresol was made. An increase in current was then observed, which reached a steady value, typically after 3 to 5 min. The cell was stirred throughout each assay. After each assay, the electrode was removed from the cell and washed in chloroform for about 60 s before being dried in air prior to the next assay.

Operational stability

The assay procedure outlined above was repeated with a final p-cresol concentration of 100/~M for a series of 16 assays.

Storage stability

Eight electrodes were constructed with dry graphite blocks and their response to p-cresol (200 /~M) was recorded. Half were then stored at room temperature and half at 5°C. At each temperature, two electrodes were stored dry, in bottles containing silica gel, and two were stored in chloroform. Their response to p- cresol (200/XM) was tested again after a few days and then after 7 weeks.

Electrode specificity

The response of a single electrode to a concentration (100 /.~M) of phenol, catechol, 4-methyl catechol, m- and p-hydroxy benzaldehyde, m-, p-, and o-cresol, p-aminophenol, and 4-chlorophenol was recorded.

Results and discussion

Electrochemical characterization of p-cresol and the product of the enzyme reaction shows that p-cresol (170 mM in chloroform) has no electrochemical activ- ity between +600 and -500 mV, versus SCE in chlo- roform, whereas, after reaction with polyphenol oxi- dase, the same solution shows an anodic peak at + 311 mV and a cathodic peak at -175 mV (Figure 2). By

544 E n z y m e M i c r o b . T e c h n o l . , 1988, vo l . 10, S e p t e m b e r

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Figure 2 Cyclic voltammograms of (a) product after reaction with polyphenol oxidase in chloroform (0.1 M TBATS) and (b) p-cresol. The potential was swept between +600 mV and -500 mV versus SCE in chloroform (0.1 M TBATS) and the current recorded using a BAS 100 Electrochemical Analyser, The sweep rate was 50 mV s -1

poising a graphite foil electrode at -275 mV versus SCE in chloroform, the product of the enzyme reac- tion can be detected via its electrochemical reduction. The response of the electrode to p-cresol was linear in the concentration range 0-100 /.¢M (Figure 3). A steady-state current was reached typically after 4 to 6 min, with 95% of the steady-state value being achieved in about 3 min. The standard deviation error bars indi- cate the good reproducibility between electrodes. The response of an individual electrode to repeated assays using p-cresol (100/.¢M) increased from 1.9 to 4.0/.cA in the first three assays and then remained stable over the next 11 assays before starting to fall more rapidly after assay 14 (Figure 4). The calibration data were col-

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Figure 3 Calibration curve of the enzyme electrode for p-cresol. The error bars represent -+1 s.d. and indicate the reproducibility between electrodes

Amperometric enzyme electrode: G. F. Hall et aL

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Assay number

Plot of electrode response to p-cresol (100 p.M) over 16

lected on assays 4 to 11. Over a period of 7 weeks, the electrodes stored at room temperature showed an av- erage loss of activity of 30% of the response after 3 days, whereas those stored at 5°C showed no signifi- cant decrease in their response to p-cresol (200/.¢M) (Table 1). A critical factor in the response of the elec- trode is the surface area of the graphite block. The graphite blocks are porous and are soaked in chloro-

Table 1 Storage stability data for the p-cresol enzyme elec- trode

Response Response Initial after after

Storage temp. Storage response 3 days 48 days (°C) method (/zA) (/~A) (/cA)

Room temp Dry 5.2 6.8 3.6 (20°C) 4.8 5.4 2.8

In CHCIz 4.0 7.0 5.5 4.2 7.2 6.5

5°C Dry 4.3 4.4 4.7 3.7 4.5 3.5

In CHCIs 4.8 8.1 10.0 3.8 9.5 7.6

Table 2 Response of the enzyme electrode to 10 phenols (100/ZM)

Phenol Electrode response (,u.A)

p-Cresol 5.6 m-Cresol 4.7 o-Cresol 0.0 Phenol 6.4 Catechol 8.6 4-Methylcatechol 6.7 p-Hydroxybenzaldehyde 0.0 m-Hydroxybenzaldehyde 0.0 p-Aminophenol 2.4 4-Chlorophenol 3.1

Enzyme M ic rob . Techno l . , 1988, vol . 10, Sep tembe r 545

Papers

form prior to use to ensure that the maximum surface area is in contact with the solution and that this area remains constant. If the graphite is not precondi- tioned, as with the electrodes used in the storage tests, then a much reduced response is obtained due to a decrease in the effective surface area of the electrode. After storage in chloroform, the electrode response returns to that which would be obtained if the elec- trode had been constructed with a presoaked graphite block (Table 1). The electrode responded to all the phenols tested, except o-cresol and p- and m-hydroxy- benzaldehyde (Table 2), indicating a potential use as a phenol sensor. This electrode is quick and easy to con- struct and demonstrates the feasibility of biosensors for use in organic solvents.

A c k n o w l e d g e m e n t s

We acknowledge SERC for financial support to G . F . H . A . P . F . T . is a Senior Fel low of the British Dia- betic Association.

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