A sulphite biosensor. Coupling of sulphite oxidase (EC 1.8.3.1) to a TTFTCNQ electrode

137

.I. Electroanal. Chem., 351 (1993) 137-143 Elsevier Sequoia S.A., Lausanne

JEC 02550

A sulphite biosensor. Coupling of sulphite oxidase (EC 1.8.3.1) to a TTFTCNQ electrode Ulrich Korell and R. Bruce Lennox

l

Department of Chemiwy, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 2K6 (Canada) (Received 31 July 1992; in revised form 27 October 1992)

An enzyme electrode used for the detection of sulphite in solution has been developed by interfacing the sulphite-specific enzyme sulphite’oxidase (EC 1.8.3.1) to the organic conducting salt electrode material tetrathiafulvalene tetracyanoquinodimethane (‘ITFTCNQ). This is the first successful coupling of a molybdohemoprotein to a TTFTCNQ electrode. The resulting electrode exhibits enzyme-limited current responses at low applied potentials (0 and 100 mV vs. Ag/AgCI), with electrochemical Michaelis constants of 5.6 PM and 27 PM respectively. Detection of 2 PM sulphite at pH 8.6 is achievable with a simple adsorbed enzyme electrode. A lack of interference from 0, and reasonable operational stability are features of this enzyme electrode.

INTRODUCTION

The analysis of sulphite in foodstuffs and pharmaceutical formulations [l] is of importance because of the health hazards associated with its ingestion or with exposure to it [2,3]. A number of approaches have been used to quantify sulphite, including redox-indicator [4], acidimetric [5], reaction-linked spectrophotometric [6] and enzyme-linked 17-111 methodologies. An enzyme-linked approach is particularly interesting, given the ready availability of sulphite oxidase, a redox enzyme with high specificity for sulphite [12]. Together with many other workers we have been very interested in interfacing redox enzymes with amperometry to produce electrochemical biosensors [13-151; in this paper we characterize a new biosensor which involves coupling sulphite oxidase and an organic conducting salt electrode

l

To whom correspondence

0022-0728/93/$06.00

should be addressed.

0 1993 - Elsevier Sequoia S.A. All rights reserved

138

SO:- + 2H+

SO;- + H,O v E - Mo(VI)/Fe(III?

‘v

E - Mo(IV)/Fe(III)

2 cyt (XII) A2

cyt C(W)

in aerobic conditions. Sulphite oxidase (EC 1.8.3.1) is a molybdohemoprotein made up of two identical 55 kD subunits. The enzyme has both sulphite : cytochrome c activity and sulphite : oxygen activity 1161. The aim of any successful intervention in the catalytic cycle would involve replacing cytochrome c or oxygen with an electrochemically accessible electron shuttle (i.e. a redox mediator) capable of transferring the reduced enzyme’s electron pair to a detector electrode. To this end, we have recently investigated the application of an interesting class of materials based on solid state charge-transfer complexes [17]. These materials, termed organic conducting salts (OCSs), exhibit relatively high conductivity, are reasonably stable at moderate applied potentials and have been shown to be excellent materials for effecting bioelectrochemistry. Specifically, it has been found that the OCS tetrathiafulvalene tetracyanodimethane (TTFICNQ) is an excellent electrode material for effecting the catalytic oxidation of the reduced form of PQQ-containing methanol dehydrogenase (EC1.1.99.8) [18]. Although previously shown to be very effective in the bioelectrocatalytic oxidation of a number of reduced flavoenzymes [19-211, the extension of OCS electrodes to other enzyme types has been limited. Kulys [22] reported that NMPTCNQ (NMP=N-methylphenazinium) could be interfaced to lactate dehydrogenase, and Hale and Skotheim [231 have recently reported a biosensor based on TIFTCNQ and a non-flavo copper enzyme, galactose oxidase. In this paper, we describe the investigation of sulphite oxidase/TIFTCNQ electrodes and show that a simple yet sensitive biosensor can be produced using the electrode-adsorbed enzyme. TI’FTCNQ is a prototypical OCS and has been characterized extensively in terms of its structural and electronic properties [24]. Its success in acting as a stable electrode material in bioelectrocatalysis is well documented and has been the subject of several mechanistic studies [25-271. The kinetic and thermodynamic conditions in which a material such as TTFICNQ will transfer electrons via either direct or indirect (i.e. mediated) processes have been studied recently [28]. EXPERIMENTAL,

SECTION

Electrochemical in.strumentation and materials Cyclic voltammetry and fixed potential studies were performed using an Oxford Electrodes potentiostat (OE PP2, Oxford Electrodes, Oxford, UK). Signals were recorded using a BBC/SE 120 chart recorder.

139

‘ITFTCNQ was prepared using the procedure of Ferraris et al. [29]. Both TTF and TCNQ (Aldrich) were used as received. Elemental analysis confirmed the 1: 1 stoichiometry of the material used. As described previously [301, TTFICNQ was mixed thoroughly with high temperature Si oil (Aldrich) in a 1: 1.6 ratio (by weight) to yield readily usable organic conducting salt paste electrodes. The TI’FTCNQ/Si oil paste was deposited in a polytetrafluoroethylene (PTFE) cavity (0.5 mm deep) whose base is a Pt electrode. The electrode system consisted of the TTFTCNQ working electrode (geometric area, 0.038 cm’), a Pt wire counter-elecreference electrode (BAS, West Lafayette, IN). trode and an Ag/AgCl/KCI(,,,, Sodium’ sulphite (98%, Anachemia, Montreal, Canada), KC1 (AnalaR, BDH, Montreal, Canada), potassium ferricyanide (Strem, USA), sodium pyrophosphate decahydrate (ACS reagent grade, Aldrich USA) and sulphite oxidase (EC 1.8.3.1) (Boehringer Mannheim, Montreal, Canada) were used as received. All solutions were made up in 18 MOhm - cm-’ Milli-Q water. The sulphite oxidase, prepared from chicken liver, was provided as a suspension in 3.2 M (NH,)pSO, (pH 7.5). The activity of the enzyme was determined by a procedure similar to that described previously [7] where ferricyanide replaces oxygen as the electron acceptor. Spectrophotometric monitoring of the loss of Fe(CN)iat 420 nm (Varian DMS300) under saturated Na,SO, (400 mM) conditions with 15 nM of enzyme leads to a sulphite oxidase : Fe(CN)i- activity of 370 U/ml. Given that the ferricyanide activity is 10.9-fold greater than the oxygen activity, then the sulphite oxidase : oxygen activity is 34 U/ml. Enzyme electrode preparation and use A sulphite oxidase/TTFTCNQ electrode was prepared by incubating 10 ml of the enzyme suspension with a TTFTCNQ electrode for 30 min. The resulting electrode was rinsed with copious quantities of water and immersed for a further 10 min to ensure that nonadsorbed enzyme was not present. In a typical experiment, the enzyme electrode was placed in 15 ml of pyrophosphate buffer (pH 8.6) along with the reference electrode and the counter-electrode. Where indicated, the electrode cell was maintained under a nitrogen blanket and the temperature was maintained at ambient (21 f 2°C) under conditions of continuous stirring. The electrode was not pretreated electrochemically either prior to, or after, enzyme adsorption. In order that the electrode could be used repeatedly, it was found to be essential that the following procedure be carried out. Once an experiment with sulphite was completed, the solution was diluted until the sulphite concentration was less than 1 PM. Only then could the electrode be switched off (i.e. to the internal circuit), withdrawn from the solution and stored etc. Maintaining the electrode in a solution containing appreciable sulphite for any period of time (> 2 s> leads to open-circuit reduction of the electrode surface [28]. If open-circuit reduction occurs, stabilization of the background current to acceptable (nanoampere) levels can take anytime from minutes to hours. The dilution precau-

140

tion ensures rapid and accurate analyte determinations and highlights the potential utility of these OCS electrodes in flow injection systems. RESULTS AND DISCUSSION

The cyclic voltammograms of sulphite (pH 8.6) at Pt and TTFCNQ/Si oil electrodes are shown in Fig. 1. The overpotential at Pt is very large, with little oxidation occurring even at 300 mV. This over-potential is substantially reduced at the organic metal electrode, with large currents arising within the window of potential stability for the electrode. Comparison with the electrochemistry of other redox couples at TI’FTCNQ [28] suggests that sulphite is oxidized by homogeneous at these moderate phase lTF+ or TCNQ’ which have been electroreleased applied potentials via surface electrochemical processes. The response of an enzyme electrode to sulphite under stirred deoxygenated conditions at 0 mV is shown in Fig. 2. The resulting response saturates and provides a good fit to the Michaelis-Menten kinetic formalism. Subsequent experiments with the same electrode 1 h and 91 h later result in the curves shown in Fig. 2. Clearly the electrode response diminishes markedly over this extended

to.5

so.:: 1

a ri \

(0)

+15

a +lO I

\ l-l

+5 0 0

+lOO

+200

+300

E/mV(Ag/AgCI) (b)

Fig. 1. Cyclic voltammograms of (a) Na,SO, (1.0 mM) at a clean Pt electrode (geometric area, 0.028 cm’) and (b) a bare TIFTCNQ electrode (geometric area, 0.038 cm’): pH 8.6 (0.1 M pyrophosphate); sweep rate, 20 mV/s; scans begin at - 100 mV.

141 60

SO r

60 40 2 \ _

30 20

-i t z

20

: 7

10

ii

10

2g

0 -10

0

-10 0

SO

60

(cl)

90

kwlfitel

120

160

160

/ wM

v

-40

0

(b)

40

60

hMite1

/ u M

120

160

Fig. 2. Current vs. sulphite concentration for a lTFTCNQ/sulphite oxidase electrode poised at 0 mV vs. Ag/AgCl. SO:- is added every 30 s (conditions as in Fig. 1, with continuous N, bubbling): (a) each curve was obtained over 30 min and there was a pause of 30 min between A and B and 88 h between B and C, (b) Hanes plots of data in (a).

time frame, with the enzyme electrode half-life being ca. 4 h. This loss of enzyme electrode activity could arise from enzyme-associated deactivation given the lability of sulphite oxidase. Hanes plots ([substrate]I-’ vs. [substrate]) of these data provide enzyme electrode Michaelis constants UC,,) and I,, values. Whereas I max values range from 50 to 6 nA over this time frame, a constant K,, value of 5.6 & 0.3 PM is observed. By comparison, the K, with oxygen as the electron acceptor is 24 PM for the chicken liver enzyme [12]. Mediator K, differences may be the origin of the difference between the K,, and the intrinsic K, of the enzyme.

0.6.

A

l

0

0

c

l

D

,

0 (a)

30

60 hlfitel

90

120 / wM

160

-40

180 (b)

1 0

40 [8Ulfihl

80

120

160

//.A M

Fig. 3. Current vs. sulphite concentration for a lTFTCNQ/sulfite oxidase electrode poised at + 100 mV vs. Ag/AgCl (conditions as in Fig. 1). (a) Curves A and C were obtained under N,-purged conditions and curves B and D were obtained under air-saturated buffer conditions. The temporal sequence of A to D is such that each curve was obtained over 20 min (30 s per sulphite increment) and there is a 30 min pause between runs. (b) Hanes plots of data in (a).

142

When the enzyme electrode is poised at + 100 mV vs. Ag/AgCl (Fig. 31, much greater sensitivity to sulphite is achieved with Ima = 300 nA. The effect of oxygen interference on the signal was investigated by performing calibration plots alternately in the presence and absence of oxygen (Fig. 3). These data reveal that enzyme deactivation dominates any oxygen interference phenomenon at the TTFTCNQ electrode. Hanes plots of these data reveal that the K, of the enzyme electrode is insensitive to the presence of oxygen. Moreover, K,, under both aerobic and anaerobic conditions is also greater at 100 mV (27 f 3 PM) than at 0 mV (5.6 f 0.3 PM). The origin of this potential dependence of K, is unknown at the present time as the details of the sulphite oxidase :TCNQ’ and sulphite oxidase : T’I’F+ activities are not available. None the less, the substantial enzyme activity in the presence of a TT’FTCNQ electrode establishes that TTF and/or TCNQ species are efficient mediators of sulphite oxidase electron transfer. This is notable given that the number of useful mediators for this enzyme is very limited 171. The sulphite oxidase/TIFTCNQ electrode can detect 2 PM of sulphite in aqueous buffer (in the presence of a background current of ca. 10 nA), has a dynamic range of ca. 50 mM at + 100 mV and is insensitive to oxygen. The ease of fabrication and operation of this system is an important factor as it involves simply dip-coating the enzyme onto the electrode surface and requires no sample pretreatment with diffusible mediators [7]. Moreover, diffusible mediator systems are usually prone to substantial oxygen interference at both the enzyme and electrochemical reaction steps. Deoxygenation procedures for sulphite-containing solutions are highly impractical given the volatility of SO,, and sulphite complexing agents must be introduced if deoxygenation via degassing is used. Electrochemical monitoring of oxidase activity (via oxygen depletion or H,O, generation) is difficult given oxygen tension variabilities and the extremely low intrinsic K, for the second substrate (sulphite). The operation of the sulphite oxidase/OCS electrode avoids all these problems and therefore seems to provide a major improvement in the quantification of sulphite in aqueous solutions. Finally, we can comment on the use of the TTFTCNQ electrode for the detection of species which are frequently coincident and are of importance in in-vivo analyses. We have shown previously that ascorbate and dopamine can be determined in each other’s presence using a bare TTFICNQ electrode [30]. Given that the TTFTCNQ electrode is capable of measuring total ascorbate, dopamine and sulphite at 0 mV, then the quantity of sulphite can be determined by using a system with two working electrodes (one coated with sulphite oxidase and the other bare). This selectivity is not otherwise readily accessible using methodologies which do not involve a prior separation step. ACKNOWLEDGEMENTS

This work was supported Faculty.

by NSERC,

Canada,

and the McGill Graduate

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