Biosensors & Bioelectronics 7 (1992) 375-380

Short communication Amperometric enzyme electrode for determination of theophylline in serum Calum J. McNeil, Jonathan M. Cooper* & Julia Al Spoors Department

of Clinical


The Medical School, University upon Tyne, NE2 4HH, UK

(Received 29 July 1991; revised version received 26 September

of Newcastle upon Tyne. Newcastle

1991; accepted 8 October 1991)

Abstract: This paper describes an amperometric enzyme electrode for the rapid determination of theophylline in serum. The method is based on the catalysed oxidation of theophylline by the haem-containing enzyme theophylline oxidase. Results are presented for two approaches. First, ferrocene monocarboxylic acid was used as a mediator. The second-order rate constant was 1.1 X 103 1 mol-’ s-l. Secondly, the organic conducting salt NMP.TCNQ was used to construct enzyme electrodes. These electrodes were employed for the rapid (60 s) measurement of theophylline in serum at a working potential of + 100 mV versus Ag/AgCl. Linear calibration curves ‘were obtained over the clinically relevant range (v = 0.13x + 0.22, n = 8). Caffeine, theobromine and 3-methylxanthine at levels up to 100 mg 1-t do not interfere and 1-methylxanthine shows cross-reactivity at concentrations greater than 50 mg 1-t. Keywords: enzyme electrode, theophylline,

INTRODUCTION Theophylline (1,3_dimethylxanthine) is a widely prescribed bronchodilator drug for the treatment of various asthmatic and pulmonary conditions (Rowe et al., 1988). Clinical studies have shown that the therapeutic control of bronchospasm is related to the maintenance of adequate serum theophylline concentrations (Jackson et al., 1964; Mitenko & Ogilvie, 1973) within a very narrow therapeutic range: lo-20 mg 1-l. If the concentration falls below this range the drug is ineffective, *Present address: Department of Electronics and Electrical Engineering University of Glasgow, Glasgow, G12 8QQ. UK 0965-5663/92/$05.00 @ 1992 Elsevier Science Publishers

serum, theophylline


whilst if it accumulates above this range the drug is toxic, causing tachycardia, nausea, arrythmia and convulsions (Jackson eral., 1973). Hence, frequent determination of theophylline in serum is recommended as the only reliable method of following the effects of therapy (Mitenko & Ogilvie, 1973). Monitoring serum theophylline is also useful following an overdose as there is a relationship between the serum concentration and the onset of potentially life-threatening side effects, which may appear with only minor previous clinical signs of toxicity (Rowe et al., 1988). Therefore, a rapid, simple method for monitoring the narrow therapeutic range for theophylline is required. Ltd.


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C. .l McNeil, J M. Cooper, J; A. Spoors

A large number of techniques are employed for serum theophylline estimation. Most of these are immunoassays which involve laborious dilution and incubation procedures and, as such, are not suitable for rapid testing. In addition, many immunoassays may show a significant degree of interference from theophylline metabolites and other xanthines (Rowe et al., 1988). Theophylline oxidase, (ThOx), a haemcontaining protein, has recently been isolated and applied to the rapid, specific measurement of the bronchodilating drug theophylline. A solution kit for the enzymatic determination of theophylline based on ThOx has been produced by GDS Technology Inc. (Elkhart, IN, USA). The enzyme reacts directly with theophylline as a substrate in the presence of ferricytochrome c as a co-substrate electron acceptor, as shown below: Theophylline

+ cyt.c(Fe3+) __* 1,3-dimethyluric acid + cyt.c (Fe*+)

In the commercial assay, the reduction of ferricytochrome c is monitored at 550 nm and is proportional to the concentration of theophylline in the sample. The enzyme has no requirement for oxygen in its catalytic action. In this preliminary study we have replaced the natural redox partner, ferricytochrome c, with non-physiological mediators in order to construct enzyme electrodes for the measurement of theophylline in serum. A theophylline enzyme electrode would provide an easy-to-use device and would enable a rapid return of biochemical information to the clinician compared with immunoassay and chromatographic methods (Rowe er al., 1988).

EXPERIMENTAL, Materials The GDS Technology Inc. enzymatic theophylline reagent kit was purchased from Impulse Clinical Diagnostics, Gwent, UK. The stock enzyme solution (20 U ml-‘) was concentrated using the Cetricon system supplied by Amicon, Gloucester, UK to give a final protein concentration of 35 mg ml-‘, as measured by the method of Lowry et al. (1951). Theophylline, caffeine, theobromine, I-methylxanthine, 3-methylxanthine, ferrocene 376

monocarboxylic acid (FcCOOH), phenazine tetracyanoquinodimethosulphate (NMP), methane (TCNQ) and bovine serum albumin (BSA) were obtained from Sigma Chemical Co., Dorset, UK AnalaR acetone was from BDH, Dorset, UK Toray carbon paper was purchased from Toray Industries, Japan. Ferrocene-mediated kinetics Direct current cyclic voltammetry experiments were performed in a two-compartment glass cell which had a working volume of 0.5 ml. The working electrode was a 4 mm diameter gold disk. The reference and, counter electrodes were a saturated calomel electrode (SCE) and a piece of platinum wire, respectively. These experiments were carried out using a Hyspec potentiostat with a Gould series 60000 XYt chart recorder. Cyclic voltammograms of FcCOOH (0.2 mM) in 20 mM phosphate buffer, pH 7.0, containing 100 mM sodium perchlorate and 1 mM theophylline were recorded at scan rates over the range l-50 mV s-’ in the presence and absence of various concentrations of ThOx in solution (7-40 PM). The ratio of kinetic to diffusion-controlled current was used to calculate the second-order homogeneous rate constant for the reaction of FcCOOH with ThOx as outlined by Davis (1987). Enzyme immobilization The organic conducting salt NMPTCNQ was prepared according to literature methods (Bartlett, 1990). An NMP*TCNQ paste electrode was constructed by mixing 100 mg of the organic salt with 40 mg of low-molecular-weight polyvinyl chloride and adding sufficient acetone to form a viscous paste. Thereafter the paste was smeared onto a 1 cm2 piece of Toray carbon paper to form a uniform layer and the electrode was dried in air. The Toray paper provided a conducting support for the organic salt electrode. Theophylline oxidase was immobilized to the electrode by passive adsorption in a solution of ThOx (35 mg ml-‘) at 4°C for 5 h. After this time, the enzyme electrode was washed with 50 mM phosphate buffer, pH 7.4, and the enzyme was reapplied for a further 5 h to maximize protein loading. Typically a 5011 aliquot of enzyme solution would make ten enzyme electrodes. Non-adsorbed enzyme was removed by repeated washing in phosphate buffer. The enzyme electrode

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was stored in the same buffer at 4°C. Electrodes with BSA immobilized instead of ThOx were prepared in an identical manner using a stock solution of 35 mg ml-’ BSA in phosphate buffer. Apparatus and procedures Electrochemical experiments using NMPeTCNQ enzyme electrodes for measurement of theophylline in buffer were performed in a two-electrode cell which contained a 5 mm diameter compacted Ag/AgCl combined reference and auxiliary electrode which surrounded a 1.5 mm diameter recessed gold contact into which the working electrode was placed (Cooper et al., l!XIla). The working electrode was poised at + 100 mV versus Ag/AgCl using either a Thompson Low Noise Ministat (Newcastle upon Tyne, UK), or using a

Amperometric enzyme electrode

custom-built bipotentiostat (McNeil et al., 1990). Measurements in serum were carried out using a dual working electrode system (Fig. 1) as described previously (McNeil et al., 1990). The results were recorded using a Goerz Metrawatt SE120 chart recorder. Enzyme electrode assembly The working electrodes were cut using a 1.5 mm diameter punch, and placed on the gold contacts. The electrodes were covered with’ a 0.05 pm pore size polycarbonate membrane (Nuclepore, High Wycombe, Bucks, UK), which was attached to a membrane holder using double-sided pressuresensitive Scotch tape. The membrane excluded serum proteins from the electrode and prevented fouling effects caused by passive adsorption on



REF ______ - ______ -

Fig. I. Electrochemical cell and membrane holderfor theophylline measurement in serum. BEE A UX,ENZ and BCK refer to the reference, auxiliary, enzyme and background electrodes, respectively. The electrodes were connected to a bipotentiostat via a DIN connector.

C. J. McNeil, J M Cooper, .I. A. Spoors

the electrode. The tension produced when ‘the membrane holder was clipped into position held the enzyme electrodes in electrical contact with the gold posts. The membrane holder formed the sample compartment in which all electrochemical measurements were made. The membrane excluded serum proteins from the enzyme electrode and ensured that passive adsorption and subsequent electrode fouling did not occur. Assembly in this manner provided a reusable electrochemical device which did not need to be dismantled for any subsequent operation. Serum measurement Blood was collected from healthy individuals immediately prior to experimentation, and was centrifuged at 800 X g for 5 min using an IEC Centra-3R Centrifuge (International Equipment Co., Dunstable, Bedford, UK). The serum was stored at 4°C and equilibrated to ambient temperature (20°C) immediately prior to use. The electrode was calibrated in serum containing known concentrations of added theophylline. Procedure A series of theophylline standard solutions (lo-100 mg 1-l) were prepared in phosphatebuffered saline, pH 7.4 (PBS), and serum, and the response of the enzyme electrode was measured in the following manner. A steady-state baseline response was first established in PBS. This was then withdrawn, and replaced by the same buffer or serum sample containing known concentrations of added theophylline. The steady-state current response was measured after 1 min and the electrode washed with aliquots of PBS to reestablish a baseline prior to addition and measurement of the next sample. The response of the ThOx electrode to various structural analogues of theophylline such as caffeine, theobromine, I-methylxanthine and 3-methylxanthine over the range O-100 mg ml-’ in PBS was measured. The enzyme electrode was stored in PBS at 4°C when not in use.

RESULTS AND DISCUSSION Studies have shown that the physiological electron acceptors for oxidases can be replaced by electron transfer mediators which are low-molecular378

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weight redox couples that shuttle electrons from the redox centre ofthe enzyme to the surface of an electrode. The most successful examples of this for biosensor technology are ferrocene (bis(n’cyclopentadienyl)iron) and its derivatives in combination with flavoproteins such as glucose oxidase (Davis, 1987). As a consequence of our interest in the development of an amperometric enzyme electrode we first examined whether the ferricinium ion would act as an electron acceptor for ThOx and whether this would be the basis of a practical approach to theophylline measurement. Direct current cyclic voltammetry was used to estimate the rate of reaction between oxidized ferrocene monocarboxylic acid (FcCOOH) and ThOx as outlined previously (Davis, 1987). The calculated second-order rate constant for the homogeneous reaction between the ferricinium ion and the reduced enzyme in phosphate buffer was 1.1 X lo3 1 mol-’ s-’ at pH 7.0. The corresponding rate constant for FcCOOH with the enzyme glucose oxidase has been measured as 2 X lo5 1 mol-’ s-t, and indeed the rate constant for ThOx was at least one order of magnitude lower than all of the oxidoreductases employed in the study by Davis (1987). As a consequence of the poor mediation of the electrochemical reaction using FcCOOH we did not pursue these investigations any further. However, it is possible that ferrocene derivatives with an overall positive or neutral charge may have shown faster mediated kinetics and proved more suitable for biosensor exploitation. This phenomenon has been observed in other enzyme systems (Cooper eral., 1991b). Numerous studies have shown that conducting organic salts, such as NMP.TCNQ, are excellent electrocatalysts for a wide range of oxidomductases with rate constants greater than 10T4 cm s-’ (Bartlett, 1990). These materials therefore have considerable advantages over mediated systems when used as electrode materials for enzymes (Albery & Craston, 1987; Hill et al., 1990). The low operating potential range of these materials (0 to +3OO mVversus Ag/AgCl) makes them extremely attractive for application to clinical analysis in plasma and blood. Over this range, many of the endogenous electroactive species in blood such as ascorbic acid and uric acid will not be oxidized and thus will not interfere. Immobilization of ThOx at a conducting salt electrode replaces ferricytochrome c as the electron acceptor and allows direct amperometric determination of theophylline concentration by oxidation of the


Biosensors & Bioelectronics

reduced form of the enzyme by the conducting salt electrode in a manner analogous to that reported previously for flavoenzymes (e.g. Albery et al., 1985; Hill et al., 1990). The applied potential used for the NMP*TCNQ electrodes (+ 100 mV versus Ag/ AgCl) was selected on the basis of the stability of the response, the oxidation signal from potential interferents in the sample, and the background current. Enzyme electrodes constructed by passive adsorption of ThOx onto the surface of an NMP.TCNQ electrode show a linear response to theophylline in buffer over the clinically relevant range 0, = 0*22x - 0.04, n = 5, r = O-99, Fig. 2, curve a). The response time was of the order of 1 min. The increase in current densities with the NMP*TCNQ electrodes compared with the ferrocene-mediated system demonstrates that the rate of the donor-mediated reaction with the enzyme is greater (5.1 /.rA cmb2 compared with 1.33 ,uA cmm2 for 20 mg 1-l theophylline) and therefore this approach is more useful for clinical measurement of theophylline levels. The linear range of the immobilized ThOx NMP.TCNQ electrode in buffer demonstrated that this system should be ideal for rapid estimation of theophylline in undiluted serum. Indeed our preliminary studies on serum ‘spiked’ with known concentrations of theophylline have shown this to be the case (Fig. 2, curve b: y = 0.13x + O-22, n = 8, r = 099). The difference in sensi-



tivity between buffer and serum measurements was presumably due to matrix effects including diffusional differences due to the protein content of serum. For clinical use it would be important to calibrate the device using solutions with a comparable composition to serum. In common with the cross-reactivities for the enzyme stated by GDS Technology Inc., ThOx electrodes show no interference from caffeine, theobromine or 3-methylxanthine at levels up to 100 mgl-*. However, I-methylxanthine interfered with the electrochemical measurement at concentrations greater than 50 mg 1-l. This would not be a problem since only trace amounts of theophylline are metabolized to 1-methylxanthine in vivo (Amaud & Welsch, 1981). The sensor described in this preliminary paper provides a rapid and sensitive assay which has a number of advantages over high-performance liquid chromatographic and immunoassay methods of serum theophylline measurement and is also much simpler to construct and operate than a previously reported amperometric enzyme electrode measurement which was based on the inhibition of bovine liver alkaline phosphatase (Foulds efal., 1990). Studies are in progress to define fully the operational characteristics, longterm stability and clinical utility of this system.

REFERENCES Albery, W. J., Bartlett, P. N. & Craston, D. H. (1985). Amperometric enzyme electrodes, Part II. Conducting organic salts as electrode materials for the oxidation of glucose ox&se. J. Electroanal. Chem.. 194,223-35.








Theophylline [mg I’]

Fig. 2. Calibration curves for NMP. TCNQ ThOx enzyme electrodes measuredat a potentialof +I00 mVvs. A&4&1: curve a, theophylline in PBS, pH 7.4; curve b, theophylline added to freshly collected human serum.

Albery, W. J. & Craston, D. H. (1987). Amperometric enzyme electrodes: theory and experiment. In Biosensors: Fundamentals and Applications, ed. A. P. F. Turner, I. Karube & G. S. Wilson. Oxford Science Publications, Oxford pp. 180-210. Amaud, M. J. & Welsch, C. (1981). Theophylline and caffeine metabolism in man. In Theophylline and Other Methykanthines, ed. N. Bietbrock, B. G. Woodcock & A. H. Staib. Friedr. Vieweg, Braunschweig, pp. 135-48. Bartlett, P. N. (1990). Conducting organic salt electrodes. In Biosensors: A Practical Approach, ed. A. E. G. Cass. Oxford University Press, Oxford, pp. 47-95. Cooper, J. M., McNeil, C. J. & Spoors, J. k (1991a). Amperometric enzyme electrode for the determination of aspartate aminotransferase and alanine aminotransferase in serum. Anal. Chim. Acta, 245, 57-62. 379

C. J. McNeil, .I M. Cooper, J. A. Spoors Cooper, J. M., Bannister, J. V. & McNeil, C. J. (1991b). A kinetic study of the catalysed oxidation of 1’,3-dimethylferrocene ethylamine by cytochrome c peroxidase. J. Electroanal. Chem., 312, 155-163. Davis, G. (1987). Cyclic voltammetry studies of enzymatic reactions for developing mediated biosensors. In Biosensors: Fundamentals andApplications, ed. A. P. F. Turner, I. Karube & G. S. Wilson. Oxford Science Publications, Oxford, pp. 247-56. Foulds, N. C., Wilshere, J. M. & Green, M. J. (1990). Rapid electrochemical assay for theophylline in whole blood based on the inhibition of bovine liver alkaline phosphatase. Anal. Chim. Acta, 229, 57-62. Hill, B. S., Scolari, C. A. &Wilson, G. S. (1990). Enzyme electrocatalysis at the TTF-TCNQ electrode. Phil. Trans. R Sot. L.ond. A, 333,63-9. Jackson, F. R., Ganido, R, Silverman, H. I. &Salem, H. (1973). Blood levels following oral administration


Biosensors t Bioekctronics of theophylline preparations. Ann. Allergy. 31, 413-19. Jackson, R H., McHenry. J. L. &Moreland, F. B. (1964). Clinical evaluation of elixophyllin with correlation of pulmonary function studies and serum theophylline levels in acute and chronic asthmatic patients. Dis. Chest, 45, 75-8. Lowry, 0. H., Rosebrough, N. J., Farr, A L. & Randall, R J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-75. McNeil, C. J., Spoors, J. A., Cooper, J. M., Alberti, K. G. M. M. & Mullen, W. H. (1990). Amperometric biosensor for rapid measurement of 3-hydroxybutyrate in undiluted whole blood and plasma. Anal. Chim. Acta, 237,99-105. Mitenko, P. & Ogilvie, R I. (1973). Rational intravenous doses of theophylline.h! Engl. .KMed. 289,600-3. Rowe, D. J. F., Watson, I. D., Williams, J. & Berry, D. J. (1988). The clinical use and measurement of theophylline. Ann. Clin. Biochem., 25, 4-26.

Amperometric enzyme electrode for determination of theophylline in serum.

This paper describes an amperometric enzyme electrode for the rapid determination of theophylline in serum. The method is based on the catalysed oxida...
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