Studies of Sol−Gel Ceramic Film Incorporating Methylene Blue on

Anal. Chem. 2002, 74, 5734-5741

Studies of Sol-Gel Ceramic Film Incorporating Methylene Blue on Glassy Carbon: An Electrocatalytic System for the Simultaneous Determination of Ascorbic and Uric Acids Soo Beng Khoo* and Fang Chen

Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543

In this work, we investigated the immobilization of methylene blue in a methyltrimethoxysilane sol-gel ceramic film on a glassy carbon electrode. Up to a certain saturation level, under our conditions, it was found that the methylene blue was tightly held and did not leach out into aqueous solutions, even with continuous immersion for up to 1 month. The electrochemical behavior of the immobilized methylene blue was then studied. pH variation revealed that there were two distinct redox couples whose existences were pH-dependent. The methylene blue/sol-gel film was also examined as an electrocatalytic system for ascorbic and uric acid oxidations. It was revealed that this system was highly sensitive for ascorbic and uric acid sensing (practical determination limits of 5.00 nM and 1.00 nM for ascorbic acid and uric acid, respectively) and also allowed simultaneous determination of these biomolecules. The simultaneous determination of these two analytes in a human urine sample was demonstrated. The stability of the methylene blue/solgel film/glassy carbon electrode sensing system was good, with up to at least a month of continual operation. Electrodes modified with thin films of sol-gel ceramic have attracted much attention in recent times1-7 because such films have advantages as hosts for dopants. Advantages that accrue include practical simplicity and convenience, because the solgel process can be easily performed at room temperature. This feature is especially useful for the fabrication of biosensors in which the active ingredients, for example, enzymes and antibodies, do not possess thermal stability. Other advantages are physical rigidity, high biodegradational, electrochemical, photochemical, and thermal stabilities and optical transparency. The main drawbacks are fragility and susceptibility to hydrolysis at high pH.1 * Corresponding author. Fax: (65) 67791691. E-mail: [email protected]. (1) Lev, O.; Tsionsky, M.; Rabinovich, L.; Glezer, V.; Sampath, S.; Pankratov, I.; Gun, J. Anal. Chem. 1995, 67, 22A. (2) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A. (3) Dunn, B.; Farrington, G. C.; Katz, B. Solid State Ionics 1994, 70/71, 3. (4) Lin, J.; Brown, C. W. Trends Anal. Chem. 1997, 16, 200. (5) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605. (6) Alber, K. S.; Cox, J. A. Mikrochim. Acta 1997, 127, 131. (7) Collinson, M. M. Mikrochim Acta 1998, 129, 149.

5734 Analytical Chemistry, Vol. 74, No. 22, November 15, 2002

Arising from the advantages noted, electrodes modified with sol-gel ceramic films have become increasingly popular as platforms for the immobilization of enzymes and other catalysts for chemical and biosensing applications.2,4,5 Chemically modified electrodes (CMEs) with electrocatalytic functions often offer improved selectivity and sensitivity, as compared to unmodified electrodes. Although enzymes are highly selective in their reactions, other catalysts, which may be used in conjunction with enzymes,8 function as electron-transfer mediators so that target analytes can undergo redox processes at lower potentials with improved sensitivity.9-16 Certain organic dyes that have reversible electron-transfer processes, for example, methylene blue (MB), are useful as electrocatalysts/electron-transfer mediators. Silicabased ceramics have been reported to be very good matrixes for some organic dyes, such as rhodamine 6G,17 cyanine dyes,18 azobenzene dyes,19 and laser dyes.20 Immobilization of organic dyes and other catalysts possesses advantages such as reusability (and therefore, cost-effectiveness) and isolation from solution impurities.4 Of particular interest for this work is MB, which is a cationic dye with reversible redox processes in aqueous solution. It has formal potential Eo′ varying between -0.10 V and -0.40 V (vs SCE) in the pH range 4-11, which makes it a useful catalytst/ mediator for biological systems, many of which have similarly located redox potentials.21,22 MB has been immobilized in different (8) Kano, K.; Ikeda, T. Anal. Sci. 2000, 16, 1013. (9) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564. (10) Andrieux, C. P.; Audebert, P.; Divisia-Blohorn, B.; Linquette-Maillet, S. J. Electroanal. Chem. 1993, 353, 289. (11) Amine, A.; Kauffmann, J. M.; Guillbault, G. G.; Bacha, S. Anal. Lett. 1993, 26, 1281. (12) Morris, N. A.; Cardosi, M. F.; Birch, B. J.; Turner, A. P. F. Electroanalysis 1992, 4, 1. (13) Chi, Q.; Dong, S. Electroanalysis 1995, 7, 147. (14) Bremle, G.; Persson, B.; Gorton, L. Electroanalysis 1991, 3, 77. (15) Ju, H. X.; Xum, Y. A.; Chen, H. Y. J. Electroanal. Chem. 1995, 380, 283. (16) Dempsey, E.; Wang, J.; Wollenberger, V.; Ozsoz, M.; Smyth, M. R. Biosens. Bioeletron. 1992, 7, 323. (17) Narang, U.; Bright, F. V.; Prasad, P. N. Appl. Spectrosc. 1993, 47, 229. (18) De Rossi, U.; Daehne, S.; Reisfeld, R. Chem. Phys. Lett. 1996, 251, 259. (19) Worsfold, O.; Malins, C.; Forkan, M. G.; Peterson, I. R.; MacCraith, B. D.; Walton, D. J. Sens. Actuators, B 1999, 56, 15. (20) Avnir, D.; Kaufman, V. R.; Reisfeld, R. J. Non-Cryst. Solids 1985, 74, 395. (21) Erdem, A.; Kerman, K.; Meric, B.; Akarca, U. S.; Ozsoz, M. Anal. Chim. Acta 2000, 422, 139. (22) Pessoa, C. A.; Gushikem, Y.; Kubota, L. T. Electroanalysis 1997, 9, 800. 10.1021/ac0255882 CCC: $22.00

© 2002 American Chemical Society Published on Web 10/15/2002

environments and utilized for the catalysis of various species. For example, it has been immobilized in Nafion polymer film on glassy carbon23 as well as on carbon fiber, where it was employed for the catalytic oxidation of hemoglobin.15 MB was immobilized in zeolites24,25 and served, together with horseradish peroxidase, as an amperometric biosensor for H2O2.25 It was also used as a mediator immobilized in β-cyclodextrin in an enzyme electrode for the determination of mercury species.26 MB was immobilized in zirconium phosphate (i) to study its electrochemical behavior when mixed into carbon paste22 (ii) together with horseradish peroxidase for the determination of H2O227 and (iii) for the photoelectrochemical determination of ascorbic acid when used in carbon paste.28 Our literature search revealed that only two works29,30 have been reported on the immobilzation of MB in sol-gel ceramic films on electrodes and none using methyltrimethoxysilane (MTMS) as precursor for the silica network. The presence of the methyl group gives a hydrophobic character to the resulting solgel ceramic film. In addition, the MB-doped sol-gel ceramic has not been investigated as an electrocatalytic system for the simultaneous determination of ascorbic acid (AA) and uric acid (UA). Because of the high electron-transfer efficiency of MB29 and the favorable characteristics of the sol-gel ceramic matrix, this electrocatalytic system is expected to provide a highly stable and sensitive sensing platform for AA and UA. As will be shown, a glassy carbon electrode modified with a sol-gel ceramic film containing MB can typically be useful for up to 1 month (a longer period may be possible) of continual measurements, and experimental (not calculated) determination limits are estimated to be 5.00 nM for AA and 1.00 nM for UA. Such low determination limits have not been previously reported for the simultaneous determinations of these two biomolecules. Our main objectives here were to investigate the electrochemical characteristics of the MB immobilized in the sol-gel ceramic film on glassy carbon and also those of AA and UA at this modified electrode. The effects of pH, electrolyte, MB loading, and scan rate were examined. Analytical aspects for the simultaneous determination of AA and UA were also studied. The modified electrode was applied to the simultaneous determination of AA and UA in a human urine sample. EXPERIMENTAL SECTION Reagents. All chemicals were of analytical reagent grade unless otherwise specified. Containers (glassware, polythene) were immersed overnight in 10% nitric acid prior to use. MTMS was purchased from Fluka (Buchs, Switzerland) and used as received. Ethanol (EtOH) and methanol (MeOH) were both HPLC grade (J.T. Baker, Phillipsburg, NJ). MB, AA (both from Merck, Darmstadt, Germany), and UA (Sigma, St. Louis) were also used without further purification. Water was obtained from a Millipore (23) Lu, Z.; Dong, S. J. Chem. Soc., Faraday Trans. 1 1998, 84, 2979. (24) Zhou, W. H.; Clennan, E. L. J. Am. Chem. Soc. 1999, 121, 2915. (25) Liu, B.; Liu, Z.; Chen, D.; Kong, J.; Deng, J. Fresenius’ J. Anal. Chem. 2000, 367, 539. (26) Han, S.; Zhu, M.; Yuan, Z.; Li, X. Biosens. Bioelectron. 2001, 16, 9. (27) Ruan, C.; Yang, F.; Xu, J.; Lei, C.; Deng, J. Electroanalysis 1997, 9, 1180. (28) Cooper, J. A.; Woodhouse, K. E.; Chippindale, A. M.; Compton, R. G. Electroanalysis 1999, 11, 1259. (29) Chen, D.; Liu, B.; Liu, Z.; Kong, J. Anal. Lett. 2001, 34, 687. (30) Leventis, N.; Chen, M. G. Chem. Mater. 1997, 9, 2621.

Alpha-Q purification system (18.2 MΩ, Millipore Corporation, Bedford, MA). Solutions and buffers were prepared employing standard laboratory procedures. Apparatus. Electrochemical experiments were performed using a Princeton Applied Research Corporation model 264A polarographic analyzer/stripping voltammeter (PARC, EG&G, Princeton, NJ) coupled to a Graphtec model WX2400 x-y recorder (Graphtec Corporation, Tokyo, Japan) or an Autolab PGSTAT 30 (Eco Chemie, Netherlands) electrochemical system. A locally made three-electrode, two-compartment glass cell (∼5 mL capacity) was used for all electrochemical experiments. The reference electrode was Ag/AgCl (saturated KCl) and the counter electrode was a platinum disk (3 mm diameter) electrode. The working electrode was either a bare glassy carbon electrode (GCE) or a GCE coated with a sol-gel ceramic film without (SGGCE) or with (MB-SGGCE) incorporation of MB. pH measurements were performed on a Hanna model 9318 (Hanna Instruments, Woonsocket, RI) pH meter. All experiments were carried out at an ambient temperature of 25(2 °C. Procedure. Electrode Modification. Prior to modification, the GCE was polished with alumina (0.3 µm)/water slurry on a polishing cloth. The electrode was then rinsed copiously with Millipore water and sonicated in a Millipore water bath for 5 min, then rinsed again with Millipore water and, finally, with EtOH. It was allowed to air-dry. For electrode modification, mixtures of EtOH/MTMS/0.10M HCl (high concentrations of HCl were studied but found to be not so suitable because of the tendencies of the films to crack after gelation and aging) in the ratio 7.50:3.75:1.00 (v/v), respectively, containing MB (various MB concentrations of 1.00, 1.53, 5.00, 10.00, 20.00, and 50.00 mM in the sol mixtures were studied) were sonicated for 1.5 min. A 5.0-µL portion of this mixture was then transferred onto the prepared GCE surface with a microsyringe. The droplet on the electrode was carefully manipulated to just cover the entire surface (GC disk plus epoxy in which it was embedded and the wall of the glass tube holder) of the electrode. The electrode, thus covered, was maintained in a vertical position facing upward and allowed to air-dry and gel at room temperature for 24 h. Following this, the coated electrode was immersed in Millipore water overnight before use. The GCE coated with sol-gel ceramic film without the addition of MB was similarly fabricated. When not in use, the MB-SGGCE and SGGCE were stored in Millipore water. Human Urine Analysis. A freshly collected human urine sample was filtered through a 0.46-µm filter disk. This was then sequentially diluted 100 times each in two steps with 1.00 M phosphate buffer, pH 6.50 to obtain a final sample of 10 000-fold dilution. Another sample was similarly prepared, but dilution was with 1.00 M phosphate buffer, pH 1.50. The AA and UA concentrations in the prepared samples were simultaneously determined by the standard addition method using differential pulse voltammetry at the MB-SGGCE. For each prepared sample, spiked experiments were also performed. All determinations were carried out in duplicate (at least). RESULTS AND DISCUSSION Cyclic Voltammograms (CVs) at the GCE and MB-SGGCE. Figure 1a, i, and ii shows the CVs (20 mV s-1) at the bare GCE and the MB-SGGCE, respectively, between 0 V and +1.00 V in Analytical Chemistry, Vol. 74, No. 22, November 15, 2002

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1.00 M phosphate buffer, pH 6.50. The latter shows a much reduced residual current due to the hydrophobic nature and the barrier of the MTMS sol-gel ceramic film. The presence of the MB in the MB-SGGCE was clearly evident by the deep blue color of the electrode surface. Because the MB was initially present in the sol solution, it is reasonable to assume that after gelation, it was homogeneously distributed throughout the bulk of the solgel ceramic matrix. At the sensitivity scale employed in Figure 1a,ii, no redox peaks could be discerned for the immobilized MB. However, at higher sensitivity, two pairs of redox peaks were apparent (Figure 1b,i-iii. At pH 1.50, the first pair of redox peaks was dominant (Ep,a,1 ) 0.199 V, Ep,c,1 ) 0.087 V, ip,a,1 ) 0.67 µA, ip,c,1 ) 0.52 µA), whereas the second pair at more positive potentials (Ep,a,2 ) 0.531 V, Ep,c,2 difficult to determine at this pH) was barely discernible. With increasing pH, up to 8.50, the second pair increased in height and became dominant, and the first pair almost disappeared at pH 8.50. The potentials of both redox couples shifted to less positive values with increasing pH. For the first redox couple, Ep,a,1 has an approximate linear relationship with pH, with Ep,a,1 (V) ) -0.056pH + 0.279, r ) 0.989 from pH 1.50 to 5.50, whereas the cathodic peak potentials vary according to Ep,c,1 (V) ) -0.061pH + 0.178, r ) 0.969 in the same pH range. Above pH 6.50, the peak potentials for the first couple could not be determined, because the peaks had decreased to insignificant levels. At pH 4.50 and above, peak heights for the second redox couple increased, allowing the measurement of peak potentials. For this second couple, Ep vs pH relationships were as follow: Ep,a,2 (V) ) -0.038pH + 0.358, r ) 0.988; Ep,c,2 (V) ) -0.042pH + 0.279, r ) 0.995. The intersection point between the linear Ep,a versus pH plots for the two redox couples occurred at about pH 5.50. Peak potentials (25 °C) can be shown to vary with pH according to23

Ep ) constant + (0.0592/n) log([H+]a3 + Kr,1[H+]a2 + Kr,2[H+]a) (1) where Kr,1 and Kr,2 are dissociation constants of MB in the reduced state, [H+]a is the proton concentration in the sol-gel ceramic film, and n is the number of electrons transferred, which for MB is 2. From this equation, it can be deduced that the Ep-pH relationship can have slopes of -90, -60 and -30 mV, depending on whether the first, second, or third term, respectively, in the bracket is predominant. For MB entrapped in Nafion,23 Ep vs pH plots exhibited two linear regions with slopes of -90 mV below pH 5.6 and -30 mV at pH above 5.6. When MB was covalently bonded to a siloxane network,30 again two regions were observed, one with a slope of -90 mV at pH below 5.0 and another region that was independent of pH at pH g5.0. In this latter case, the existence of two regions was attributed to proton availability or lack of it. Indeed, proton availability was cited to explain the presence of two oxidation peaks, with the more positive peak being dominant at lower scan rates. Our observations for MB immobilized in MTMS-sol-gel film contrast with these two earlier studies. The slopes for the Ep vs pH plots of the oxidation and reduction peaks at -56 and -61 mV, respectively, for the first redox couple, at lower pH values where the first couple was dominant, suggest that the second term in the bracket in eq 1 5736 Analytical Chemistry, Vol. 74, No. 22, November 15, 2002

Figure 1. CVs (20 mV s-1) in 1.00 M phosphate buffer: (a) (i) at GCE and (ii) at MB-SGGCE, both in pH 6.50 buffer; (b) (i, ii, iii) all at MB-SGGCE in pH 1.50, pH 6.50 and pH 8.50 buffers, respectively. The recordings in (a) were at much lower sensitivity than those in (b).

Figure 2. Redox reactions of MB.

was the dominant factor for this couple. This behavior was not seen in the earlier works. On the other hand, the second redox couple had slopes of -38 and -42 mV for oxidation and reduction peak potentials, respectively. These values, closer to the theoretical value of -30 mV, imply that the third term in the bracket in eq 1 was controlling. Although -30 mV slope was obtained for MB in Nafion, at higher pH, it was not observed for covalently bonded MB. Further, the relative importance of the two oxidation peaks found here depended on only pH and not on scan rate, as was found for covalently bonded MB.30 MB redox reactions occur according to Figure 2. Nishikiori et al.31 have shown that MB entrapped in tetraethyl orthosilicate sol-gel ceramic can exist in three forms: the protonated form, MBH+; MB; and the dimer, (MB)2. Our conditions were not acidic enough for MBH+ formation (pKa -0.258), which has a short lifetime anyway.31 The first redox couple observed in this work can be attributed to the reaction in Figure 2 involving a two-proton, two-electron transfer corresponding to the predominance of the second term in the bracket in eq 1. The second redox couple with a slope of -30 mV could be assigned to a two-electron, one-proton process corresponding to the third term in eq 1, according to Leventis and Chen.30 This could arise from a singly deprotonated form of MB at higher pH, which was not mentioned by Nishikiori et. al. As noted earlier, the changeover from -60 mV slope to -30 mV slope was centered at about pH 5.50. Although the dimer, (MB)2, was shown to exist in TEOS-sol-gel ceramic, its existence and (31) Nishikiori, H.; Nagaya, S.; Tanaka, N.; Katsuki, A.; Fujii, T. Bull. Chem. Soc. Jpn. 1999, 72, 915.

Figure 3. Plots of total oxidation and reduction charges (both couples) of MB versus pH. Qa ) total anodic charge, Qb ) total cathodic charge. Obtained from the areas under the cyclic voltammetric (20 mV s-1) peaks.

role in the present system could not be determined on the basis of the present studies. At pH 1.50, 6.50, and 8.50, where peaks were measurable, plots of peak currents versus scan rates were linear at least up to 50 mV s-1, indicating surface rather than diffusion-controlled behavior. Peak potentials for the two redox couples generally did not shift significantly except at scan rates >50 mV s-1 where shifts of 30-50 mV were observed at 200 mV s-1. Therefore, for scan rates at 50 mV s-1 or lower, no kinetic parameters are available. For scan rates from 50 to 200 mV s-1, the source of the potential shifts is difficult to ascertain under the present conditions. We attribute this to film resistance because of the relatively high resistances of the films, as shown below. Figure 3 shows that the total anodic (Qa) and cathodic (Qc) charges (sum of both redox couples) decreased with pH. Qa was generally higher than Qc, with the difference being lower at the low pH end (e.g., at pH 1.50, Qa ) 11.2 µC, Qc ) 10.2 µC) but increasing at the high pH end (pH 8.50, Qa ) 6.9 µC, Qc ) 4.4 µC). Considering the narrower peaks at lower pH for oxidation, we ascribe the higher charge with decreasing pH to increased conductivity/electroactivity. Both Qa and Qc are essentially constant, with scan rates up to ∼50 mV s-1 at all pH values but decreasing slightly at higher scan rates. As for the peak currents, these also imply the absence of diffusional charge transport at least up to 50 mV s-1. The claim of higher film conductivity with lower pH can be substantiated by impedance data. Figure 4 shows plots of impedance against potential from -1.10 to -0.20 V (each point on the plot represents a potential step of 50 mV) at pH values of 1.50, 6.50, and 8.50 for the MB-SGGCE (Figure 4a) and SGGCE (Figure 4b), all at frequencies of 20 kHz (sine waveform with 10 mV rms amplitude). In this potential region, there was no faradaic reaction, so the film-covered electrode can be represented by a film resistance and capacitance. Further, at the high frequency of 20 kHz, the capacitance contribution to the impedance is small, and the measured impedance can be largely attributed to film resistance. As can be seen, in going from pH 1.50 to 6.50 for the MB-SGGCE, there is a significant increase in film resistance (∼250 Ω). Further increase in resistance from pH 6.50 to 8.50 is also observed, but relatively smaller (about 50 Ω). For the SGGCE, the trend of increasing film resistance from pH 1.50 to 8.50 is maintained. However, it is noted that film resistances are much higher in the absence of MB. For the 5.0-µL droplet of the sol placed on each electrode, the amount of MB (based on 10.00 mM MB in the sol used for MBSGGCE fabrication) spread over the electrode surface was 0.050

Figure 4. Plots of impedance versus potential at different pH (a) MB-SGGCE. (b) SGGCE. Each point represents a potential step of 50 mV. A sinusoidal waveform with root-mean-square amplitude of 10 mV at 20 kHz was applied for each measurement.

µmol (1.60 × 10-5 g). Because the total electrode surface, inclusive of the GC, epoxy and glass, had a diameter of 5.5 mm, and the GC electrode diameter was 3.0 mm, the amount of MB in the sol-gel film covering the GC disk was 0.015 µmol, assuming the film had uniform thickness (4.80 × 10-6 g). If this were fully accessible for electron transfer through the thickness of the film (for a 5.0-µL drop, on gelling, the mass was found to be 0.000798 g; the density of the dry gel was estimated to be 0.88 g ml-1; thus, the thickness of the sol-gel film can be estimated to be ∼38 µm) for a two-electron process, the expected charge would be 2.90 × 10-3 C. The consideration of the MB in the film directly above the GC electrode but not outside the perimeter of the electrode contributing to the charge is thought to be a good first approximation. But the actual charge, Qa at pH 1.50 was only 11.2 µC, which corresponds to only 0.39% of the expected value. This rough estimation shows that only a very small fraction of the MB was available for electron transfer. Therefore, we have to conclude that the vast majority of the immobilized MB was isolated and strongly held in the pores of the sol-gel ceramic matrix with low mobility. Additional evidence of this restricted mobility of the MB is given by the fact that films formed from the sol containing 10.00 mM MB and lower exhibited no sign of leakage of MB, noted by the absence of any blue coloration in water, over periods of 1-2 months immersion in water/electrolyte solutions. Further support is provided by the good stability of the MB-SGGCE over up to 1 month applications, as will be discussed later. We had also observed that when the MB-SGGCE was fabricated from a 20.00 or 50.00 mM MB-containing sol, the initially prepared electrode had a higher current, which decreased with time. When such electrodes were immersed in water/solutions, the aqueous media turned blue after 1 or 2 days. We monitored the leakage into the water from a MB-SGGCE prepared from a 50.00 mM MB sol over a period of 28 days, when the leakage was judged to have stopped. Since 5.0 µL of the original mixture was used, the amount of MB deposited on the electrode surface was 8.06 × 10-5 g. After 28 Analytical Chemistry, Vol. 74, No. 22, November 15, 2002

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Figure 5. CVs (20 mV s-1) for the oxidation of AA and UA in 1.00 M phosphate buffer, pH 6.50: (a) 1.00 mM AA (i) at the GCE and (ii) at the MB-SGGCE; (b) 1.00 mM UA (i) at the GCE and (ii) at the MB-SGGCE.

days, 5.84 × 10-5 g leached into the water, as determined by visible spectrophotometry, leaving 2.22 × 10-5 g, which was thought to be tightly held. In comparison, for a mixture containing 10.00 mM MB, 5.0 µL for MB-SGGCE fabrication deposited 1.60 × 10-5 g on the electrode surface, and this amount was shown to be tightly immobilized without leakage. The observations above suggest that under the conditions of the MB-SGGCE fabrication, MB amounts above a threshold value of ∼2.22 × 10-5 g are quite mobile. Therefore, there is an apparent saturation level for the immobilization by the MTMS-sol-gel ceramic matrix. Oxidation of AA and UA at the GCE, MB-SGGCE, and SGGCE. Figure 5a,b compares the oxidations of AA and UA (both irreversible), respectively, in 1.00M phosphate buffer, pH 6.50 at the bare GCE and MB-SGGCE electrodes. These CVs established the electrocatalytic effects of the MB-SGGCE arising from MB acting as an electrocatalyst. This is clear from the increases in peak heights and shifts to less positive potentials at the MBSGGCE relative to the bare GCE. For the CVs in Figure 5a and b, for AA, GCE, Ep,a ) +0.63 V, and ip,a ) 2.1 µA; for MB-SGGCE, Ep,a ) +0.27 V and ip,a ) 6.0 µA; for UA, GCE, Ep,a ) +0.75 V and ip,a ) 4.0 µA; for MB-SGGCE, Ep,a ) +0.46 V and ip,a ) 10.0 µA. Figure 6a,b provides proofs that the peak current enhancements and potential shifts for the oxidations of AA and UA found in Figure 5a,b were largely due to the presence of MB. At the SGGCE, no AA oxidation was observed in the absence of MB (Figure 6a,i). When the SGGCE was immersed in the 0.50 mM AA solution over time, an oxidation peak appeared, the height of which increased with time of immersion, and attained a constant height after ∼25 min (Figure 6a,ii). Thus, AA was able to diffuse slowly through the sol-gel film and reached the electrode surface for oxidation but not on an instantaneous basis, as seen for MBSGGCE (Figure 6a,iii). Additionally, the height for this intrafilm diffusion peak did not reach the level achieved for MB-SGGCE. For the SGGCE, there was some potential shift as compared to the bare GCE but less so than for MB-SGGCE. Similar results were found for UA (Figure 6b), for which at the SGGCE, a maximum peak height was achieved after 55 min of immersion 5738 Analytical Chemistry, Vol. 74, No. 22, November 15, 2002

Figure 6. CVs (20 mV s-1) in 1.00 mM phosphate buffer, pH 6.50, (a) 0.50 mM AA at (i) SGGCE, no time delay, (ii) SGGCE after immersion in the AA solution for 25 min, and (iii) Mb-SGGCE, no time delay; (b) 0.50 mM UA at (i) SGGCE, no time delay, (ii) SGGCE after immersion in the UA solution for 55 min, and (iii) MB-SGGCE, no time delay. Table 1. Cyclic Voltammetric Peak Currents and Potentials for Oxidations of AAa and UAa AA

UA

electrode

Ep,a (V)

ip,a (µA)

Ep,a (V)

ip,a (µA)

b

0.63 0.24 0.36

2.10 2.13 0.44c

0.75 0.46 0.50

4.00 4.80 0.88d

GCE MB-SGGCE SGGCE

a AA and UA solutions were 0.50 mM in 1.00 M phosphate buffer at pH 6.50 unless otherwise noted. b For GCE, AA and UA solutions were 1.00 mM in 1.00 M phosphate buffer at pH 6.50. c Peak current after immersion in AA solution for 25 min; increasing immersion time beyond this did not increase the current. d Peak current after immersion in UA solution for 55 min; increasing immersion time beyond this did not increase the current.

in 0.50 mM UA (Figure 6b,ii). The data from Figure 6a,b is summarized in Table 1. It should be noted that in pH 6.50 solution, AA is essentially fully mono-deprotonated (pK1 ) 4.01, pK2 ) 11.3432). For UA (pK1 ) 5.40, pK2 ) 5.5333), the distribution of the various forms are in the following ratios: UA ) 0.0076, UA) 0.0960, UA2- ) 0.8963. Influence of the Amount of Immobilized MB on AA Oxidation Peak. The influence of the concentration of MB in the sol mixture (i.e., also the amount of MB deposited on the electrode) on the oxidation peak for 1.00 mM AA in 1.00 M phosphate buffer, pH 6.50, was investigated. Under these conditions, the CV anodic peak height for AA increased approximately linearly with increasing concentration of MB used in the sol up to 10.00 mM. From 10.00 mM MB upward, the peak current for AA remained constant. Thus, 10.00 mM MB was taken as the optimum concentration, bearing in mind that at concentrations (32) The Combined Chemical Dictionary on CD-ROM [electronic resource]; Chapman & Hall, Electronic Publishing Division, Boundary Row: London, 1997. (33) Lange’s Handbook of Chemsitry, 13th ed.; Dean, J. A., Ed.; McGraw-Hill: New York, 1985; pp 5-23, 5-60.

Figure 7. (a) Plots of CV (20 mV s-1) anodic peak current versus pH for AA and UA (1.00 mM each in 1.00 M phosphate buffer): (i) AA, (ii) UA. (b) Plots of CV (20 mV s-1) anodic peak potential versus pH for AA and UA (1.00 mM each in 1.00 M phospahte buffer): (i) AA, (ii) UA.

above this, significant leakage of MB was encountered. All further studies employed MB-SGGCE fabricated from sols containing 10.00 mM MB. Effects of Scan Rate and pH on AA and UA Oxidation Peaks at MB-SGGCE. Plots of peak currents against square root of scan rate, up to 200 mV s-1, for 1.00 mM AA and UA in 1.00 M phospahte buffers, pH 1.50, 6.50, and 8.50, gave straight lines through the origin. Furthermore, for these buffers, oxidation peak potentials for AA and UA increased approximately linearly in a logarithmic manner with scan rate up to 200 mV s-1. These data suggest that oxidation of AA and UA at the MB-SGGCE were fast (with high electrocatalyic efficiency).34,35 Figure 7a,b shows the variation of peak currents and potentials, respectively, with pH of 1.00 M phosphate buffer for AA and UA (each 1.00 mM). From Figure 7a, it is seen that anodic peak currents increased with increasing pH up to 6.50, after which the current fell sharply. The initial increase is more pronounced for AA than for UA, which is slight (Figure 7a,ii). It is interesting to note that in both cases, the point of commencement of the sharp decrease occurred at pH 6.50. The electrochemical oxidations of AA36 and UA37 are usually reported to involve net losses of two electrons and two protons. This being the case, the increase in pH is expected to increase peak currents, as is seen here, at least up to pH 6.50. However, because the MB-sol-gel film acts as a conduit for electrons between the electrode and the analyte in solution, any limitation in the electrocatalytic system will also affect the analyte. Therefore, at higher pH, the low proton availability for MB redox processes (see earlier discussion) has a retardation effect on AA and UA oxidations with increasing pH. The fact that (34) Andrieux, C. P.; Saveant, J. M. J. Electroanal. Chem. 1978, 93, 163. (35) Wang, J.; Wu, Z.; Tang, J.; Teng, R.; Wang, E. Electroanalysis 2001, 13, 1093. (36) Han, X.; Tang, J.; Wang, J.; Wang, E. Electrochim. Acta 2001, 46, 3367. (37) Dryhurst, G. In Comprehensive Treatise of Electrochemistry; Srinivasan, S., Chizmadshev, Y. A., Bockris, J. O., Conway, B. E., Yeager, E., Eds.; Plenum Press: New York, 1985; Vol. 10, Bioelectrochemistry, pp 131-188.

the commencement points of the sharp current decreases (Figure 7a)for both AA and UA occurred at pH 6.50 points to a factor common to both AA and UA, that is, the MB-sol-gel film electrocatalytic system. The causes of the Ep,a variations with pH in Figure 7b are uncertain. Although we expect the peaks to shift to less positive values with increasing pH as shown, we have no explanation for the relationship between peak potential and pH. For example, for AA, two linear sections are obtained (Figure 7b,i) with equations Ep,a (V) ) -0.058pH +0.062 (r ) 0.998) at lower pH and Ep,a (V) ) -0.029pH + 0.435 (r ) 1.000) at higher pH. The transition between these two linear sections occurred at pH 5.50, the same as for the two sets of redox processes observed for MB. It seems that the variations of peak potentials with pH for AA oxidation is controlled by the variations of MB oxidations with pH in the sol-gel film. For UA, a single linear region is observed with Ep,a ) -0.036pH + 0.716 (r ) 0.999) (Figure 7b,ii) for the whole pH range, in contrast to AA. The oxidations of AA and UA could be explained by the MBsol-gel ceramic film’s functioning either as an electron-transfer mediator or as a true electrocatalyst. For the former, we expect the oxidation potentials of AA and UA to track those of the redox couples of the immobilized MB. The data for MB, AA, and UA, as given above under our conditions, and the differences in potentials between AA and UA, which resulted in their simultaneous determinations (see below), favor an electrocatalytic mechanism. Therefore, in the absence of any other data, we invoke an electrocatalytic mechanism. The role of the MTMS-sol-gel ceramic film is uncertain. Our experiments indicate that the film in the absence of MB did give rise to some catalytic effect on AA and UA (not as strong as with MB), but there was a time delay, presumably for the analytes to reach the electrode surface. However, the presence of MB in the film resulted in instantaneous electrocatalytic oxidations of AA and UA. This is another feature that is difficult to resolve at this point. Nevetheless, the rapid electron transfers to AA and UA add to the efficacy of the electrocatalytic system. Analytical Characteristics and Simultaneous Determination of AA and UA at the MB-SGGCE. For analysis, differential pulse voltammetry (DPV) is superior to CV, and for this reason, we chose DPV as the main technique for analytical studies of AA and UA at MB-SGGCE. The analytical characteristics are summarized in Table 2. pH 1.50 and 6.50 were chosen for these studies because they gave good sensitivities as well as separations of the DPVs for AA and UA (see below). Figure 8b,c depicts the DPVs for AA and UA, respectively, at their determination limits. The determination limits found here are, as far as we are aware, among the lowest reported in the literature. From Table 2, it is also observed that the precisions at the determination limits are good, ranging from 1.94 to 2.35%. In addition, linear ranges were respectable, typically 4-4.5 orders of magnitude. Above the upper limit of the linear region, the calibration plots gave straight lines with much gentler slopes. The electroanalysis of AA and UA in real samples (e.g., urine,38 body fluids39) gives rise to mutual interferences because of overlapping oxidation peaks. Because of this, simultaneous de(38) Zen, J. M.; Jou, J. J.; Ilangovan, G. Analyst 1998, 123, 1345. (39) Xiao, L.; Chen, J.; Cha, C. S. J. Electroanal. Chem. 2000, 495, 27.

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Table 2. Analytical Data for AA and UA at the MB-SGGCEa AA

determination limit, nM RSD (%)b linear range regression eq detection limit, nMe

UA

pH 1.50

pH 6.50

pH 1.50

pH 6.50

5.00 2.07c 5.00 nM-0.10 mM ip,a (µA) ) 4.214C (µM) + 0.0057 (r ) 0.9995, 19 points) 0.45

5.00 1.94c 5.00 nM-0.10 mM ip,a (µA) ) 4.519C (µM) + 0.0067 (r ) 0.9996, 19 points) 0.37

1.00 2.35d 1.00 nM-0.010 mM ip,a (µA) ) 8.308C (µM) + 0.0020 (r ) 0.9999, 19 points) 0.20

1.00 2.34d 1.00 nM-0.050 mM ip,a (µA) ) 6.137C (µM) + 0.0017 (r ) 0.9998, 19 points) 0.25

a The MB-SGGC was prepared from the sol containing 10.00 mM MB. b rsd ) relative standard deviation; based on 6 consecutive measurements. Estimated based on 5.00 nM solution in 1.00 M phosphate buffer. d Estimated based on 1.00 nM solution in 1.00 M phosphate buffer. e S/N ) 3, estimated using the standard deviations at the determination limits and slopes of the calibration plots.

c

Table 3. Simultaneous Determination of AA and UA in Urinea AA

UA

pH 1.50

human urine a

pH 6.50

pH 1.50

pH 6.50

spiked (µM)

found (µM)

recovery (%)

spiked (µM)

found (µM)

recovery (%)

spiked (µM)

found (µM)

recovery (%)

spiked (µM)

found (µM)

recovery (%)

0 0.100

0.107 0.102

102

0 1.00

0.115 0.102

102

0 0.200

0.233 0.209

1.04

0 0.200

0.226 0.211

106

MB-SGGC fabricated from sol containing 10.00 mM MB.

Figure 8. DPVs for AA and UA at the MB-SGGCE in (a) 1.00 M phosphate buffer, pH 1.50 phosphate buffer background (b) 5.00 nM AA (c) 1.00 nM UA. Scan rate, 10 mV s-1; pulse amplitude, 50 mV.

termination requires a separation step40,41 or electrocatalytic surfaces that allow sufficient resolution of the oxidation peaks.42,43 In some cases, chemically modified electrodes have been used for the selective determination of either AA or UA alone in the presence of the other.36,38 Here, because of the electrocatalytic effect of MB in the MB-SGGCE and the strong dependence of the anodic peaks of AA and UA on pH, a good opportunity arises that allows the simultaneous determination of AA and UA. This is further aided by the high catalytic efficiency of the MB-SGGCE, which afforded excellent determination limits for the two analytes. The DPVs for a mixture of AA and UA at pH 1.50 and 6.50 (1.00 M phosphate buffer) are shown in Figure 9a and b, respectively. On the basis of principles borrowed from chromatography, for Gaussian peaks, adjacent peaks have baseline separation for an estimated resolution, Rs of 1.6, whereas for Rs < 0.75, >50% overlap would result.44 The Rs values in Figure 9a,b (40) Cheng, M. L.; Liu, T. Z.; Lu, F. J.; Chiu, D. T. Y. Clin. Biochem. 1999, 32, 473. (41) Wang, J.; Chatrathi, M. P.; Tian, B.; Polsky, R. Anal. Chem. 2000, 72, 2514. (42) Zhang, L.; Lin, X. Analyst 2001, 126, 367. (43) Strochkova, E. M.; Tur’yan, Ya. I.; Kuselman, I.; Shenhar, A. Talanta 1997, 44, 1923.

5740 Analytical Chemistry, Vol. 74, No. 22, November 15, 2002

Figure 9. DPVs for a mixture of AA and UA (each 1.00 × 10-7 M in 1.00 M phosphate buffer) at the MB-SGGCE (a) pH 1.50 (i) phosphate buffer background (ii) AA + UA; (b) pH 6.50 (i) phosphate buffer background (ii) AA + UA. Scan rate, 10 mV s-1; pulse amplitude, 50 mV.

are 0.9 and 1.2, respectively. Although both pHs gave sufficient separation for analysis, pH 1.50 would be preferred for better accuracy. In addition, peak sensitivities are slightly better at this pH. However, other factors may force one to choose pH 6.50. For example, note that at pH 1.50, both AA and UA peaks were pushed ∼200 mV more positively, as compared to pH 6.50; i.e., the electrocatalytic effect was not as strong at pH 1.50. The MB-SGGCE was utilized for the simultaneous determination of AA and UA in a human urine sample. No sample (44) Wang, J.; Kuo, D. B.; Freiha, B. Talanta 1986, 33, 397.

pretreatment was employed except for filtration and dilution (by a factor of 10 000). For comparison, the analysis was performed at both pHs 1.50 and 6.50 using the standard addition method. The results are summarized in Table 3. As can be seen, the results for the two pH values are comparable and in all cases, recoveries were good. Using the results at pH 1.50, the concentration of AA and UA in the original urine sample were 1.07 mM and 2.33 mM, respectively. MB-SGGCE Stability. Electrode stability is an important consideration in the development of an electrochemical sensor. For the present case, MB-SGGCE fabricated from sol containing 10.00 mM MB typically were stable for 1 month or more. As an indicator of its stability, we tracked the performance of a particular MB-SGGCE over a period of 1 month with measurements of the oxidation peak current for 1.00 mM AA in 1.00 M phosphate buffer, pH 6.50, every 2-5 days. A total of 13 measurements of current were taken, which gave a relative standard deviation of 1.65%.

CONCLUSION In this work, we have demonstrated that the MB-SGGCE is a good electrocatalytic system for the oxidation of biomolecules, such as ascorbic and uric acid. It is a fast system with apparently high catalytic efficiency leading to very low determination limits (experimental) in the region of nanomolar concentrations. The pH dependence of the oxidations of AA and UA can be exploited for their simultaneous determination in a real sample, such as human urine, with little sample pretreatment. The stability of the MB-SGGCE of 1 month or longer further adds to its efficacy as a biosensor. ACKNOWLEDGMENT This work was supported by a grant from the National University of Singapore. Received for review September 17, 2002.

February

19,

2002.

Accepted

AC0255882

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