Anal. Chem. 1994,66, 1595-1599
Preparation and Characterization of Octadecylamine-Containing Carbon Paste Electrodes Azlz Amine, Jamal Denl, and Jean-Michel Kauffmann' Institut de Pharmacie, Universith Libre de Bruxelles, Campus Plaine, CP 205/6 1050 Bruxelles, Belgium
An effectivestrategy for circumventingproblems of interference by electroactive species during the detection of hexacyanoferrate at the carbon paste electrode (CPE) is described. The concept relies on the incorporation of octadecylamine within the carbon paste. The electrochemical characterization of the modified electrode (potential range, background current, stability, permselectivity)has been investigatedby amperometry and linear scan and cyclic voltammetry in aqueous media with different ionic strengths and in hydroalcoholic media. The interference of analytes such as ascorbic acid, uric acid, and dopamine can be eliminated totally by appropriate adjustment of the amount of octadecylamine within the carbon paste matrix. The modified electrodewas highly selectiveto hexacyanoferrate. Opticalmicroscope observationsof the CPE and modified CPE showed a higher density of graphite particles covered with liquid paraffin-octadecylamine layers at the latter. Development of biosensors using such modified electrodes is discussed. Mixed enzyme-carbon paste electrodes are receiving considerable attention for the preparation of fast-responding reagentless biosensors.1-8 Recently, carbon paste based biosensors for glucose, sucrose, lactose, lactate, and ethyl alcohol have seen successful commercial applications (Orion Research Inc., Boston, MA, and Dosivit SA, Nantes, France). The response of these biosensors is often affected by the presence of electroactive species in complex samples. Several strategies have been proposed in order to minimize the amperometric signal resulting from these species, such as covering the electrodesurfacewith lipid,'"afion? or Eastman polyme+ or mixing stearic acid within the carbon paste matrix.' In a previous work on enzyme-modified carbon paste electrodes (CPE), we noted that addition of octadecylamine (OA) into the electrode allowed a better stability of the resulting bio~ensor.~ In this paper and in other works devoted to the study of carbon paste based biosensors, we have been able to point out a very high selectivity of the OA-CPE with respect to hexacyanoferrate detection. The objective of the present study is to describe an efficient and yet simple strategy to eliminatethe interference of electroactivespecies in sensing systems using hexacyanoferrate detection (oxidation and reduction). The latter is widely applied as a mediator in ~~
(1) Wang, J.; Wu, L.; Lu, Z.; Li, R.; Sanchez, J. Anal. Chim. Acta 1990, 228, 251. (2) Amine, A.; Kauffmann, J.-M.; Patriarche, G. P. Talonra 1991, 38, 107. (3) Amine, A.; Kauffmann, J.-M. Bioelectrochem. Bioenerg. 1992, 28, 117. (4) Amine, A.; Kauffmann, J.-M.;Guilbault, G. G.; Bacha, S.Anal. Letf. 1993, 26, 1281. ( 5 ) Kulys, J.; Schuhmann, W.; Schmidt, H. L. Anal. Lett. 1992, 25, 1011. (6) Bremle, G.; Persson, B.; Gorton, L. Elecfroanalysis 1991, 3, 77. (7) Yabuki, S.; Mizutani, F.; Katsura, T.Biosens. Bioelecfron. 1992, 7, 695. (8) Bonakdar, M.; Vilchez, J. L.; Mottola, H. A. J . Electroanal. Chem. 1989. 266, 47.
0003-2700/94/0386-1595$04.50/0 @ 1994 American Chemical Society
numerous enzymatic reactions and in the field of biosensor technology.%16
EXPERI MENTAL SECT I ON Apparatus. Voltammograms were taken with a CV 27 voltammograph (BAS, West Lafayette, IN) connected to a Hewlett-Packard 7090A X-Y recorder. Amperometric measurements were made with a Bruker E-100 polarograph connected to a ServagorX-Yrecorder. All experimentswere performed with a three-electrodecell configuration comprising the working electrode, the saturated calomel reference electrode (SCE), and a platinum wire auxiliary electrode. The pH of the solution was measured with a Tacussel Mini 80 pH meter. Microscope observations have been made by using an optical microscope (Reichert-Jung MeFsA). Reagents. All reagents were of analytical grade and supplied by Sigma or Merck. Potassium salts of hexacyanoferrate(I1) and -(III) and of iridium(II1) hexachloride were used. Octadecylamine was obtained from Aldrich. The carbon paste (CP) was from Metrohm (EA 207). It consisted of spectroscopic graphites particles mixed with liquid paraffin (binder). Electrode Preparation and Procedures. The modified carbon paste electrode was prepared by thoroughly mixing in a mortar carbon paste and an appropriate amount of octadecylamine (OA) in the presence of a minimum amount of chloroformand allowing the solvent to evaporate overnight at room temperature. Quantities of OA higher than 10% gave a mechanically unstable paste. OA-CP refers to the 5% octadecylamine-containing carbon paste and St-CPE refers to the 5% stearate-containing carbon paste electrode. The St-CPE was prepared the same way as the OA-CPE. A portion of the resulting paste was packed into the well of the body of the BAS electrode (3 mm diameter, 2 mm deep). The amperometric measurements were performed in a thoroughly stirred solution (10 mL). All measurements were performed at room temperature. Solutions were prepared from reagent-grade chemicals using deionized water. The supporting electrolyte was 0.1 M phosphate buffer (pH 7.0) Amine, A.; Kauffmann, J.-M.; Patriarche, G. J.; Christian, 0 . D. Talonta 1993, 40, 1157. Ruiz, B. L.; Dempsey, E.; Hua, C.; Smyth, M. R.; Wang, J. Anal. Chim. Acto 1993, 273, 425. Attiyat, A. S.;Christian, G. D. Fresenius 2.Anal. Chem. 1975, 295, 157. Xic, X.; Kuan, S.S.;Guilbault, G. G. Bioses. Bioelectron. 1991, 6, 49. Racck, J.; Musil, J. Clin. Chim. Acfn 1987, 167, 59. Comtat, M.; Galey, M.; Goulas, P.; Suope,J. Anal. Chim. Acta 1988, 208, 295. Kitagawa, Y.; Kitabatake, K.;Kubo, I.; Tamiya, E.; Karube. I. Anal. Chim. Acta 1989, 218, 61. Cosgrove, M.; Moody, G. M.; Thomas, J. D. R. Analyst 1988, 113, 1811.
AnalvticaIChemistry, Vol. 66, No. 9, May 1, 1994 1195
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POTENTIAL (VI Flgure 2. Unear scan voltammogramsat the (a)CPE and @) OAICPE: phosphate buffer 0.1 M (pH 7.0); sweep rate 10 mV s-l.
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POTENTIAL (VI Flgwe3. Unearscanv~mmogramsatthe(a)CPE,@)OAICPE,and (c) St-CpE: 5 X lo4 M ascorbicacid; phosphatebuffer 0.1 M(pH 7.0); sweep rate 10 mV s-l. Figure 1. Optical micrographs of the electrode surfaces: (A, top) CPE; (B, bottom) OA-CPE.
RESULTS AND DISCUSSION Previous works have shown that optical microscopy (OM) of the CPE surface is preferred instead of electron microscopy for highlighting the graphite particles not covered with the insulating hydrophobic binder.” The optical microscopy images of the CPE and OA-CPE are shown in Panels A and B of Figure 1, respectively. A good contrast was obtained by OM, and the images were particularly informative due to the possible comparison between CPE and OA-CPE. It appeared clearly in Figure 1 that the OA-CPE presented a lower amount of “active” graphite (the white regions are the particles not covered by the binder) (Figure 1B) than the CPE (Figure 1A). This effect may be attributed to the presence of the synthetic amphiphile(OA) which allowed a better dispersion, thus a more efficientcovering, of the graphite particles (giving to the paste also a more fatty-like aspect). The available potential range and the magnitude of the background currents were compared by linear scan voltammetry at the CPE and OA-CPE. As shown in Figure 2, the solvent breakdown was shifted toward more positive potentials at the OA-CPE. The lower residual current observed at the
OA-CPE may be related to its lower density of active graphite particles. Interestingly, the oxidation of a 5 X l0-C M solution of ascorbate at pH 7.0 shifted toward more positive potentials at the stearate-modifiedCPE and at the OA-CPE (Figure 3, curves c and b, respectively). It is established from literature data that permselectivity of the CP modified with stearic acid (octadecanecarboxylic acid) is based on electrostatic repulsion.’* Surprisingly, the CP modified with octadecylamine (positively charged) showed no increase of the response of ascorbic acid (AA) as would be expected from electrostatic attraction, but rather a drastic current decrease and potential shift (of about 0.3 V) toward more positivevalueswasobsewed (Figure 3, curve b). Note also that the magnitude of the shift at theOA-CPEwasmuchmoreimportant than at thestearatemodified CPE. In order to characterize the OA-CPEbehavior, several molecules differing in terms of charge, size, and hydrophilicity at pH 7.0 have been investigated by linear scan voltammetry at the CPE and OA-CPE. The voltammogram of urine diluted 10 times with 0.1 M phosphate buffer at pH 7.0 was also recorded comparatively at the CPE and OA-CPE. The results showed that all the compounds tested, namely, tyrosine, tryptophan, promethazine, paracetamol, uric acid, dopamine, catechol, 8-hydroxyquinoline~5-sulfonic acid, ferrocenecarboxylic acid, and hydrogen peroxide (Chart l), showed a
(17) Digua, K.; Kauffmann, J. M.; Delplancke, J. L. Efectroanafysis,in press.
(18) Blaha, C. D.; Lane, R. E BrufnRes. Bull. 1983, 10,861.
and was prepared from a mixture of 0.1 M Na2HP04 and 0.1 M NaH2PO4 solutions.
1596 AnalyticalChemk@y, Vol. 66, No. 9, May 1, 1994
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Flguro 4. Linear !scan vdtemmograms at the (a) CPE and (b) OA-CPE. Analyte: (A) 5 X 5 X 10-4 M cabchol, and (D) wlne diluted 1\10. Other conditions, as in Figure 3.
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considerable signal decrease and a marked potential shift toward positive values at the OA-CPE (some examples are given in Figure 4). Quite unexpected, hexacyanoferrate(I1) showed a higher current and its response shifted toward less
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positive potentials at the OA-CPE (Figure 5, curve b). To the contrary, the CPE modified with an additional amount of paraffin ( 5 % ) showed no current decrease, but a marked shift toward more positive values was obtained (Figure 5 , curve c). Analytical Chemism, Vol. 00, No. 9, my 1, 1994
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POTENTIAL ( V I Figure 5. Linear scan voltammogramsat the (a) CPE, (b) OA-CPE, and (C)CPEmodifiedwith paraffin@%): 5 X 104M hexacyanoferrate(I1). Other condkbns, as In Figure 3.
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Potential ( V I Figure 7. Cyclic voltammetric curves at CPE (dotted ilne) and at CPE modified with OA: (a) 1, (b) 5, and (c) 10%: 5 X lo4 M hexacyanoferrate(II1). Other conditions, as in Figure 3.
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POTENTIAL (VI Figure 8. Cyclic voltammetric curves at the OACPE: 5 X lo4 M hexacyanofente(II1);solkl ilne, 0.1 M phosphatebuffer (pH 7.0); dotted line, 0.01 M phosphate buffer (pH 7.0); sweep rate 10 mV s-'.
The latter observations suggested that increase in the hydrophobicity of the CPE impaired the electron-transfer rate of hexacyanoferrate. . In order to point out any electrostatic effect, cyclic voltammograms (CV) of 5 X 10-4 M hexacyanoferrate(II1) have been recorded at the OA-CPE in phosphate buffer (pH 7.0) at different ionic strengths. No significant change, within the experimental error, was observed using either 0.1 or 0.01 M phosphate buffer (Figure 6, solid and dotted lines, respectively). Likewise, by stirring a 5 X lo4 M solution of hexacyanoferrate(II1) at pH 7.0 for 5 min and recording the CV, no increase of the peak currents was observed. It should be pointed out that the cyclic voltammetry of hexacyanoferrate(II1) at the CPE exhibited an irreversible response (Figure 7, dotted line) in agreement with previous ~ o r k . ~ The , ' ~ incorporation of OA into the CP matrix improved considerably the reversible character of the hexacyanoferrate(II/III) couple (Figure 7, curves a-c). The effect of potential scan rate (u) within the range 5-400 mV s-l on the cyclic voltammetric response of 5 X l o " M hexacyanoferrate at OA-CPE was determined. A linear i, vs v1I2 plot over the range of 25-400 mV S-I (characteristic of diffusional control) was obtained (result not shown). The heterogeneous (19) Dhesi, R.; Cotton, T. M.; Timkovich, R. J. Electround. Chem. 1983, 154, 129.
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AnalytcalChemisby, Vol. 66,No. 9, May 1, 1994
rate constant (k")was estimated from AE, by the method of Nicholson.2o The average value (N= 4) obtained at 100 mV s-l scan rate was (2.2 f 0.3) X cm se1. This value is well within the range of values (1.5 X lo"4.20 cm s-l) that have been obtained at various pretreated glassy carbon electrodes.21 Considering the low amount of Yactivengraphite (see OM observations) and the rapid electrochemical response of the hexacyanoferrate(III/II) couple at the OA-CPE, we may postulate that hexacyanoferrate diffusion through thin layers of binder (paraffin OA) governed to a large extent the electrochemical response at the OA-CPE.19 This may also be inferred from recent resultsI7showing that the electrochemical area of the CPE, determined by chronoamperometry in the presence of hexacyanoferrate(II), was distinct and higher than its active carbon surface area. Owing to these considerations, we may suppose that OA facilitates the diffusion of hexacyanoferrate(I1) through thin layers of liquid paraffin. The cyclic voltammogram of iridium(III)/(II) hexachloride, a species that is more similar to hexacyanoferrate than the other species tested above, has also been studied at the CPE and OA-CPE. The CV results indicated that iridium (III)/(II) hexachloride already exhibited rapid electron transfer at the CPE (Figure 8), and the currents were diminished at the OA-CPE but the peak potentials were not shifted. Thus, in contrast to hexacyanoferrate, the diffusion rate of iridium hexachloride is limited at OA-CPE. It is interesting to note that iridium hexachloride and hexacyanoferrate exhibited similar electrostatic binding effects at protonated poly(viny1pyridine)-coated graphite electrodes,22 but in our work, their electrochemical behavior was different. Ion-pair formation between OA and hexacyanoferrate might be responsible for the results obtained; however, the exact mechanism of the observed process is still unclear since all the molecules investigated, negative and positively charged, hydrophilic,and hydrophobic, except hexacyanoferrate showed restricted diffusionthrough the binder to the graphite particles.
+
(20) Nicholson, R. S. Anal. Chem. 1965, 37, 1351. (21) Hu, I. F.; Karweik, D. H.; Kuwana, T. J . Electround. Chem. 1985,188,59. (22) Oyama, N.; Anson, F. C. Anal. Chem. 1980, 52, 1192.
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POTENTIAL (VI Flgure 8. Cycilc voltammetric curves at CPE (dotted line) and at OACPE (solid line): 5 X 1Ow M lridium(II1)hexechlorkle. Other conditions, as in Flgure 3.
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Interestingly from a practical point of view, by poising the OA-CPE at low potentials, around +0.3 V, interferences of electroactive species present in biological media (e.g., in urine) were not observed. The amperometric response of urate and hexacyanoferrate(I1) at the unmodified CPE and CPE modified with different amounts of OA is shown in Figure 9. It appeared clearly that a high response of hexacyanoferrate(I1) and an effective exclusion of the urate species was obtained at the CPE modified with 5% OA. The study of the influence of the amount of OA in the CPE also showed that the highest response for hexacyanoferrate(I1) oxidation was obtained for OA amounts in the CPE comprised between 2.5 and 5%. Higher amounts gave current decreases and slight (23) Wang, J.; Fang, L.; Lopez, D.; Tobias, H.Anal. art.1993, 26, 1819.
peak potential shifts probably related to the increase in the diffusion layer in the OA-CPE and in the electrode resistance (Figure 9 and Figure 7, curve c). Calibration curves ( N = 5 ) of hexacyanoferrate(I1) were made using the OA-CPE poised at +0.32 V. Linearity was observed from 20 nM to 1 mM (sensitivity 1.4 f 0.2 nA pM-l, correlation coefficient 0.999). The limit of detection (S/N = 3) was very low, 20 nM. The coefficient of variation of the slope of the calibration curves ( N = 5 ) using the same electrode was 15%. The stability of the OA-CPE in buffer-acetonitrile solutions was evaluated by recording the amperometric response of 1 pM hexacyanoferrate(I1) after 7-h contact with 0.1 M phosphate buffer-acetonitrile (60/40% v/v). The results showed that the intensity of the response and the magnitude of the residual current were unchanged. Similar stability results were obtained with the CPE modified by hexadecanesulfonic acid.17 The stability of the OA-CPE in bufferacetonitrile mixture may be related to the presence of polar head groups facing the sample solution and preventing the organic solvent from solubilizing the liquid paraffin.17 The high selectivity of the OA-CPE with respect to hexacyanoferrate(I1) may be of particular interest for biosensor construction. It is well-known that hexacyanoferrate is a very effective mediator for several enzyme ele~trodes.~-l~ Electrochemical analyzers for lactate based on hexacyanoferrate(I1) detection at platinum electrodes are already commerciallized (Microzym-L from SGI, Toulouse, France). The enzyme may likely remain active in the OA-CPE, as we have shown previously using a glutamate dehydrogenase immobilized OA-CPE,3 and the proposed electrode may be of general use for enzyme systems using hexacyanoferrate. It is worth mentioning that recently a mixed glucose oxidaseruthenium OA-CPE was reported by Wang et al., but no comment was given with regard to the role of OA in the paste.23 In conclusion, the present work showed that a highly selective and sensitive (in terms of S/N) amperometric detection of hexacyanoferrate may be obtained by modifying the carbon paste electrode with a small amount of octadecylamine. The improved selectivity resulted from a considerable intensity decrease and potential shift toward positive values of the response of interfering species along with a current increase and a shift toward less positive potentials of hexacyanoferrate(I1) oxidation. This interesting property of the OA-CPE may be advantageously exploited for the construction of OA-CPEs with an immobilized enzyme for which hexacyanoferrate acts as redox mediator, as reported previ~usly.~
ACKNOWLEDGMENT Thanks are expressed to the Fonds National de la Recherche Scientifique (FNRS) for financial support. Received for review November 3, 1993. Accepted February 8, 1994."
* Abstract published in Aduance ACS Abstracfs, March 15, 1994. AnalyticalChemistry, Vol. 66, No. 9, May 1, 1994
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