Prussian Blue Based Nanoelectrode Arrays for H2O2 Detection

Anal. Chem. 2004, 76, 474-478

Prussian Blue Based Nanoelectrode Arrays for H2O2 Detection Arkady A. Karyakin,* Elena A. Puganova, Igor A. Budashov, Ilya N. Kurochkin, Elena E. Karyakina, Vladimir A. Levchenko, Vladimir N. Matveyenko, and Sergey D. Varfolomeyev

Faculty of Chemistry, M. V. Lomonosov Moscow State University, 119992, Moscow, Russia

We propose to form nanoelectrode arrays by deposition of the electrocatalyst through lyotropic liquid crystalline templates onto inert electrode support. Whereas Prussian Blue is known to be a superior electrocatalyst in hydrogen peroxide reduction, carbon materials used as electrode support demonstrate only a minor activity. We report on the possibility for nanostructuring of Prussian Blue by its electrochemical deposition through lyotropic liquid crystalline templates, which is noticed from atomic force microscopy images of the resulting surfaces. The resulting Prussian Blue based nanoelectrode arrays in flow injection analysis mode demonstrate a sub-part-per-billion detection limit (1 × 10-8 M) and a linear calibration range starting exactly from the detection limit and extending over 6 orders of magnitude of H2O2 concentrations (1 × 10-8 to 1 × 10-2 M), which are the most advantageous analytical performances in hydrogen peroxide electroanalysis. Monitoring of low levels of hydrogen peroxide is of great importance for modern medicine, environmental control, and various branches of industry. H2O2 is a chemical threat agent; its excessive concentration as a product of industry and atomic power stations affects the environment.1,2 At the other hand, H2O2 is the most valuable marker for oxidative stress, recognized as one of the major risk factors in progression of disease-related pathophysiological complications in diabetes, atherosclerosis, renal disease, cancer, aging, and other conditions.3-7 Recently, we reported on Prussian Blue as the most advantageous hydrogen peroxide transducer.8-10 Compared to the most widely used platinum, Prussian Blue modified electrodes are 3 orders of magnitude more active in H2O2 reduction and oxidation in neutral media and 3 orders of magnitude more selective for (1) Wang, Y.; Huang, J.; Zhang, C.; Wei, J.; Zhou, X. Electroanalysis 1998, 10, 776-778. (2) Nowall, W. B.; Kuhr, W. G. Electroanalysis 1997, 9, 102-109. (3) MacCarthy, P. A.; Shah, A. M. Coron. Artery Dis. 2003, 14, 109-113. (4) Rodrigo, R.; Rivera, G. Free Radical Biol. Med. 2002, 33, 409-422. (5) Sohal, R. S.; Mockett, R. J.; Orr, W. C. Free Radical Biol. Med. 2002, 33, 575-586. (6) Yang, T. T. C.; Devaraj, S.; Jialal, I. J. Clin. Ligand Assay 2001, 24, 13-24. (7) Yorek, M. A. Free Radical Res. 2003, 37, 471-480. (8) Karyakin, A. A.; Gitelmacher, O. V.; Karyakina, E. E. Anal. Chem. 1995, 67, 2419-2423. (9) Karyakin, A. A.; Karyakina, E. E. Sens. Actuators, B 1999, B57, 268-273. (10) Karyakin, A. A.; Karyakina, E. E.; Gorton, L. Anal. Chem. 2000, 72, 17201723.

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hydrogen peroxide reduction in the presence of oxygen.11 The latter allows low-potential H2O2 detection with advantageous analytical characteristics: (i) the sensitivity of 1 A M-1 cm-2, which is at the upper limiting sensitivity level,12 (ii) the detection limit of 10-7 M in flow injection analysis (FIA) mode, and (iii) linear calibration range starting from the detection limit and extending over more than 3 orders of magnitude of hydrogen peroxide concentration.9-11 The attractive performance characteristics of the electrochemically deposited Prussian Blue allowed it to be denoted as artificial enzyme peroxidase.9,10 Low-potential detection of hydrogen peroxide is also possible with peroxidase enzyme modified electrodes.12-15 However, despite the low detection limits achieved, peroxidase electrodes demonstrate saturation with the substrate, which affects linear calibration range. In addition, peroxidase electrodes are usually less selective relative to oxygen. Moreover, the use of the enzyme obviously deteriorates transducer properties because of its instability, high cost, etc. Despite the possibility to detect hydrogen peroxide down to 10-7 M, both clinical diagnostics and environmental control in certain cases require monitoring of lower H2O2 levels. The decreased detection limit is possible with the use of microelectrodes instead of conventional ones. The profile of substrate diffusion to a microelectrode surface is semispheric. For such diffusion type the inversed current density has linear dependence on the electrode radius; the slope is positive and is inversely proportional to substrate concentration.16-18 This leads to an improved signal-to-noise ratio for low analyte concentrations and, as a result, to a decreased detection limit. However, single microelectrodes generate low currents, which are hardly detectable with the conventional electrochemical technique. For this aim, different microelectrode arrays with quite similar analytical characteristics have been elaborated.19,20 The most common procedure to produce microelectrode arrays involves photolytography and/or electronic beam techniques. (11) Karyakin, A. A. Electroanalysis 2001, 13, 813-9. (12) Ruzgas, T.; Cso¨regi, E.; Emne´us, J.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1996, 330, 123-138. (13) Lindgren, A.; Ruzgas, T.; Gorton, L.; Csoregi, E.; Ardila, G. B.; Sakharov, I. Y.; Gazaryan, I. G. Biosens. Bioelectron. 2000, 15, 491-497. (14) Ferapontova, E. E.; Grigorenko, V. G.; Egorov, A. M.; Borchers, T.; Ruzgas, T.; Gorton, L. Biosens. Bioelectron. 2001, 16, 147-157. (15) Kenausis, G.; Chen, Q.; Heller, A. Anal. Chem. 1997, 69, 1054-1060. (16) Dong, S.; Che, G. J. Electroanal. Chem. 1991, 309, 103-14. (17) Lyons, M. E. G.; Bannon, T.; Rebouillat, S. Analyst 1998, 123, 1961-6. (18) Mori, V.; Bertotti, M. Talanta 1998, 47, 651-8. (19) Feeney, R.; Kounaves, S. P. Electroanalysis 2000, 12, 677-684. (20) Fiaccabrino, G. C.; Koudelka-Hep, M. Electroanalysis 1998, 10, 217-222. 10.1021/ac034859l CCC: $27.50

© 2004 American Chemical Society Published on Web 12/09/2003

However, except for their cost and complexity, they are not suitable for structuring of needle-type electrode supports with the working lateral surface as well as for microelectrodes. Recently, an alternative way for material nanostructuring has been proposed.21 Deposition of different metals (Pt, Pd, Co, Sn) through lyotropic liquid crystalline phases resulted in mesoporous nanostructured surfaces.22-24 Nanostructuring of platinum microelectrodes prolonged calibration range of the corresponding sensor to decimolar analyte concentrations.25 We propose to use nanostructuring to improve analytical performances of the hydrogen peroxide sensors, and, in particular, to decrease its lower detection limit. We believed that the electrocatalyst nanostructured on inert electrode support would behave as a random nanoelectrode array concerning the detection of the corresponding analyte. As electrocatalyst, Prussian Blue deposited on glassy carbon has been chosen. Whereas the former serves as a superior electrocatalyst in hydrogen peroxide reduction (see above), the latter demonstrates only a minor activity. In the present article we report on the possibility for nanostructuring of Prussian Blue by its electrochemical deposition through colloid templates. Compared to the conventional electrodeposited Prussian Blue, the resulting nanostructured electrodes demonstrate an order of magnitude decreased detection limit (10-8 M). The linear calibration range of the Prussian Blue based nanoelectrode arrays in FIA mode starting exactly from the sub-part-per-billion detection limit and extending over 6 orders of magnitude of H2O2 concentrations (10-8-10-2 M), demonstrates the most advantageous analytical performance in hydrogen peroxide electroanalysis. EXPERIMENTAL SECTION Materials. Experiments were carried out with MilliQ water from a Millipore MilliQ system. All inorganic salts and hydrogen peroxide (30% solution) were obtained at the highest purity from Reachim (Moscow, Russia) and used as received. Brij-56 (polyoxyethylen (n ) 10) cetyl ether), Tween-60 (polyoxyethylen (n ) 20) sorbitan monostearate), and AOT (2-ethylhexylsodium sulfoxinate) were purchased from Sigma-Aldrich (Steinheim, Germany). Instrumentation. Electrochemical experiments were made in a three-compartment electrochemical cell containing a platinum net auxiliary electrode and a Ag|AgCl reference electrode in 1 M KCl. The cell construction allowed deaeration of the working electrode space. Glassy carbon disk electrodes (2 mm in diameter) were used as working electrodes. Prior to use, the glassy carbon electrodes were mechanically polished with alumina powder (Al2O3, 0.03 µm) until a mirror finish was observed. The flow injection system consisted of a Cole Parmer (Vernon Hills, IL) peristaltic pump (7519-10), homemade flow-through walljet cell with 0.5-mm nozzle positioned 1-2 mm from the surface (21) Attard, G. S.; Bartlett, P. N.; Coleman, N. R. B.; Elliott, J. M.; Owen, J. R.; Wang, J. H. Science 1997, 278, 838-40. (22) Attard, G. S.; Bartlett, P. N.; Coleman, N. R. B.; Elliott, J. M.; Owen, J. R. Langmuir 1998, 14, 7340-2. (23) Bartlett, P. N.; Birkin, P. N.; Ghanem, M. A.; de Groot, P.; Sawickib, M. J. Electrochem. Soc. 2001, 148, C119-C123. (24) Whitehead, A. H.; Elliott, J. M.; Owen, J. R.; Attard, G. S. Chem. Commun. 1999, 331-2. (25) Evans, S. A. G.; Elliott, J. M.; Andrews, L. M.; Bartlett, P. N.; Doyle, P. J.; Denuault, G. Anal. Chem. 2002, 74, 1322-6.

of the disk electrode, (Ag|AgCl|1 M KCl) reference, homemade injector, and Metrohm potentiostat (641-VA) or Solartron electrochemical interface (model 1286) interfaced to an IBM PC. Flow rates used were in the range 0.5-1 mL min-1. In FIA experiments, the peak current values were taken for data treatment, sample volume was 50 µL, and working electrode potentials were 0.000.05 V, allowing hydrogen peroxide reduction on Prussian Blue modified electrodes. Concentration of hydrogen peroxide in stock solutions was controlled by optical density at 230 nm with an LKB-Ultraspec UII spectrophotometer (Broma, Sweden). Atomic force microscopy (AFM) images were obtained in contact regime on a scanning sonde microscope, Solver P47 (NTMDT, Moscow, Zelenograd). Silicic cantilever with constant of resilience 0.03 N/m (Ultrasharp CSG, NT-MDT was used as a sonde. To control the hexagonal structure of the lyotropic liquid crystalline layers, both an Axiolab Pol polarizing microscope by Zeiss (Germany) and a transmission electron microscopy (TEM) technique using a JEM-100 electron microscope in the microdiffraction mode were used. The image was displayed on a monitor via a videocamera. The texture hexagonal LLC dimensions were determined with the aid of a Linkam VTO 232 adapter. Hexagonal liquid crystalline phase was deposited onto glass. Electrodeposition of Prussian Blue was made in cyclic voltammetric conditions with switching potentials of 0.3-0.4 V (cathodic) and of 0.7-0.8 V (anodic) at a sweep rate of 40 mV s-1 or by applying a constant potential of 0.4 V as described elsewhere.10 Growing solution contained 4 mM K3[Fe(CN)6] and 4 mM FeCl3. A solution of 0.1 M HCl and 0.1 M KCl was used as supporting electrolyte. After deposition, Prussian Blue films were electrochemically activated in the same supporting electrolyte by cycling in the range -0.05 to 0.35 V at a rate of 40 mV s-1 until a stable voltammogram was obtained. Then the electrodes were heated at 100 °C for 1 h. Total electrode coverage with Prussian Blue was of 6-8 nmol cm-2. Hexagonal lyotropic liquid crystals from nonionic surfactants Brij-56 or Tween-60 were prepared by mixing with the supporting electrolyte solution in the ratios 56:44 and 63:37 wt %, respectively.23 These mixtures were equilibrated at room temperature at least for 2 h and then were deposited onto the electrode surface (3.5-4.5 mg cm-2, layer thickness was 40-50 µm). A gentle heating was applied to melt the surfactant phase in order to achieve a homogeneous layer. Hexagonal liquid crystal phase on the basis of anionic surfactant AOT was prepared by adding water to the AOT-octane mixture to a final content:26 15 wt % AOT, 52 wt % octane, and 33% of aqueous solution of 0.1 M HCl and 0.1 M KCl. The mixture was equilibrated for 3 h. Afterward it was deposited onto the electrode surface (4.5-5.5 mg cm-2 to the final layer thickness of 40-60 µm). Synthesis of nanostructure Prussian Blue was made by its electrochemical deposition (as described above) through a hexagonal liquid crystal template. The total time for electrochemical deposition was did not exceed 4 min in order to preserve the liquid crystalline structure upon exposing to the growing solution. After deposition, modified electrodes were electrochemically (26) Tamamushi, B.; Watanabe, N. Colloid Polym. Sci. 1980, 258, 174-178.

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activated in supporting electrolyte and washed with ethanol and distilled water to remove the surfactant. Second activation was made to check the remaining electroactivity. Final electrode coverage with Prussian Blue was of 0.6-1.6 nmol cm-2. RESULTS AND DISCUSSION Liquid Crystalline Structure of the Surfactant Layer. The templating mixtures used in our experiments were the ternary system for an anionic surfactant consisting of AOT (2-ethylhexylsodium sulfoxinate), octane, and aqueous supporting electrolyte (0.1 M KCl and 0.1 M HCl), and the two binary systems for a nonionic surfactants consisting of either Brij-56 (polyoxyethylen cetyl ether) or Tween-60 (polyoxyethylen sorbitan monostearate), and similar aqueous supporting electrolyte. Hexagonal lyotropic liquid crystalline phases of all surfactants were prepared in accordance with the phase diagrams known to yield hexagonal liquid crystalline structure. However, it was independently investigated whether the resulting films are indeed of hexagonal structure because of supporting electrolyte and, which is the most improtant, whether the hexagonal phase is remained upon exposing the templates to growing solution. The anisotropy of lyotropic layers was investigated by both polarizing microscopy and transmission electron microscopy. Figure 1 displays the corresponding images obtained for lyotropic liquid crystalline phase of Brij-56 obtained in the presence of supporting electrolyte rather than of pure water. As seen from the polarizing microscopy image, the texture corresponds to the hexagonal phase. An independent confirmation concerning formation of hexagonal phase was obtained using transmission electron microscopy. As seen (Figure 1b), the reflexes correspond to the hexagonal structure of lyotropic liquid crystal. Similarly, a formation of hexagonal lyotropic liquid crystalline phase was confirmed, when Tween-60 and AOT were used as surfactants. It was important to check whether the obtained liquid crystalline structures remained upon exposure to the growing solution during deposition of Prussian Blue. Since exposure time was rather short (less than 4 min), we expected no dramatic changes in liquid crystalline phase, especially taking into account that these phases are solid. Indeed, as was shown for all surfactants, even 10 min of exposure to the growing solution causes only a minor changes in anisotropy of templates noticed from polarized light microscopy images. Thus, liquid crystalline templates preserve their hexagonal structure during deposition of the electrocatalyst. Nanostructuring of Prussian Blue. Among the known methods for nanostructuring of the electrodeposited material, we have chosen the electrochemical synthesis through lyotropic liquid crystalline hexagonal phases. The advantage of conducting the synthesis in a single phase system is that it is possible to exploit the rich lyotropic polymorphism exhibited by surfactants. This provides nanostructures of the deposited materials, which are in effect casts of the hexagonal structures of the liquid crystalline phases in which they were formed. The morphology of platinum deposited through lyotropic liquid crystalline phases contains in large scale separate islands of submicrometer dimension.27 The latter gives promise to form separate (27) Elliott, J. M.; Attard, G. S.; Bartlett, P. N.; Coleman, N. R. B.; Merckel, D. A. S.; Owen, J. R. Chem. Mater. 1999, 11, 3602-9.

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Figure 1. Polarizing microscopy image (a) and electron diffraction pattern (b) of a hexagonal lyotropic liquid crystalline texture Brij-56 on the glass surface.

islands of electrocatalyst onto inert surfaces, which would result in a nanoelectrode array. The templating mixtures used were the ternary system for an anionic surfactant AOT, octane, and the two binary systems for a nonionic surfactants Brij-56 and Tween-60. Electrodeposition of Prussian Blue onto the polished glassy carbon through liquid crystalline plating mixtures was carried out similarly to its growing onto blank electrodes. Compared to the pure glassy carbon, the deposition rate through the liquid crystalline template was 5-10 times lower, and the total amount of the deposited inorganic polycrystal was also 5-10 times less. After deposition through liquid crystalline templates, the electrodes were cycled in supporting electrolyte solution. The recorded cyclic voltammograms has shown broad peaks of Prussian Blue|Prussian White redox activity with peak separation of 70 mV and half peak width of >100 mV. This is dramatically higher compared to the conventional Prussian Blue displaying a set of peaks with separation of 15-25 mV.11 It was supposed that electroactivity of Prussian Blue grown through colloid templates is suppressed by the surfactant remaining at the electrode surface. Indeed, after thorough washing with ethanol and water, the cyclic voltammograms have shown an improved redox activity with peak separation of 22 mV.

Figure 3. Calibration plot for hydrogen peroxide detection in flow injection mode with nanostructured Prussian Blue as a detector: Prussian Blue electrodeposited through Brij-56 liquid crystalline template; operating potential, 50 mV; flow rate, 0.7 mL min-1.

Figure 2. AFM images of Prussian Blue modified monocrystalline graphite: (a) conventional Prussian Blue deposited without surfactants; (b) Prussian Blue electrochemically deposited through liquid crystalline phase of nonionic surfactant Brij-56; (c) Prussian Blue grown through liquid crystalline phase of anionic surfactant AOT.

The morphology of Prussian Blue electrodeposited onto a monocrystalline graphite surface was investigated by AFM. Figure 2a presents an AFM image of Prussian Blue deposited without templates. As seen, inorganic film is of polycrystalline structure; however, the layer covers the surface completely. A conventionally deposited Prussian Blue covers the electrode surface uniformly, and the AFM image as in Figure 1a can be taken all over the film. When Prussian Blue has been growing through the liquid crystalline template of nonionic surfactant, the corresponding AFM image (Figure 2b) displays an archipelago of new structures, which may be attributed only to ferric hexacyanoferrate. These structures of sub-micrometer dimensions are surrounded by very smooth areas, which, taking into account polycrystalline Prussian Blue morphology (Figure 2a), obviously display an unmodified graphite surface. Electrodeposition of Prussian Blue through hexagonal liquid crystals formed by anionic surfactant AOT resulted in quite similar AFM images (Figure 2c): islands of submicrometer dimension surrounded with blank surface. It is important that deposition of Prussian Blue onto glassy carbon through lyotropic liquid crystalline templates resulted in nonuniform layers of the electrocatalyst throughout the surface. This is noticed from AFM images similar to those shown in parts

b and c of Figure 2 taken at different positions of the sonde. Moreover, the density and morphology of Prussian Blue structures in the case of the films deposited through AOT are similar in different surface areas. As mentioned, Prussian Blue is a superior electrocatalyst of hydrogen peroxide reduction, whereas graphite materials possess in this reaction only a minor activity. Hence, the resulting nanostructured Prussian Blue on carbon (parts b and c of Figure 2) can be considered as a nanoelectrode array in relation to H2O2 electrochemical reduction. Analytical Characteristics of Nanostructured Prussian Blue. Analytical performances of Prussian Blue modified electrodes in hydrogen peroxide detection were investigated in a flow injection system equipped with wall-jet cell. The working electrode potential was 50 mV rather than 0 mV used in our previous study.9,10 An increased electrode potential was chosen to decrease the background oxygen reduction; the corresponding cathodic current is decreased 2-2.5 times. At the same time, the response to hydrogen peroxide remains almost unchanged within 3 orders of magnitude of H2O2 concentrations. The calibration plot for hydrogen peroxide detection with the conventional Prussian Blue modified electrodes in FIA mode is linear at least over 4 orders of magnitude of H2O2 concentrations. The lower limit of the linear calibration range referred to as the detection limit is of 10-7 M. At lower H2O2 concentrations either a significant deviation form the linearity appears or a response to analyte injection becomes hardly recognizable. Nanostructured Prussian Blue modified electrodes demonstrate a significantly decreased background, which resulted in improved signal-to-noise ratio. Compared to the uniformly covered electrodes, this parameter is decreased approximately five times. Nanostructuring also resulted in a decreased detection limit. The latter chosen as the lower limit of the linear calibration range is of 10-8 M (Figure 3). Since among the main disadvantages of the enzyme peroxidase based H2O2 sensors is the saturation of the enzyme with the substrate affected linear calibration range, and as a result the latter never reaches 4 orders of magnitude of H2O2 concentration, we decided to investigate the upper detection limit in the case of Analytical Chemistry, Vol. 76, No. 2, January 15, 2004

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nanostructured Prussian Blue. Figure 3 presents a typical calibration graph for hydrogen peroxide detection in FIA mode. It is seen that the linear calibration range is extended over 6 orders of magnitude of H2O2 concentration (10-8-10-2 M). The significant deviations from the linearity are observed only at decimolar (0.1 M) hydrogen peroxide content. The sensitivity of the nanostructured Prussian Blue modified electrode was approximately 0.06 A M-1 cm-2. The observed linear calibration range (Figure 3) is extended at least 2 orders of magnitude as compared with the known hydrogen peroxide sensors. Moreover, the detection limit of 10 nM (10-8 M) was declared only for gene-engineered peroxidase electrode under continuous flow (more sensitive mode compared to FIA),14 where as successful detection of centi- and decimolar H2O2 is the property of the recently reported mesoporous platinum microelectrode.25 Thus, nanostructured Prussian Blue accumulates the properties of the best H2O2 sensors concerning both high and low concentration diapasons. CONCLUSION We conclude that nanostructuring gradually improves analytical performances of the corresponding sensors compared to the

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uniformly covered electrodes. Nanostructuring of electrocatalyst is possible by its deposition through lyotropic liquid crystalline templates. Prussian Blue based nanoelectrode arrays in FIA mode demonstrate a sub-part-per-billion detection limit (1 × 10-8 M) and a linear calibration range starting exactly from the detection limit and extending over 6 orders of magnitude of H2O2 concentrations (1 × 10-8-1 × 10-2 M). The observed linear calibration range is extended at least 2 orders of magnitude as compared with the known hydrogen peroxide sensors. ACKNOWLEDGMENT We thank Professor Pankaj Vadgama (Queen Mary, University of London, U.K.) and Professor Philip Bartlett (Southampton University, U.K.) for kind help and fruitful discussions. Financial support through a NATO Linkage grant and an INTAS grant (00273) is greatly acknowledged.

Received for review July 28, 2003. Accepted October 22, 2003. AC034859L