Advances in amperometric enzyme electrodes in reversed micelles

ANALYTICA

CHIMICA ACTA ELSEVIER

Analytica

Chimica Acta 315 (1995) 93-99

Advances in amperometric enzyme electrodes in reversed micelles A.J. Reviejo a, C. Fernhdez a Department

a, F. Liu a, J.M. Pingarrbn ay*, J. Wang b

of Analytical Chemistry, Faculty of Chemistry, Complutense Universily of Madrid, 28040 Madrid, Spain b Department of Chemistry, New Mexico State University, Las Cruces, NM 88003, USA Received 29 March 199.5;

revised 30 May 1995;accepted30 May 1995

Abstract Some new trends in the development of amperometric enzyme electrodes in reversed micelle systems are shown. The possibility of using these media for on-line applications as well as for enzyme inhibition measurements both in the batch and the flow-injection modes is demonstrated. Reversed micelles were formed of ethyl acetate as organic solvent, dioctyl sulfosuccinate as emulsifying agent and a 4% of 0.05 mol 1-l phosphate buffer of pH 7.4 as aqueous phase. An Eastman AQ/enzyme immobilization approach was used. Flow-injection analysis of various phenolic compounds using the reversed micelle as the carrier solution was accomplished. Despite the hydrodynamic conditions, the immobilized enzyme layer displayed a good stability in the reversed micelle flowing stream. Phenolic compounds with all o&o-positions substituted gave no analytical signal. The possibility of developing reversed micelle enzyme inhibition biosensors is illustrated by monitoring inhibitors of tyrosinase such as benzoic acid, thiourea or propyl gallate, and using phenol as the substrate. The steady-state response towards phenol rapidly decreases upon additions of these compounds at a low concentration level. A reversible inhibition process was shown in all cases, and the type of enzyme inactivation in the reversed micelle of each inhibitor studied. The rapid response of the tyrosinase electrode to the changes of inhibitor concentration in the reversed micelle can be also exploited for on-line applications. Keywords:

Reversed micelles; Biosensors;

Flow injection; Inhibitors

1. Introduction Biosensing in predominantly non-aqueous systerns is becoming a new trend in analytical biochemistry because of the great number of particular applications that can be undertaken in these media [1,2]. Recently, we have illustrated the possibility of using

* Correspondingauthor. 0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(95)00312-6

reversed micelle systems as working media for atnperometric enzyme biosensors [3,4]. Reversed micelles or water-in-oil emulsions consist of an organic solvent as the continuous phase, water as the dispersed phase and a surfactant acting as emulsifying agent. The most important features of the use of these media for enzymatic reactions are three: these systems can be considered as universal solubilization media for both hydrophilic and hydrophobic analytes; second, the amount of water necessary for the hydration of the enzyme, which is an essential point

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in order that enzymes keep biocatalytic activity in non-aqueous media, is very easy to control, which means an important advantage with respect to organic phase enzyme electrodes where the organic solvent is immiscible with water; finally, a greatly simplified enzyme immobilization scheme onto the electrode surface is achieved if working in reversed micelles where enzymes are scarcely soluble. On the other hand the concept of organic-phase biosensors has been recently expanded toward measurements of impo~~t enzyme inhibitor. Wang et al. [S] exploited the ~hibition of tyrosinase and horseradish peroxidase by various organic compounds for their highly sensitive amperometric monitoring in a flow-injection operation with an acetonitrile carrier solution. Regarding enzyme-based flowinjection analysis (FIA), it has been employed for the determination of substrates using enzymes as catalyst, for the determination of enzyme activity where the enzyme itself is the analyte to be determined, and for the dete~ination of enzyme inhibitors [6]. This work has a double objective; on the one hand, to demonstrate that reversed micelles can be used as the carrier in flow-injection analysis, and consequently to exploit all the advantages of these media for on-line monitoring applications; second, to show that reversed micelle systems can be also employed for enzyme inhibition measurements both in batch and in flow-injection modes. The determination of phenolic compounds included in the US Environmental Protection Agency List of priority pollutants 131has been chosen to accomplish the first objective, whereas the behaviour of some inhibitors of tyrosinase (benzoic acid, thiourea and propyl gallate) was used for the second aim.

Chimica Acta 315 (15%) 93-99

enzyme electrode was prepared as described previously [3]. The flow-injection arrangement consisted of an Isco Model Wiz peristaltic pump and an Omnifit Model 1106 injection valve with variable injection volumes. A large-volume (50-ml) glass wall-jet cell [7] was used, and electrode potentials were controlled by means of a Metrohm 641 VA detector, the signals being registered on a Linseis L 6512 recorder. All potentials are reported vs. the Ag/AgCl reference electrode (Model RE-1, BAS). 2.2. Reagents and solutions The reagents used were: tyrosinase (EC 1.14.18.1, activity 2100 units/mg solid, Sigma); dioctyl sulfosuccinate (AOT, Sigma); phenol (Sigma); 4-chloro3-methylphenol, 2,4_dimethylphenol, 3,5dimethylphenol, 2,6-dimethylphenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, pentachlorophenol (Aldrich); benzoic acid (Sigma); thiourea (Sigmas propyl gallate (~edel-de-banns Eastman AQ 55s (Kodak) and ethyl acetate (Panreac). Tyrosinase stock solutions were prepared by dissolving 1 mg of enzyme in 100 ~1 of a solution containing 0.05 M phosphate buffer (pH 7.4) and 1.4% of the poly(ester-sulfonic acid) polymer Eastman AQ. A 0.10 M AOT stock solution in ethyl acetate was also prepared. Stock solutions of the phenolic compounds as well as of the inhibitor compounds tested (0.10 M) were water-in-oil microemulsions prepared daily, formed with ethyl acetate as organic solvent, 0.10 M AOT as emulsify~g agent and 0.05 M phosphate buffer (pH 7.4) as aqueous phase (4%). More dilute standards were prepared by suitable dilution with the same components of the emulsions.

2. Experimental 2.3. Procedures 2.1. Apparatus, electrodes and electrochemical cells Batch measurements were performed on an EG& G PAR Model 362 Scanning Potentiostat, in connection with a Linseis LY 16100 A recorder. The electrochemical cell was a BAS Model VC-2 cell with a BAS RE-1 Ag/AgCl reference electrode and a platinum wire counter electrode. Measurements were carried out by amperometry in stirred solutions. The

Activation of the tyrosinase electrode was accomplished in accordance to reference [3]. Enzyme-based flow-injection analysis of phenolic compounds were carried out by using the above mentioned reversed micelle system as the carrier. ~alib~tion plots were obtained at -0.30 V with a flow rate of 0.40 ml min-‘. Reversed micelle enzyme inhibition measure-

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AJ. Reviejo et al. /Analytica Chimica Acta 315 (1995) 93-99

ments were accomplished in the same reversed micelles containing a constant 0.3 mM phenol substrate concentration. The volume of the reversed micelle for batch experiments was always 5.0 ml. The potential applied to the enzyme electrode as well as the flow rate for FIA experiments were the same as mentioned above.

~::-L_,I: 1: 0%

0.01 0.0

3.1. Enzyme-based FL4 of phenols in reversed micelles Although reversed micelles have been demonstrated to be suitable as working media for developing enzyme amperometric biosensors [3,4], they had never been used in flowing systems in connection with amperometric biosensor detectors. Furthermore, although for batch work in these media direct adsorption of the enzyme on the electrode surface can be used as immobilization procedure, such scheme results in low stability when used for detection in flowing systems. Consequently, the EastmanAQ/enzyme immobilization approach was used taking into account the well-demonstrated stability of Eastman AQ coatings in various organic solvents [8]. The composition of the reversed micellar systems employed is the same as described for batch studies [3], i.e., ethyl acetate as organic solvent, 0.10 mol 1-l AOT as emulsifying agent and a 4% of 0.05 mol

.._ 0.6. 0.6.

a 8

0.4.

d

o.2. Oh -1.2

T

0.6

0.6

.lO.O

0.3 Flow

3. Results and discussion

s

-50.0

1.2.

0.3

0.0

0.3

E,V

Fig. 1. Influence of the applied electrode potential on peak current for 50 PM phenol in reversed micelles formed with ethyl acetate, 0.10 mol 1-l AOT, and 4% 0.05 mol 1-l phosphate buffer of pH 7.4. Flow rate 0.40 ml min-‘.

0.6

0.Q

TO.0 1.2

rate, mL mine1

Fig. 2. Effect of the reversed micelle flow rate on the peak height (O), and the peak width at half-height (M) for 50 /.LM phenol; applied potential, - 0.30 V.

1-l phosphate buffer of pH 7.4 as dispersed aqueous phase. Moreover, the content of Eastman AQ polymer in the solution used for coating the electrode surface, a 1.4%, was that recommended by Wang et al. [5]. Fig. 1 shows the influence of the applied electrode potential between + 0.40 and - 1.00 V on the peak current, for injections of 110 ~1 of a 50 PM phenol stock water-in-oil microemulsion into the same reversed micelle system used as the carrier. A well-defined plateau, at potentials between - 0.30 and -0.60 V was obtained, followed by a decrease in the current at more negative potentials (which may be attributed to polymerization of the o-quinone produced at these negative potentials [3]). It must be noticed that the current remains practically constant within a potential range much larger than that observed for the steady-state amperometric response in batch experiments 131,which may be due to a more effective washout of the quinone product from the electrode surface under flowing conditions. A potential of -0.30 V was chosen for subsequent studies. At this potential, the enzyme electrode responds rapidly to injections of phenol microemulsions, the peak half-width being 18 s. The short response time and rapid return to the baseline indicates facile transport of the substrate in the reversed micelle system used. The flow-injection peak height dependence on the reversed-micelle flow rate showed a gradual decrease as the carrier flow rate increased (Fig. 2). This same effect has also been observed, in organic

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AJ. Reuiejo et al./Analytica

Fig. 3. Thirty sucessive injections of 50 PM phenol in the reversed micelle formed with ethyl acetate, AOT, and phosphate buffer of pH 7.4. Flow rate, 0.40 ml min-’ ; sample volume, 110 ~1; EaPP = - 0.30 v.

streams [2], indicating that slower passages of the sample plug are required to produce higher rates of the enzymatic reaction. Moreover, Fig. 2 also shows that the peak width increased with decreasing the flow rate. Accordingly, as a compromise between sensitivity and sampling frequency, all subsequent assays were carried out with a reversed micelle flowing at 0.40 ml min-‘. No significant dependence of the peak height on the sample volume injected was found between 110 and 680 ~1, but, as usual, the peak width increased as such a volume increased. Consequently, a sample loop of 110 ~1 volume was chosen. Under these experimental conditions, about 30 samples h-’ can be analysed. The reproducibility of the response was estimated from a series of 30 repetitive injections of 50 PM phenol (Fig. 3). A relative standard deviation of 5.1% was obtained for i, measurements, thus demonstrating that, despite the hydrodynamic conditions, the stability of the enzyme layer on the graphite electrode surface in the reversed micelle stream is sufficiently good to allow a correct operation of the amperometric biosensor. A linear calibration graph for phenol was obtained over the 5 and 100 PM range (I = 0.99911, the slope and intercept being 0.011 PA PM-’ and -0.002 ,uA, respectively. A levelling off was observed for phenol concentrations higher than 100 PM, as expected for enzymatic reactions. The limits of determination and detection were calculated according to the 10 X standard deviation and 3+,/m criteria, where m is the slope of the calibration graph and sb is estimated as the standard deviation (n = 10) of the signal from 10 PM phenol. The values obtained, 3.1 PM and 0.9 PM, respectively, are considerably lower

Chimica Acta 315 (1995) 93-99

(approximately six-fold) than those obtained by amperometry under batch conditions. Finally, the behaviour of other phenolic compounds scarcely soluble in water, such as 2,4-dimethylphenol, 4-chloro-3-methylphenol and 3,5-dimethylphenol, was tested in the same experimental conditions used for phenol, with the aim of ascertaining the suitability of reversed micelles as working media in flowing systems for hydrophobic substrates. All of them exhibited well-defined flow injection response peaks at -0.30 V. Linear calibration graphs were obtained within the 25-700 PM range (r = 0.998) for both 4-chloro-3methylphenol and 2,4_dimethylphenol. The slopes and intercepts were 8.2 X 10m4 PA PM-’ and -0.001 PA, and 3.8 X 10m4 PA PM-’ and 0.008 PA, for 4-chloro-3-methylphenol and 2,4-dimethylphenol, respectively. In the case of 3,5-dimethylpheno1 linearity could only be obtained between 1 and 6 mM (r = 0.9991, with a slope of 0.02 PA mM_’ and an intercept of -0.0002 PA. The determination and detection limits, calculated from the standard deviation (n = 10) of the signals from 100 PM 4-chloro-3-methylphenol or 2,4_dimethylphenol, and from 2 mM 3,5_dimethylphenol, were 27 PM and 8 PM for 4-chloro-3-methylphenol, 33 PM and 10 PM for 2,4_dimethylphenol, and 0.7 mM and 0.2 mM for 3,5_dimethylphenol. As can be observed, and just like in batch work [3], the sensitivity decreased as the water solubility of these compounds decreased in the sequence phenol > 4-chloro-3methylphenol > 2,4_dimethylphenol > 3,5dimethylphenol, which has been attributed to the enzymatic reaction taking place in the aqueous microdomains at the electrode surface [9]. A very important aspect from an analytical point of view is that it has been described that only o&o-diphenols and phenols having at least one free ortho-position can undergo oxygenase activity with tyrosinase [lo]. This point was confirmed by testing several phenolic compounds with all ortho-positions substituted (2,6_dimethylphenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol and pentachlorophenol). Under the experimental conditions mentioned above, none of them gave an analytical signal indicating that they would not interfere with the determination of phenolic compounds with free ortho-positions.

A.J. Reviejo et al./Analytica

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Chimica Acta 315 (1995) 93-99

‘L Y 0

1 min.

Benzoic acid

t Phenol

Fig. 4. Current-time recordings at the tyrosinase electrode, immersed in the reversed micelle formed with 5 ml ethyl acetate, 0.10 M AOT and 4% phosphate buffer (pH 7.4), for 0.3 mM phenol followed by 10 PM successive additions of the inhibitors. Eapp = - 0.20 V.

3.2. Reversed micelle enzyme inhibition biosensors Reversed micelle systems can be also used for enzyme inhibition measurements, thus taking advantage of the inherent properties of these media. The possibility of developing reversed micelle enzyme inhibition biosensors is illustrated by monitoring the behaviour of some inhibitors of tyrosinase in connection with the Eastman AQ-enzyme immobilization approach 151,and using phenol as the substrate. Fig. 4 displays the response of the tyrosinase electrode immersed in the reversed micelle formed with ethyl acetate, AOT and phosphate buffer of pH 7.4, to a 0.3 mM phenol concentration, followed by 10 PM successive additions of the inhibitors propyl gallate, thiourea and benzoic acid. As can be seen, the steady-state response towards phenol rapidly decreases upon additions of these compounds at a low concentration level. The inhibition percentage caused for an inhibitor concentration of 0.4 mM, is of 31, 63 and 90% for propyl gallate, thiourea and benzoic acid, respectively. The enzyme electrode responds very rapidly to the changes in the inhibitor concentration with no need for an incubation period, steady-state signals being achieved within lo-20 s. Furthermore, the initial response for phenol, i.e., restoration of the enzyme activity, could be obtained again by a short incubation period (2 min) in a substrate microemulsion, indicating a reversible inhibition process in all cases. Fig. 5 displays calibration plots for the three inhibitor compounds tested; the different inhibition

Fig. 5. Calibration curves for (0) benzoic acid, ( n ) thiourea, and (A) propyl gallate at the tyrosinase electrodes in reversed micelles. Conditions as in Fig.4.

processes resulting in curves of different shapes. Linearity was observed within the ranges l-7 PM (r = 0.995), 5-40 PM (I = 0.997) and lo-110 PM (r = 0.997) for benzoic acid, thiourea and propyl gallate, respectively. The slopes and intercepts of these linear ranges were: 0.067 PA PM-’ and 0.10 PA for benzoic acid, 0.014 PA PM-’ and 0.06 PA for thiourea, and 0.004 PA PM-’ and 0.04 PA for propyl gallate. The determination and detection limits, calculated from the standard deviation (n = 10) of the signal from 1 PM benzoic acid, from 10 FM thiourea and from 20 PM propyl gallate, were 1.6 PM and 0.5 PM for benzoic acid, 8.3 PM and 2.5 PM for thiourea, and 17.0 PM and 5.0 PM for propyl gallate. The type of enzyme inactivation in the reversed micelle of each inhibitor was studied by plotting the steady-state current for successive additions of 10 ~1

0.0 0.0

0.3

0.6

0.6

1.2

1.6

cpLad,-

Fig. 6. Calibration plot for phenol in the absence (A), and presence of (0) 2 mM propyl gallate, (W ) 0.05 mM benzoic acid, and (+) 0.2 mM thiourea, at a tyrosinase electrode in reversed micelles. Conditions as in Fig. 4.

98

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0.0

1.0

10.016.020.0

IKkmM

d.0 Ok

610 16.016.62i.O lE9~

6.0 C /

.

intersect on the base line for propyl gallate indicating a fully non-competitive inhibition, whereas for thiourea and benzoic acid the lines intersect on the vertical axis which is indicative of fully competitive inhibitors [ll]. The Lineweaver-Burk plots can be also used to determine K, (inhibitor constant) from the values of the appropriate intercepts in the presence and absence of inhibitor [ll]. The calculated Ki values were 3.31, 0.063 and 0.017 mM for propyl gallate, thiourea and benzoic acid, respectively. The rapid response of the tyrosinase electrode to the changes of inhibitor concentration in the reversed micelle can be also exploited for on-line applications. For example, Fig. 8a displays flow injection measurements for the inhibitor propyl gallate when the reversed micelle containing a 0.1 mM concentration of the phenol substrate was employed as the carrier. The analytical signal in this flow system relies on

4.0 0.6 6.0 10.016.020.0

Fig. 7. Lineweaver-Burk plots for (a) propyl gallate, (b) benzoic acid, and (c> thiourea; (A> plots for phenol in the absence of inhibitors. Conditions as in Fig. 6.

of a 25 mM phenol stock microemulsion, in the presence and absence of inhibitor, until a subsequent addition did not appreciably increase that current. As it can be deduced from Fig. 6, approximately the same value for the apparent ~chaelis-Menten constant (.K~,,,,>, but different V, values (the maximum rate of the reaction), were obtained in the absence and presence of propyl gallate, which is consistent with a non-competitive inhibition process [II]. However, from thiourea and benzoic acid plots, it can be estimated that similar V, values will be obtained in the absence and presence or inhibitor, although quite different K, constants are also evident, indicating, in this case, a competitive inhibition behaviour ill]. The qualitative predictions commented above were confirmed from the plots of l/i vs. l/c, where i and c are the steady-state current for phenol and its concentration, respectively, without and with each inhibitor (Fig. 7). As it can be observed, the lines

b

0.3..

Fig. 8. (a) how-injection peaks for increasing ~ncen~ations of the inhibitor propyl gatlate. The reversed micelie containing 0.1 mM phenol was employed as the carrier. EqP = -0.30 V, flow rate, 0.40 ml min-‘. (b) Calibration plot for propyl gaIlate. Conditions as in Fig 8a.

A.J. Reviejo et al. /Analytica Chimica Acta 315 (1995) 93-99

the appearance of “negative” peaks from the continuous signal corresponding to the constant phenol substrate. Twenty five repetitive injections of 40 ,uM propyl gallate yielded reproducible flow-injection responses with a relative standard deviation of 4.6%. As an example, Fig. 8b displays the calibration plot for propyl gallate over the 20-180 PM range. The range of linearity obtained for the three inhibitors tested were: l-6 PM (r = 0.9990), 4-40 PM (r = 0.9971, and 20-100 PM (r = 0.9994) for benzoic acid, thiourea and propyl gallate, respectively. As it can be observed, the range of linearity obtained for each inhibitor is different, as expected because of the quite different Ki values. As the inhibition effect increases, the loss of linearity is produced earlier, as a consequence of the larger effect on the enzyme activity. The slopes and intercepts of the above mentioned calibration graphs were: 0.028 PA ,uM-’ and 0.02 PA for benzoic acid, 0.0047 PA PM-’ and 0.0009 FA for thiourea, and 0.002 PA PM-’ and 0.014 /_LAfor propyl gallate. Detection limits (0.5, 1.6, and 4.3 PM for benzoic acid, thiourea and propyl gallate, respectively) were calculated from the standard deviation (n = 10) of the signal from 2 PM benzoic acid, 8 /.LMthiourea, and 20 PM propyl gallate.

4. Conclusion The above results demonstrate that reversed micelles can be used in flow-injection analysis, and

99

consequently exploited for on-line applications, as well as suitable working media for monitoring inhibitors both in the batch and the flow-injection modes. Advantage is taken of the inherent characteristics of these systems, i.e., the possibility of performing enzymatic determinations of compounds scarcely soluble in water. Acknowledgements Financial support from the Spanish CICYT (project ALI92-0049) is gratefully acknowledged. References ill S. Saini, G.F. Hall, M.E. Downs and A.P.F. Turner, Anal. Chim. Acta, 249 (1991) 1. Wang, A.J. Reviejo and S. Mannino, Anal. Lett., 25 (1992) 1399. 131 F. Liu, A.J. Reviejo, J.M. Pingarr6n and J. Wang, Talanta, 41 (1994) 455. [41 A.J. Reviejo, F.Liu, J.M. Pingm6n and J. Wang, J. Electroanal. Chem., 374 (1994) 133. 151J. Wang, E. Dempsey, A. Eremenko and M. Smyth, Anal. Chim. Acta, 279 (1993) 203. [61R. Kindervater, W. Kiirmecke and R.D. Schmid, Anal. Chim. Acta, 234 (1990) 113. [71 J. Wang and B. Freiha, Anal. Chem., 57 (1985) 1776. Bl J. Wang, Y. Lin and Q. Chen, Electroanalysis, 5 (1993) 23. [91 K. Larsson, Thesis, Enzyme Catalysis in Microemulsions, University of Lund, Sweden, 1990. 1101C. Walsh, Enzymatic Reaction Mechanisms, Freeman, New York, 1979, p. 461.

121J.

Ml M. Dixon and E.C. Webb, Enzymes,

3rd edn., Academic Press, San Diego, CA, 1979, pp. 344-350.