Anal. Chem. 2000, 72, 3369-3373
A Binderless, Bulk-Modified, Renewable Surface Amperometric Sensor for NADH and Ethanol P. Ramesh and S. Sampath*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India
Graphite particles are exfoliated and subsequently functionalized with toluidine blue. The resulting covalently modified graphite particles are restacked without any binder to form a surface-renewable, bulk-modified electrode. Electrocatalytic oxidation of NADH and its application in the amperometric biosensing of ethanol using alcohol dehydrogenase enzyme have been demonstrated with this material. Chemically functionalized electrodes that lead to improved electrocatalytic properties have been actively researched, especially toward the development of matrixes for catalysis, sensing, and other applications.1-5 In this direction, covalent modification of metals, metal oxides, and carbon has already been reported.3 This paper deals with a new renewable surface material based on the covalent functionalization of exfoliated graphite with toluidine blue and its subsequent use as a bulk-modified, binderless electrode. Amperometric sensing of dihydronicotinamide adenine dinucleotide (NADH), a cofactor for the dehydrogenase enzymes, is demonstrated using this material. The electrochemical detection of NADH is of considerable interest2,6-8 since there is a large number of dehydrogenases that require this cofactor for their enzymatic reaction. The dehydrogenase enzymes catalyze the oxidation of a variety of families, such as alcohols, aldehydes, and carbohydrates, that are of immense interest from the analytical point of view. However, the electrochemistry of NADH suffers from a high over voltage at most of the bare-electrode surfaces.9-11 Use of mediators has been proposed to reduce the over-potential requirements. Nevertheless, electrode fouling is one of the main concerns due to radical intermediates generated during the one-electron steps and the subsequent polymerization products.11 Hence, the surface is to * Corresponding author. Email:
[email protected]. (1) Wring, S. A.; Hart, J. P. Analyst (Cambridge, U.K.) 1992, 117, 1215. (2) Gorton, L. J. Chem. Soc., Faraday Trans. 1986, 82(1), 1245. (3) Murray, R. W. Chemically Modified Electrodes. In Electroanalytical Chemistry, A Series of Advances; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13. (4) Wang, J.; Brennsteiner, A.; Sylwester, A. P. Anal. Chem. 1990, 62, 1102. (5) Tsionsky, M.; Lev, O. Anal. Chem. 1995, 67, 2409. (6) Coughlin, R. W.; Aizawa, M.; Alexander, B. F.; Charles, M. Biotechnol. Bioeng. 1975, 17, 515. (7) Jaegfeldt, H.; Torstensson, A.; Johansson, G. Anal. Chim. Acta 1978, 97, 221. (8) Silber, A.; Brauchle, C.; Hampp, N. J. Electroanal. Chem. 1995, 390, 83. (9) Braun, R. D.; Santhanam, K. S. V.; Elving, P. J. J. Am. Chem. Soc. 1975, 97, 2591. (10) Moiroux. J.; Elving, P. J. Anal. Chem. 1979, 51, 346. (11) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 47, 1337. 10.1021/ac000049n CCC: $19.00 Published on Web 06/01/2000
© 2000 American Chemical Society
be renewed after every use. In this direction, polishable carbon paste, sol-gel derived ceramic carbon, and epoxy-based electrodes have been reported2,12-14 for various analytes. However, these materials use a binder that will deteriorate the electrode performance with time. The drawbacks associated with carbon paste electrodes for biosensing were reported earlier.15,16 It is desirable to explore the possibility of a renewable surface electrode, preferably without any binder, to have unlimited storage stability. Exfoliated or expanded graphite (EG) is a low dense material with high-temperature resistance.17 The process of exfoliation involves the formation of graphite intercalation compounds (GICs) that undergo subsequent thermal dissociation. SbCl5 and FeCl3 are two examples of substances that can penetrate graphite by diffusion through edge planes to form layered structured crystals. When these particles are suddenly heated, the layers explode apart, leading to an enhanced c lattice parameter. The expanded material can be compressed or restacked without any binder. Additionally, EG has better homogeneity of the surface than any other form of graphite.18 It is used as seals, high-temperature gaskets, catalyst supports, and adsorption substrates, owing to the near perfect crystallographic face that EG provides.18 Very few studies deal with the use of EG based electrodes for electrochemical applications.19-21 Fukuda and co workers21 have reported the use of foliated natural graphite as anode material for rechargeable lithium-ion cells. The specific capacities obtained were very close to the theoretically predicted values. Recently, Frysz and Chung19 reported cyclic voltammetric data on the exfoliated graphite electrodes and deduced the electron transfer kinetics, capacitance, and electrochemical area. Further, EG was found to be better than carbon paste in terms of electrochemical kinetics. In the present studies, we have exploited some of the properties of EG to prepare a covalently functionalized, bulkmodified electrode without the use of any binder and utilized it for amperometric sensing of NADH. Subsequently, the modified (12) (13) (14) (15) (16) (17) (18) (19) (20) (21)
Gorton, L. Electroanalysis 1995, 7, 23. Walcarius, A. Electroanalysis 1998, 10, 1217. Wang, J; Varughese, K. Anal. Chem. 1990, 62, 318. Motta, N.; Guadalupe, A. R. Anal. Chem. 1994, 66, 566. Amine, A.; Patriarche, G. J.; Kauffman, J.-M.; Kaifer, A. E. Anal. Lett. 1991, 24, 1293. Chung, D. D. L. J. Mater. Sci. 1987, 22, 4190. Gilbert, E. P.; Reynolds, P. A.; White, J. W. J. Chem. Soc., Faraday Trans. 1998, 94(13), 1861. Frysz, C. A.; Chung, D. D. L. Carbon 1997, 35, 5(6), 858-860. Kao Corp. Jpn. Kokai Tokkyo Koho JP 59 78 204, 1984; Chemical abstract 101:131358z, 1984. Fukuda, K.; Kikuya, K.; Isono, K.; Yoshio, M. J. Power Sources 1997, 69, 165.
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electrode was used to sense ethanol using alcohol dehydrogenase enzyme and NAD+. EXPERIMENTAL DETAILS The expansion and subsequent functionalization of the graphite particles were carried out as given below: Natural graphite (particle size 300-400 µm, Stratmin Graphite, ?) particles were intercalated by immersing the particles in a mixture of concentrated H2SO4/HNO3 (3:1 by volume) for 24 h at ambient conditions. The volume of the material was found to increase by approximately twice due to intercalation. The material was washed with distilled water and air-dried. The exfoliation was then carried out by introducing the material in a furnace preheated to 800 °C. Exfoliation occurred within several tens of seconds. The material was then cooled to room temperature. The EG particles were subsequently covalently functionalized with toluidine blue through cyanuric chloride modification. Cyanuric chloride (CC) can react with a variety of substances including hydroxyl and amino compounds. Toluidine blue is chosen for the present study since it is a good mediator for NADH oxidation at low over potentials and consequently reduces the interferences and fouling of the electrode surface.22 CC modification was carried out through the hydroxyl groups present on the graphite. One gram of EG was stirred in a 2 wt % solution of cyanuric chloride in dry benzene for about 24 h at ambient conditions. Soxhlet extraction with dry benzene was carried out for 24 h to remove the physisorbed CC. The graphite particles were dried and stored in a vacuum at 4 °C. Subsequently, toluidine blue was attached to the CC modified EG by refluxing the powder (1 g) in a solution containing 4 mg of the dye in 100 mL of benzene for 12 h. The amino group present in the toluidine blue reacts with the Cl of cyanuric chloride, effecting covalent attachment. The resulting material was Soxhlet extracted for a week to remove the physisorbed mediator. The powder was then dried and pressed in the form of pellets without any binder. Approximately 400 mg of the modified material was used at a pressure of 9 tons/cm2 for about 10 hours. This resulted in a very compact, hard pellet. This was cut into small pieces and made into electrodes using conducting silver epoxy. The electrodes were subsequently polished well with SiC paper of various grades. The electrode surface was roughened by scratching the surface against different grades of emery sheets (400-grit in most of the cases) in one direction. The samples for the IR measurements were in the form of KBr pellet containing the graphite particles. XPS measurements were carried out using the pellets as such. All the chemicals used were of analytical grade. Yeast alcohol dehydrogenase, NAD+, and NADH are products of Sigma, St. Louis, MO. The electrochemical cell contained platinum foil as the counter and calomel as the reference electrode. Electrochemical measurements were carried out using a CHI 660A Electrochemical Analyzer from CH instruments, Austin, TX. IR studies were carried out using a Bruker Equinox FT-IR spectrometer, and XPS studies were done using the spectrometer from VSW Scientific Instruments, UK.
Figure 1. Cyclic voltammograms of toluidine blue modified EG electrode in phosphate buffer, pH 7 at (1) 10 mV/s; (2) 20 mV/s. Inset: voltammogram of an unmodified EG at 20 mV/s.
RESULTS AND DISCUSSION The modified graphite particles were chemically characterized at every stage by XRD, IR, XPS, elemental analysis, and the
morphology was followed by SEM. The data on the XRD, SEM, and elemental analyses will be reported elsewhere. Electrochemical oxidation of NADH was carried out using pellets pressed without the use of any binder. Figure 1 shows the cyclic voltammograms of the unmodified and toluidine blue modified EG electrodes in a phosphate buffer of pH 7. The unmodified EG did not show any electrochemical activity, while the functionalized EG showed one set of peaks in the available potential range. The formal potential, Ef0, was observed to be -0.280 V vs SCE, and this is very close to the value (-0.285 V vs SCE) reported for the toluidine blue modified graphite electrode.23 The currents due to the toluidine blue redox process were found to vary with the scan rate. The peak current-scan rate plot (not shown) was linear up to a scan rate of 40 mV/s, passing through the origin, showing that the redox species is in the immobilized state.3 The peaks were, however, not very sharp and well-defined. This may be attributed to a distribution of redox states23 and, to some extent, resistive effects. It should also be pointed out that the toluidine blue loading was not very high. The modification was carried out using natural graphite particles, and the number of available surface OH groups for cyanuric chloride reaction and, subsequently, for toluidine blue modification was low, as observed in XPS studies. The surface coverage of toluidine blue based on the geometric area of the electrode was calculated to be 1.7 × 10-9 mol/cm2. However, it should be pointed out that the actual area is larger than the geometric area due to surface roughness. The electrochemical response of EG based electrodes depends on the surface roughness, and it is found that modified electrodes with smooth surfaces do not give rise to any peak, while an increase in surface roughness leads to better definition of the response. Roughening the surface leads to an increased amount of edge planes getting exposed to the electrolyte. This implies that the activity of the edge planes is higher than the basal planes. It is known that, for highly oriented pyrolitic graphite, the activity of
(22) Schlereth, D. D.; Katz, E.; Schmidt, H.-L. Electroanalysis 1994, 6, 725.
(23) Persson, B. J. Electroanal. Chem. 1990, 287, 61.
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Figure 2. Electrocatalytic oxidation of NADH using toluidine blue modified EG electrode at pH 7, in phosphate buffer, at a scan rate of 5 mV/s; (1) Toluidine blue modified EG; (2) with 1, (3) with 5.55, and (4) with 11.2 mM of NADH. Inset: plot of catalytic current at -0.05 V vs concentration of NADH. Steady-state response of toluidine blue modified EG at -0.05 V; 1-4 are additions of 2 mM and 5&6 are additions of 4 mM, each.
the edge planes is higher than that of the basal planes. Edge planes do have unsaturated, dangling bonds and are very reactive. The activity and the immobilization are much higher on the edge plane than the basal plane in the case of EG as well. This is confirmed by ESCA results that show variations depending on the roughness. The data shown in Figure 1 is obtained using the electrodes having roughness created with a 400-grit SiC paper. The SEM picture (not shown) reveals a very rough surface as well. The fact that the modifier is chemically attached to the graphite particles and not physically adsorbed was confirmed by electrochemistry as well as spectroscopy. Nonmodified EG particles were intentionally adsorbed with toluidine blue, and the resulting material was subjected to electrochemical measurements. The cyclic voltammograms showed two sets of redox processes corresponding to two different kinds of physisorbed species. The reduction potentials were found to be -0.325 and -0.535 V at a scan rate of 20 mV/s, and these two peaks were inferred to be due to the adsorption of toluidine blue at the edge (rough) and basal (smooth) planes of the graphite, respectively. This is based on the increase in respective peak currents as a function of roughness. On the other hand, the covalently functionalized material resulted in only one reduction peak for the rough electrodesat -0.385 V. The difference in the peak potentials between the physisorbed and the covalently functionalized material may be attributed to the presence of NH2 and physical adsorption. Second, the change in the peak potentials with scan rate was found to be much higher (25-30 mV for a change in scan rate from 10 to 40 mV/sec for both the peaks) for the physisorbed material as opposed to the chemically bound moiety (7-9 mV for the same change in scan rate). This reveals that the kinetics of physically adsorbed toluidine blue is sluggish compared
with that of the covalently functionalized dye. The smooth surface of the functionalized EG did not result in any redox peak, showing that the covalent modification has occurred only on the edge planes of the graphite. The catalytic oxidation of NADH using the toluidine blue modified EG electrode is shown in Figure 2. The catalytic currents start at -0.200 V and get saturated around -0.050 V vs SCE. The fact that the catalytic current starts at a potential very close to the peak potential of the mediator reveals that the reaction rate between NADH and the mediator is rather sluggish. It is already known that there is a charge-transfer complex formed between the reduced nicotinamide coenzymes and the oxidized form of the mediator.23 This complex decomposes in a rate-limiting step analogous to the Michalis-Menten type reaction and this may be represented as k1
k2
NADH + M (ox) y\ z CT 98 NAD+ + M (red) k -1
where M is the mediator. The rate constant, k2, associated with the decomposition of the complex is probably small. Similar observations in the potentials of mediator and NADH oxidation processes were reported by Schmidt and co workers22 for gold electrodes covalently modified with toluidine blue. The catalytic currents observed in the present studies were found to be proportional to the concentration of NADH, and a linear range of ∼10 mM and a dynamic range of 35 mM were observed. The steady-state response characteristics carried out at -0.05 V show that the response of the sensor is rather fast, of the order of 15 s (Figure 2, inset). The steady-state measurements show a linear range of up to 15 mM. Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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One of the main advantages of the bulk-modified EG electrode is that the surface of the electrode can be reused after every use. Mechanical polishing using emery sheets exposes a new reactive surface whenever required. This is especially useful for NADH based hydrogenase enzyme sensors since the regeneration of NAD+ is known to be a complicated process and leads to electrode fouling. The surface renewability resulting from the bulk modification of the material shows reproducible electrochemical data for different exposed electrode surfaces. The standard deviation in the peak currents for five successive polishings works out to be
