Anal. Chem. 1998, 70, 2596-2600
Bioelectrochemical Monitoring of Phenols and Aromatic Amines in Flow Injection Using Novel Plant Peroxidases Florentina-Daniela Munteanu,† Annika Lindgren,*,‡ Jenny Emne´us,‡ Lo Gorton,‡ Tautgirdas Ruzgas,‡ Elisabeth Cso 1 regi,§ Anton Ciucu,† R. B. van Huystee,| Irina G. Gazaryan,⊥ and L. Mark Lagrimini+
Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, Sos. Panduri, Bucharest Sector 5, Romania, Department of Analytical Chemistry and Department of Biotechnology, Lund University, P.O. Box 124, S-22100 Lund, Sweden, Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7, Department of Chemical Enzymology, Chemical Faculty, Moscow State University, Moscow 119899 GSP, Russia, and Department of Agriculture and Crop Science, The Ohio State University, Columbus, Ohio 43210-1096
An amperometric flow system combined with a glucose oxidase-mutarotase reactor was optimized and used to determine aromatic amines and phenols using peroxidase-modified graphite electrodes. An increase in currents upon injection of the analyzed substrate was shown to be approximated by a Michaelis-Menten type dependence. The detection limit was calculated as 3 times the noise, and the sensitivity was calculated as Imax/K app m . Commercially available horseradish peroxidase was compared with tobacco anionic and peanut cationic peroxidases for determination of aromatic amines and phenols. Detection limits of 10 nM for determination of o-aminophenol and o- and p-phenylenediamine achieved with a tobacco peroxidase-modified electrode give a promise for further improvements in sensitivities and detection limits of biosensors.
Phenolic compounds are widely spread in nature due to industrial pollution.1,2 Moreover, existing processes of paper bleaching result in the presence of phenolic compounds in wrapping paper, thus making phenol determination equally vital for environmental and food control. Aromatic amines belong to pollutants with a high mutagenic/carcinogenic activity and therefore they have to be thoroughly controlled and determined at very low concentrations. Various enzyme-based detection units were described in literature for measuring phenols3-7 and aromatic
amines;8,9 however, most of the sensors do not assure monitoring at low concentration ranges. Commercially available horseradish peroxidase (HRP) is the peroxidase usually used for construction of biosensors.10 This enzyme is highly specific for hydrogen peroxide, which is its main oxidation substrate, but it is rather unselective for its reducing agents, among which phenols, amines, aminophenols, etc., are found. However, the efficiency (in terms of turnover rate) as electron donor substrates, e.g., between various phenols and aromatic amines, varies substantially between different plant peroxidases. Therefore, the use of different peroxidases or genetically engineered ones has the promise of resulting in biosensors highly active for a subgroup of donor substrates. Peroxidase (POD) catalyzes the oxidation of donor substrates by hydrogen peroxide in three steps:
POD + H2O2 f Cpd I + H2O Cpd I + S f Cpd II + S•
(k1)
(1)
(k2)
Cpd II + S + 2H+ f POD + S• + H2O
(2) (k3)
(3)
In the first step H2O2 is reduced under formation of an oxidized enzyme intermediate called compound I (reaction 1). Compound I is rereduced in two steps; in each step a donor substrate (S) is oxidized giving a radical product (S•) (reactions 2 and 3). Usually reaction 3 is considered as rate-limiting under steady-state conditions. If the enzyme is immobilized on an electrode surface, the electrode can donate electrons to the oxidized enzyme, in a
* Corresponding author: (phone) +46-46-222 8164; (fax) +46-46-222 4544; (e-mail)
[email protected]. † University of Bucharest. ‡ Department of Analytical Chemistry, Lund University. § Department of Biotechnology, Lund University. | University of Western Ontario. ⊥ Moscow State University. + The Ohio State University. (1) Crompton, T. R. Determination of organic substances in water; John Wiley & Sons: New York, 1985. (2) Health, A. G. Water pollution and fish physiology; CRC Press: Boca Raton, FL, 1987. (3) Ortega, F.; Domı´nguez, E.; Jo ¨nsson-Pettersson, G.; Gorton, L. J. Biotechnol. 1993, 31, 289. (4) Skladal, P. Collect. Czech. Chem. Commun. 1991, 56, 1427.
(5) Connor, M. P.; Sanchez, J.; Wang, J.; Smyth, M. R.; Mannino, S. Analyst 1989, 114, 1427. (6) Lindgren, A.; Emne´us, J.; Ruzgas, T.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1997, 347, 51-62. (7) Ruzgas, T.; Emne´us, J.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1995, 311, 245. (8) Kulys, J.; Bilitewski, U.; Schmid, R. D. Biochem. Bioenerg. 1991, 26, 277286. (9) Dominguez-Sanchez, P.; O’Sullivan, C. K.; Miranda-Ordieres, A. J.; TunonBlanco, P.; Smyth, M. R. Anal. Chim. Acta 1994, 291, 349. (10) Ruzgas, T.; Cso ¨regi, E.; Emne´us, J.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1996, 330, 123.
2596 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
S0003-2700(98)00022-5 CCC: $15.00
© 1998 American Chemical Society Published on Web 05/09/1998
Figure 1. Principle of the determination of p-aminophenol using a peroxidase-modified electrode.
nonmediated direct electron-transfer mode;11 this is represented by reaction 4. In the presence of a donor substrate, the peroxidase
Cpd I + 2e- + 2H+ f POD + H2O
(ks)
(4)
electrode can also work in mediated electron transfer. Mediated electron transfer is when the enzyme is rereduced according to reactions 2 and 3, and the formed radicals (S•) are reduced by the electrode (at a potential of -50 mV vs Ag/AgCl), resulting in a reduction current proportional to the concentration of substrate (reaction 5). The direct electron transfer is known to be less
S• + e- + H+ f S
(ks,m)
(5)
efficient than the mediated due to lower values of ks.11 All enzyme molecules on the electrode are not equally involved in the two processes. Ruzgas et al.11 have shown that only around 40% of the peroxidase molecules adsorbed on graphite and still active after immobilization is able to work in direct electron transfer. If the peroxidase-modified electrode is used as a sensor in the flow injection mode, a continuous supply of H2O2 results in a steadystate current due to the direct reduction of peroxidase. If a sample containing a donor substrate is injected, the peroxidase molecules only available for mediated electron transfer are switched on, and electrochemical response peaks can be observed on top of the steady-state current (see Figure 1). In previous work, the fundamentals of amperometric detection of phenolic compounds using HRP-modified electrodes, as well as different immobilization techniques, were studied.6,7 The present work extends the study to include, with the exception of phenols, aromatic amines and aminophenols, using novel plant peroxidases. EXPERIMENTAL SECTION Chemicals. Sodium dihydrogen phosphate monohydrate, potassium chloride, phenol, catechol, and hydrogen peroxide, (11) Ruzgas, T.; Gorton, L.; Emne´us, J.; Marko-Varga, G. J. Electroanal. Chem. 1995, 391, 41.
30%, were purchased from Merck (Darmstadt, Germany). Glutaraldehyde, 25%, aniline, o-phenylenediamine, m-phenylenediamine, and p-phenylenediamine were from Sigma Chemical Co. (St. Louis, MO), and 1,2-aminophenol and 1,4-aminophenol were from Fluka (Buchs, Switzerland). D-Glucose was obtained from AnalaR, BDH Chemicals Ltd. (Poole, England). The phenolic and aromatic amine compounds were of the highest purity available. All substrate solutions were prepared daily from 0.1 M stock solution in methanol (Merck). All aqueous solutions were prepared using water purified with a Milli-Q system (Millipore, Milford, CT). Enzymes. Peroxidase from horseradish, RZ 1.5, was purchased from Sigma. Tobacco peroxidase overexpressed in transgenic tobacco plants Nicotiana sylvestris (TOP), RZ 3.0, was purified as described earlier.12 Peroxidase from peanut cell culture (PNP), RZ 2.5, was purified according to ref 13. Glucose oxidase from Aspergillus niger, 314 units/mg, and mutarotase from porcine kidney were from Serva (Heidelberg, Germany) and Merck, respectively. Methods. (1) Preparation of Enzyme Electrodes. Solid graphite electrodes were prepared by the following procedure. Rods of solid spectroscopic graphite (Ringsdorff Werke GmbH, Bonn, Germany, type RW001, 3.05-mm diameter) were cut, polished on wet fine emery paper (Tufbak Durite, P1200), carefully rinsed with deionized water, and additionally polished with ordinary white paper to obtain a flat mirrorlike surface. An 8-µL aliquot of the enzyme solution (5-10 mg/mL) was added to the polished ends of the graphite rods, and the electrodes were then placed at 4 °C for 20 h in a glass beaker covered with sealing film, to prevent rapid evaporation of the droplet of enzyme solution. The enzyme electrodes were thoroughly rinsed with 0.1 M sodium phosphate buffer at pH 7.0 and were stored in this buffer at 4 °C. (2) Glucose Oxidase-Mutarotase Reactor. In our previous work,6 a glucose oxidase reactor was used to achieve a constant hydrogen peroxide concentration in the carrier. Glucose oxidase is active only toward β-D-glucose, and thus, only about 64% of D-glucose could be oxidized.14 In this work, glucose oxidase was coimmobilized with mutarotase to achieve a complete conversion. Glucose oxidase (9.5 mg) and 2 mg of mutarotase were dissolved in 1.4 mL of 0.1 M phosphate buffer, pH 7.0, and added to 0.15 g of glutaraldehyde-activated, silanized, controlled-pore glass (CPG 10, pore size 500 Å, 200-400 mesh, Serva).6 The coupling was performed overnight at 4 °C under reduced pressure. The biocatalyst obtained was then washed with phosphate buffer and filled into a 235-µL reactor made of Plexiglas (i.d. 2.7 mm). When not in use, the reactor was stored filled with 0.1 phosphate buffer, pH 7.0, at 4 °C. (3) Electrochemical Measurements. The enzyme electrode was fitted into a Teflon holder and inserted into a flow-through wall-jet amperometric cell.15 The enzyme electrode was used as the working electrode, an Ag/AgC1 (0.1 M KC1) electrode was used as the reference electrode, and a platinum wire served as the auxiliary electrode. The electrodes were connected to a three(12) Gazaryan, I. G.; Lagrimini, L. M. Phytochemistry 1996, 41, 1029-1034. (13) Sesto, P. A.; van Huystee, R. B. Plant Sci. 1989, 61, 163-168. (14) Pigman, W.; Anet, E. F. L. J. In The Carbohydrates; Pigman, W., Ed.; Academic Press: Orlando, FL, 1972; Vol. 1A, p 168. (15) Appelqvist, R.; Marko-Varga, G.; Gorton, L.; Torstensson, A.; Johansson, G. Anal. Chim. Acta 1985, 169, 237.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
2597
(
)
1 1 1 1 + ) Ienz nFEDET k1c* ks
Figure 2. Schematic picture of the used two-channel flow injection system.
electrode potentiostat (Za¨ta Elektronik, Lund, Sweden) and the current was recorded on a recorder (Kipp and Zonen). All measurements were performed at an applied potential of -50 mV vs Ag/AgCl.6,7,16 The flow cell was connected to the two-channel flow injection system6 described in Figure 2. A flow rate of 0.25 mL/min in each line was maintained by a peristaltic pump (Alitea). In one line, the samples were injected (30 µL injection loop) into a flow of 0.1 M sodium phosphate buffer, pH 7.0. In the other line, with the glucose oxidase-mutarotase reactor, D-glucose was added to the buffer (100 µM) to give a hydrogen peroxide concentration of 100 µM. The two lines were connected using a mixing T, followed by a knotted coil (200 µL). To check the performance of the reactor, the steady-state currents obtained using the reactor and freshly prepared hydrogen peroxide solutions; respectively, were compared. To measure steady-state current for H2O2, the reactor was disconnected and the H2O2 solution was pumped through that line. The differences in the currents obtained were negligible, confirming 100% conversion of glucose into hydrogen peroxide and gluconic acid in the reactor. The concentration of the H2O2 solution was determined spectrophotometrically using a molar absorption of 72.7 M-1 cm-1 at 230 nm.17 RESULTS AND DISCUSSION The behavior of a wall-jet electrode flow system in the absence of added substrate is described by the following equation:
1 1 1 + ) I Ilim Ienz
(6)
where I is the registered current, Ilim is the diffusion limited current, and Ienz is the kinetically limited current. The diffusion limited current for a wall-jet system is expressed as follows:18
Ilim ) 1.38nFc*D /3ν- /12V /4a- /2R 2
5
3
1
3/ 4
R>a
(7)
where n is the number of electrons transferred, F is the Faraday constant, c* is the hydrogen peroxide concentration, D is the diffusion coefficient for hydrogen peroxide, ν is the kinematic viscosity of the flow carrier, V is the volume flow rate, a is the radius of the capillary nozzle, and R is the radius of the electrode. The kinetically limited current due to direct electron transfer can be expressed as11 (16) Cso ¨regi, E.; Jo ¨nsson-Pettersson, G.; Gorton, L. J. Biotechnol. 1993, 30, 315. (17) Maethly, A. C. Methods Enzymol. 1953, 2, 801. (18) Yamada, J.; Matsuda, H. Electroanal. Chem. Interfacial Electrochem. 1973, 44, 189-198.
2598 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
(8)
where EDET is the amount of enzyme active in direct electron. In the presence of saturating concentrations of a donor substrate, one can assume that all peroxidase molecules work in mediated electron transfer, and thus, the kinetically limited current is expressed as follows:
(
1 1 1 1 + ) Ienz 2nFE k1c* k3[S]
)
(9)
where E is the total amount of active enzyme on the electrode surface. This assumption allows us to calculate the rate constants of the enzymatic reaction. However, under the experimental conditions used in this work, at the moment of injection of the donor substrate the enzyme works in both direct and mediated electron transfer and the enzyme distribution between the two mechanisms is unknown. Moreover, the donor substrate is injected by small portions into a flow of 0.5 mL/min (in the cell), and thus, diffusion limitations also affect the current measured in a wall-jet flow system. The final equation for the current should include both these components, i.e., diffusion-limited and kinetically limited current, but since the bioelectrocatalytic reduction of H2O2 at a peroxidase electrode is a second-order reaction, these equations are only valid at high flow rate. Taking into account the unknown portions of the enzyme working in direct and mediated electron transfer, under these conditions, there is no currently available way to calculate the real parameters corresponding to the enzymatic reaction from the currents measured in the system presented here. However, current work in the laboratory is directed toward evaluation of kinetic rate constants and the fraction of peroxidase molecules available for direct electron transfer for different plant peroxidases. Nevertheless, the wall-jet flow system can be successfully used for applied purposes of donor substrate determination. Figure 1 illustrates the phenomenon used to determine the concentration of phenols. The peak heights obtained in this system are related to the phenol concentration injected as shown in Figure 3. The dependence of the registered currents vs concentration of the donor substrate (p-aminophenol) resembles Michaelis-Menten kinetics, and apparent values for the Michaelis-Menten constant, K app m , can be calculated by fitting the data to the MichaelisMenten equation:
I)
Imax[S] [S] + K app m
(10)
This manipulation corresponds to the assumption that at the moment of injection the enzyme molecules only available for mediated electron transfer are switched on and work in mediated electron transfer only. Undoubtedly, such an assumption is an oversimplification of the real process; however, it can be used to found the basis of calibration graphs and to calculate detection limits and sensitivities.
Table 1. Sensitivities for Aromatic Amines and Phenols Using Different Peroxidasesa sensitivity/nA µM-1 substance
HRP
TOP
P NP
phenol catechol aniline o-aminophenol p-aminophenol o-phenylenediamine m-phenylenediamine p-phenylenediamine
2.0 ( 0.1 8.1 ( 0.2 1.1 ( 0.1 34.5 ( 1.5 37.0 ( 1.5 132 ( 10 10.0 ( 0.8 74 ( 4
0.76 ( 0.08 21 ( 1 0.64 ( 0.09 521 ( 15 214 ( 10 960 ( 20 6.4 ( 0.5 860 ( 20
1.4 ( 0.1 8.0 ( 0.2 0.62 ( 0.09 443 ( 15 20 ( 1 115 ( 6 10.0 ( 0.7 147 ( 7
a
Figure 3. Calibration graph for p-aminophenol determination with a HRP-modified electrode at 100 µM hydrogen peroxide. The curve is obtained by fitting the experimental data to the Michaelis-Menten equation.
a
Table 2. Detection Limits for Aromatic Amines and Phenols Using Different Peroxidasesa detection limit/µM substance
HRP
TOP
PNP
phenol catechol aniline o-aminophenol p-aminophenol o-phenylenediamine m-phenylenediamine p-phenylenediamine
3.6 ( 0.5 1.3 ( 0.1 6.5 ( 0.5 0.1 ( 0.01 0.1 ( 0.01 0.05 ( 0.004 1.2 ( 0.1 0.05 ( 0.004
10.1 ( 0.8 0.45 ( 0.05 10.0 ( 0.7 0.01 ( 0.001 0.04 ( 0.005 0.01 ( 0.001 3.0 ( 0.6 0.01 ( 0.001
7.0 ( 0.6 0.75 ( 0.05 10.0 ( 0 7 0.01 ( 0.001 0.25 ( 0.01 0.05 ( 0.004 0.75 ( 0.05 0.05 ( 0.005
a
b
Figure 4. (a) Calibration graphs for phenol determination with HRPmodified electrodes (mean value of two electrodes) at (×) 20, (]) 40, (0) 80, and (O) 160 µM hydrogen peroxide, and (b) dependence of sensitivity on hydrogen peroxide concentration.
Hydrogen Peroxide Concentration. It can be seen from eqs 8 and 9 that the concentration of hydrogen peroxide plays a key role in terms of the current dependencies on direct electron transfer or mediated electron transfer. The hydrogen peroxide concentration is very crucial in this type of system, since the steady-state current due to the direct electron transfer is always present. The noise is highly dependent on this continuous
Mean value of three injections.
Mean value of three injections.
background current; the higher the background current, the higher the noise. Figure 4a presents the calibration curves for phenol determination obtained using different hydrogen peroxide concentrations. The sensitivity was calculated as Imax/K app m (eq 10). When the sensitivities were plotted vs the hydrogen peroxide concentrations (Figure 4b), 100 µM H2O2 was found optimal, since no further increase in sensitivity was obtained above this concentration. All the concentrations presented in this work are before dilution (50%) by mixing in the system. If nothing else is stated, the presented peak data are the mean values of three injections. Stability and Reproducibility. The stability of HRP electrodes was studied in our previous work.6 The electrodes showed initially a rapid decrease in steady-state current for H2O2; meanwhile the flow injection response for p-cresol was approximately constant for one or two weeks, when the electrodes were stored in buffer solution at 4 °C between the experiments. The short time stability for continuous injections is also good; the response is only decreased by 3.7% if 50 µM phenol is injected every third minute for 60 min. The relative standard deviations (RSD) between injections are 1.6% (n ) 7), 2.1% (n ) 7), and 3.6% (n ) 7) for HRP, PNP, and TOP electrodes, respectively. Determination of Phenolics and Aromatic Amines. At the optimal concentration of hydrogen peroxide, different phenolic compounds and aromatic amines have been tested using peroxidases of different origin. The sensitivities and detection limits obtained for a number of phenols, aminophenols, and phenylenediamines are presented in Tables 1 and 2. One can see from Table 1 that peroxidases of different origin differ in their sensitivities and, thus, in activities toward various phenolic compounds. Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
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demonstrates that the substrate structure affects the enzyme reactivity and this can be used to discriminate between ortho- and para-substituted amines and meta and nonsubstituted ones. The difference in sensitivities for ortho- and para-substituted amines and nonsubstituted ones for tobacco peroxidase is even more pronounced, varying in the range of 3 orders of magnitude. Tobacco peroxidase shows very high response to aromatic diamines and therefore shows the better detection limits among the peroxidases tested (Table 2). Thus, the newly isolated tobacco peroxidase is a promising reagent for electrochemical determination aminophenols and aromatic diamines at nanomolar range.
Figure 5. Correlation of flow injection responses with Hammett coefficients for 10 µM aniline, p-aminophenol, and p- and m-phenylenediamine, respectively: (]) peanut peroxidase; (0) tobacco peroxidase; (O) horseradish peroxidase.
Phenol and aniline have the lowest sensitivities, probably due to the absence of substituents increasing the electron density. In these terms, aromatic diamines except m-phenylenediamine are much better substrates than catechol. The fact that m-phenylenediamine is a worse substrate than its ortho analogue could be explained if one considers its ortho and para analogues’ ability to give two-electron-oxidized products in contrast to m-phenylenediamine. When the responses obtained are plotted vs the corresponding Hammett coefficients19 for substituted anilines (Figure 5), a correlation between the response and the substituent effect on electron donation is observed. The lower the Hammett coefficient, the higher is the response achieved. For HRP, all sensitivities are in the range 1-10 nA/µM except those for o- and p-phenylenediamine and aminophenols. It (19) Jaffe´, H. H. Chem. Rev. 1953, 53, 191-261.
2600 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
CONCLUSIONS The optimized amperometric flow system combined with a glucose oxidase-mutarotase reactor was used to determine aromatic amines and phenols with a peroxidase-modified graphite electrode. The newly isolated peroxidases were shown to have a potential for analytical application in this area. The detection limits of 10 nM for determination of o-aminophenol and o- and pphenylenediamine achieved with tobacco peroxidase-modified electrode give a promise for further improvements in sensitivities and detection limits of biosensors for determination of phenolic pollutants. The further efforts will be focused on array detection using different peroxidases which will allow us to discriminate between structures of the pollutants. ACKNOWLEDGMENT The authors thank the Royal Swedish Academy of Science (Project 1532), the Swedish Natural Science Research Council (NFR), the European project (INCO-Copernicus IC15CT96-1008), and the Tempus program N°S JEP-09227-95 for financial support.
Received for review January 6, 1998. Accepted March 19, 1998. AC980022S
