J. Phys. Chem. 1995,99, 11896- 11900
11896
Local Detection of Photoelectrochemically Produced H202 with a “Wired” Horseradish Peroxidase Microsensor Hideki Sakai, Ryo Baba, Kazuhito Hashimoto, and Akira Fujishima* Department of Applied Chemistry, Faculty of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
Adam Heller Department of Chemical Engineering, The University of Texas at Austin Austin, Texas 78712-1062 Received: November 2, 1994; In Final Form: April 21, 1 9 9 9
A hydrogen peroxide (H202) micro sensor has been prepared on a 7 pm carbon fiber microdisk, using horseradish peroxidase (HRP) immobilized in a poly(4-vinylpyridine) based redox hydrogel having [O~(bpy)2Cl]~+’~+ redox centers. Because of radial diffusion of the electrons through the hydrogel, the sensor’s steady state current density for H202 electroreduction was 0.17 mA cm-2 at 1.0 x M H202, more than 10 times that of a 3 mm macroelectrode. With the microsensor positioned 50 pm from the surface, H202 production by photogenerated hole oxidation of water and photogenerated electron reduction of molecular oxygen was monitored independently on a partially Ti02 coated IT0 (indium tin oxide) glass. H202 was produced primarily by photoelectroreduction of dissolved oxygen on the IT0 glass, with a lesser amount of H202being produced by photoelectrooxidation of water on the Ti02 film.
Introduction Photocatalytic reactions on Ti02 particles and films with cocatalysts, such as Pt, Pd, or Au, were extensively studied in the past decade.‘-6 Islands of the cocatalyst act as reduction sites, increasing the efficiency of reduction of dissolved 0 2 by the photogenerated electrons. Photoelectrochemical cleavage of water is significantly accelerated on Pt-loaded Ti02 relative to Ti02 without the cocatalyst.’ Wang et ala6reported that modification of the Ti02 surface by palladium increases the efficiency of electron transfer to oxygen, thereby reducing the steady state surface concentration of electrons and thus the recombination rate of holes, increasing the efficiency of photoassisted oxidation of organic compounds. In the previous studies, however, only the sum of the chemistries at the oxidation (TiO2) and reduction (metal) sites was detected. This has left unanswered questions about the mechanism by which the cocatalysts act, e.g., whether they catalyze predominantly oxidation or reduction reactions. Recently we reported that the reaction products formed at the oxidation and reduction sites of Ti02 photocatalyst films can be monitored independently by microelectrode^.^^^ We coated polycrystalline Ti02 onto half of a transparent IT0 (indium tin oxide) electrode and monitored the products on each of the solution-exposed films using a carbon microelectrode positioned 50 pm from the Ti02 or the I T 0 surface. Hydrogen peroxide (H202) formation in Ti02 photocatalyzed reactions has been studied e ~ l i e r . ~ -In I ~an oxygenated Ti02 aqueous suspension H202 can be formed either at the oxidation or at reduction sites or at both, through reactions 1 and/or 2,
-
+ 2h’ H202+ 2H’ 0, + 2H’ + 2e- - H,02
2H20
(1)
(2)
h+ and e- representing, respectively, the photogenerated hole and electron.
* To whom correspondence should be addressed. E-mail address:
[email protected]. Abstract published in Advance ACS Abstracts, July 15, 1995. @
The aim of the present study was to monitor the relative H202 production rates at the oxidation and the reduction sites of the Ti02-IT0 film independently. Some analytical schemes exist to detect H202. These techniques can monitor the sum of the chemistries at the oxidation and reduction sites; however, they cannot monitor the reaction products on each site separately. Moreover, when optical methods like fluorometry are applied for this system, the photogenerated holes created on the irradiated Ti02 surface may oxidatively decompose the indicators. In the present study, for such local monitoring of H202, produced with the photocatalytic reactions, we fabricated a 7 pm diameter H202 microsensor by immobilizing horseradish peroxidase in an electron conducting redox hydrogel on the surface of a carbon fiber microelectrode.I6 With the electrode positioned 50 pm from the surface, H202 formation on the Ti02 photoanode and on the I T 0 cathode was monitored independently. It was found that H202 was generated much more rapidly by the cathode reaction than it was by the reaction on the photoanode.
Experimental Section Chemicals. Horseradish peroxidase (HRP) (Type VI, 260 unitdmg) was purchased from Sigma. Poly(ethy1ene glycol 600 diglycidyl ether), (PEGDGE) and titanium oxyacetylacetonate were purchased from Polysciences and Tokyo Kasei, respectively. All experiments were performed in phosphate-buffered saline (PBS, pH 7.4) purchased from Sigma. The poly[(vinylpyridine)Os(bipyridine)2Cl] derivative-based redox hydrogel (Polymer I, shown in Figure la) was synthesized as rep01ted.I~ Horseradish peroxidase was electrically “wired” to the electrode surface via the redox hydrogel;I8Le., electrons diffused from the carbon electrode to the enzyme reaction centers through the electron conducting enzyme hydr0ge1.I~ Microelectrode Fabrication. The procedure for beveled carbon-fiber microdisk electrode fabrication was similar to that previously described.20 A 7 pm diameter carbon fiber (Asahi Nippon Carbon Fiber Co.) was inserted into a 2 mm 0.d. glass tube. The tube was pulled using a capillary puller (Narishige,
0022-36S4/95/2099-11896$09.00/0 0 1995 American Chemical Society
J. Phys. Chem., Vol. 99, No. 31, I995 11897
Local Detection of Photoelectrochemically Produced H202
Side view
I
I
j
Glass Substrate
IT
102
TODview
(bPY)zCI
CHz
I I
CHz m = I , n = 3.35, o = 0.6
II -1 I Potentiostat
NH2
Low viscosity
Micro H202 Sensor (WE) Ti02 film IT0 Glass
--
Pyrex glass tube (0.d = 2 mm)
Phospate Buffered Saline (pH = 7.4)
Inverted Microscope
ca. 20~lrni Enzyme ;ontaining Hydrogel
I
Figure 2. Schematic illustration of (a) the Ti02-IT0 structure used and (b) the experimental setup for the photoelectrochemical measurements.
Figure 1. (a) Structure of the redox polymer electrically connecting
t
the horseradish peroxidase (HRP) redox centers to the carbon fiber microtip. (b) Schematic drawing of the H202 microsensor.
PN-3) to form a glass tip of about 30 p m 0.d. The small gap between the glass and the fiber was sealed with a low-viscosity epoxy resin (Polysciences). The tip was then polished with a pipette beveler (Narishige, EG-6) to produce a carbon disk. Electrical contact between the carbon fiber and copper wire was made with mercury. Enzyme Immobilization onto the Microelectrode. The wired HRP H202 sensing layer on the microelectrodewas made by the method used in making 3 mm macroelectrodes.21Thus, HRP (13 mg/mL) and sodium periodate (4 mg/mL) were dissolved in 0.1 M sodium bicarbonate solution and incubated in the dark for 3 h (solution I). The redox polymer solution (10 mg/mL), solution I, and PEGDGE (1 mg/mL) were mixed thoroughly at a volume ratio of 5:1 :6. The enzyme containing hydrogel was formed of 1 pL of the premixed solution on the microelectrode surface at ambient temperature, the gelling being observed with a microscope.19 After the droplet was dried, the residual film was cured in air for 2 days. A schematic drawing of the microsensor is shown in Figure lb. Electrochemical Measurements in Photocatalytic System. The microcrystalline anatase Ti02 film was deposited on about one half of the I T 0 glass by spray pyrolysis of an ethanol solution of titanium oxy acetylacetonate (0.1 M) at 400 "C (Figure 2a).22 The experimental setup for the local monitoring of the photoproduced H202 is shown in Figure 2b. The electrochemicalcell was set on the stage of an inverted optical microscope (Olympus, IMT-2) and the microsensor was positioned 50 pm from the Ti02 or the I T 0 surface using a micromanipulator. The irradiating light source was a 150 W Hg-Xe lamp, the light of which was filtered so as to remove the short wavelength photons that might have excited the ITO. The potential of the fiber sensor was poised at 0.0 V vs SCE, and the increase in the current of the microsensor with time, resulting from the local increase in H202 concentration, was monitored. Phosphate-bufferedsaline (PBS, pH 7.4) was used as the electrolyte solution. A platinum wire and a saturated calomel electrode were used as counter and reference electrodes, respectively. A Hokuto Denko HP150-A potentiostavgal-
400.00
scan rate; 50 mV/s
200.00 --
B E \
Y
0.00--
3
0
-200.00--
-400.OO
t 4 0.00
I 1
1
I
I
-I 0.50
Potentialvs. SCE / V
Figure 3. Current-voltage characteristics of the H202 microsensor in phosphate buffered saline (PBS) pH 7.4 (a) without H202 and (b) with M H202. Scan rate 50 mV/s.
vanostat was used for cyclic voltammetry and for the constantpotential experiments.
Results Characterization of the Microsensor. Figure 3a shows a cyclic voltammogram for the H202 microsensor in PBS in the absence of H202 at a scan rate of 50 mV/s. The observed redox peak is attributed to electron self-exchange between osmium sites (eq 3).'*
os3+ + e-
-
os2+
(3)
The shape of the voltammograms was that expected for a microcarbon electrode operating in a diffusion-limited mode. With the only diffusing and current-limiting species in the system being electrons, diffusing radially through the redox hydrogel, the voltammograms represent the kinetics of electron diffusion and reaction on the electrode. A voltammogram M H202 is shown in obtained in the presence of 1.0 x Figure 3b. An H202 reduction current was observed at potentials starting as high as 0.45 V vs SCE. The current
Sakai et al.
11898 J. Phys. Chem., Vol. 99, No. 31, 1995 loo0
m
(a) Cathodic
above I T 0
Light off
4
4 I
Light on I
0.1
1 10 100 Concentration of HzOz / pM
O5
I
Figure 6. Time evolution of the H202 electroreduction current over Ti02 contacting IT0 and I T 0 surfaces (a) microsensor positioned 50 p m above the I T 0 surface; (b) microsensor positioned 50 pm above the Ti02 surface. 0.0 V vs SCE, PBS.
I
not show any decrease of current output in an aqueous solution containing H202 for at least 10 h. Because the microsensor was used under W light, the effect of W irradiation (unfiltered Hg-Xe lamp) on its sensitivity was evaluated. The drop in the H202 electroreduction current was plotted as a function of time when the microsensor was irradiated in air. The current output was slightly reduced upon W irradiation. After 10 and 30 min irradiation, the current was reduced by 4 and 9%, respectively. In the previous study we have shown that the TiO2-IT0 film acts as an photoelectrochemical cell with Ti02 as the anode and IT0 as the cathode using the microelectrode technique.' Here the H202 microsensor was positioned using a micromanipulator 50 pm from the Ti02 or IT0 surface, and more than 5 mm away from the boundary between the Ti02 coated and the uncoated I T 0 glass. Its potential was poised at 0.0 V vs SCE in the PBS solution. I T 0 itself can be also photoexcited with the light whose energy was greater than 4.0 eV (corresponding to the light with 310 nm), thus the Ti02 exciting UV light of the Hg-Xe lamp was now filtered to prevent excitation of the I T 0 by a W - D 2 filter (Toshiba Glass). The change in the H202 electroreduction current with time was monitored above the Ti02 (oxidation site) and the IT0 (reduction site) surfaces under irradiation. As seen in Figure 6, an increase of the current with time was observed above both the Ti02 and the IT0 surface, even though the sensor's sensitivity was decreased because of the W damage. The calculated diffusion distance of H202 during the irradiation period (5 min) was much shorter (on the order of lo-' mm) than the distance between the measurement sites (-10 mm), wherefore H202 formed on one surface could not have contributed to the current measured at the other. Furthermore, with the sensor being insensitive to variation from the initial pH of 7.4and irradiation not causing an increase of the sensor output (Figure 5), the observed increase in current above the Ti02 and IT0 sites could have been caused only by the HzOz formation via the reactions 1 and 2, respectively, with Ti02 being the oxidizing electrode and IT0 the reducing electrode. On the contrary, when a Ti02 film was coated on the entire IT0 surface and the structure was irradiated, no H202 could be detected (data not shown). Thus, unless the IT0 "back" electrode was discharged by electron transfer to dissolved molecular oxygen, the photogenerated carriers recombined quickly and the H202 formation through both reactions 1 and 2 is negligibly small. As seen in Figure 6, H202 was formed much more efficiently on the I T 0 surface than on the Ti02 surface. This observation showed that in a photoelectrochemical cell, with a Ti02 on I T 0 electrode and an uncoated IT0 electrode, H202 was produced mostly at the reduction site, i.e. by reduction of dissolved oxygen (eq 2), rather than by oxidation of water (eq l), even in the absence of an electron donor. After 5 min of UV irradiation,
' : 1 O
3.0
4.0
5.0
6.0 7.0 8.0 Solution pH
Figure 5. pH dependence of the current,
9.0 10.0
M H202,O.O V vs SCE.
reached its plateau at a potential of 0.3 V (SCE). The electrocatalytic reduction of H202 can be described by eqs 4 and 5.2' 20s"
1 min.
1000
Figure 4. Dependence of the steady state H202 electroreduction current on the H202 concentration. The microsensor was poise at 0.0 V vs SCE. Magnetic stimng, PBS pH 7.4. 50
*
+ H202+ 2H' HRP_ 20s3+ + 2 H 2 0 os3++ e- - os2+
(4)
(5)
The steady state current density of the microsensor at 0.0 V vs SCE in a 1.0 x M H202 solution was 0.17 mA cm-2, about 10 times larger than that observed in a 3 mm macroelectrode,2' consistent with the increase in the current density of a wired glucose oxidase microsensor.20 The higher current density of the microsensor is attributed to increased flux of hydrogen peroxide by radial rather than semiinfinite planar diffusion between the microelectrode surface and the enzyme in the The dependence of the current of the microsensor on the H202 concentration at 0.0 V vs SCE in a magnetically stirred cell is shown in Figure 4. The current increased linearly with H202 concentration over a range of 2 orders of magnitude, from 5 x M. The H202 concentration at which the lo-' to 1 x noise equivalent current was observed was about 5 x M. At 1 x M H202, the 0-95% and 0-80% rise times were about 30 s and 15 s, respectively. In the detection of (photo)electrochemically generated H202, the solution pH often changes with the H202 concentration, because protons are consumed or released in the H202 generating reaction. When H202 is produced by water oxidation (eq 1) the pH decreases. When the H202 is produced by the reduction of dissolved oxygen through the reaction of eq 2, two protons are consumed, resulting in an increase in pH. Figure 5 shows the dependence of the current density on the solution pH. As found by Vreeke et al in macroelectrodes2' at 1.0 x M H202 concentration, the current density was almost insensitive to pH between 4.0 and 8.5, indicating that the sensor can be used without correcting its output for variation in sensitivity with pH through this range. Monitoring of the HZOZ Fonned at Discrete Oxidation and Reduction Sites on UV-Irradiated Ti02 on IT0 and Contacting IT0 Films. The H202 microsensor fabricated here did
Local Detection of Photoelectrochemically Produced H202
J. Phys. Chem., Vol. 99, No. 31, 1995 11899
the H202 concentration 50 p m from the IT0 surface was 1.2 x M and from the Ti02 surface only 2.5 x M. Taking into account that the detection limit of the microsensor for H202 was 5 x lo-* M, both values were significant; i.e., the efficiency of H202 formation on the I T 0 surface was more than 20 times faster than that on the Ti02 surface.
Discussion In the Ti02 photocatalyzed reactions in an oxygenated aqueous solution, H202 can be produced by either an oxidation (eq 1) or a reduction (eq 2) reaction. In reaction 1 water is oxidized by a hole, and the resulting OH radical combines with a second OH radical to form H202. In reaction 2, photogenerated electrons reduce dissolved molecular oxygen to H202 and possible sequences of this reaction are as follows.
0,’-
20,’-
0,’-
+ HO,’
-
+ H+
-
HO,’
10,2-12H+ JH20,1 IHO,-I
(7)
-
IH,041
H,O,
+ 0,
H,O,
+ 0,
(8b) (8c)
H202 formation was earlier measured in a suspension of Ti02 particles by its quenching of the fluorescence of a dye.IO No H202 was detected in the deaerated suspension, even in the presence of an efficient electron acceptor, Ag+. It was concluded that H202 was formed only by reduction of dissolved oxygen via the reaction of eq 2. There were, nevertheless, reports mentioning that H202 was produced by the oxidation of water via the reaction of eq l.I5 Reaction 2 has been studied in detail by Harvour et and Hong et a l . I 2 They concluded that H202 was produced by the reaction 2 only when electron donors were present in the Ti02 suspension. Nevertheless, in a phosphate buffer solution H202 was detected even in the absence of an electron donor.IO In all of these reports the evidence for the mechanism of H202 formation was indirect, and their results could have been affected by the added electron donors or acceptors. By directly monitoring the H202 formation at the oxidation and reduction sites we show here that H202 is formed by both the oxidation and reduction reactions, but mostly by the 0 2 reduction (Figure 6 ) . We note that on the irradiated Ti02 surface photogenerated holes form hydroxyl radicals, that oxidize H202 to H20 and molecular oxygen (eq 9). Thus H202 formed on the Ti02
+
H 2 0 2 20H’
-
2H20
+ 0,
(9)
surface (oxidation site), readily consumed by reaction 9, and even if Ti02 were an adequate electrocatalyst for 0 2 reduction to H202, the observed H202 concentration near the irradiated surface would not have represented the actual H202 formation by 0 2 electroreduction. Photogenerated electrons reaching the Ti02 surface, i.e., those electrons that did not drift or diffuse to the I T 0 layer, would have recombined through the reactions of eqs 2 and 9. The H202 generation seen represents the difference between the rate of its generation (reactions 1 and 2) and its consumption (reaction 9). Because H202 was not detected when the entire I T 0 surface was Ti02 coated, but was
detected when part of the I T 0 was solution-exposed, we conclude that the 0 2 electroreduction on I T 0 was much more efficient than the 0 2 electroreduction on Ti02. H202 can be catalytically decomposed with noble metals such as Pd and Pt. I T 0 may also catalyze the degradation of H202 which was formed by the reduction of the dissolved oxygen. The decomposition of H202 on the I T 0 surface, however, is considered to be negligibly small as compared to the H202 formation via eq 2. The efficiency of 0 2 electroreduction is highly dependent on the Orreducing cocatalyst used. Both the nature of the electron-hole separating junction formed and intrinsic overpotential for 0 2 electroreduction contribute to this dependence. For example, Wang et aL6 reported that palladium deposited onto the Ti02 surface promoted the rate of photodegradation of organic compounds catalyzing the reduction of the dissolved oxygen by the photogenerated electrons. This reduced the steady state electron density on the Ti02 particles, and thereby the rate of recombination of the photogenerated holes with electrons. In this case, H202 formation through 0 2 reduction on the Pd islands of the Ti02 crystallites was much faster than on their bare Ti02 surface. Thus, one expects that on a Ti02 film, part of which is coated with palladium, H202 will be observed mostly on the cocatalyst coated area in experiments similar to those described here. Furthermore, better spatial resolution than that in the described experiments will be attained by scanning electrochemical microscopy (SECM).’6,23 Such experiments are currently in progress.
Conclusion Using a 7 p m diameter H202 microsensor, made by wiring horseradish peroxidase through a hydrogel to a carbon electrode, the relative rates of H202 evolution were determined on electrically contacting Ti02 and I T 0 (indium tin oxide) films. It was found that in an oxygenated solution H202 was produced mainly through reduction of dissolved oxygen by photogenerated electrons on the I T 0 surface.
Acknowledgment. We thank David Schmidtke and Mark Vreeke for useful discussions. We are also grateful to Dr. L. A. Nagahara for careful reading of the manuscript. This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. A.H. acknowledges support of the U.S. Office of Naval Research and of the U.S. National Science Foundation. References and Notes (1) Dunn, W. W.; Aikawa, Y.; Bard, A. J. J . Am. Chem. SOC. 1981, 103, 3456. (2) Kobayashi, T.; Yoneyama, H.; Tamura, H. J . Electrochem. SOC. 1983, 130, 1706. (3) Nishimoto, S.;Ohtani, B.; Kagiya, T. J . Chem. Soc., Faraday Trans. 1985, 81, 2467. ( 4 ) Wold, A. Chem. Mater. 1993, 81, 280. (5) Papp, J.; Shen, H.-S.; Kershow, R.; Dwight, K., Wold, A. Chem. Mater. 1993, 5, 284. (6) Wang, C.-M.; Heller, A.; Gerischer, H. J . Am. Chem. SOC. 1992, 114, 5230. (7) Sakai,H.; Baba, R.; Hashimoto, K.; Fujishima, A. J . Electroanal. Chem. 1995, 379, 199. (8) Sakai, H.; Cai, R.-X.; Baba, R.; Hashimoto, K.; Kubota, Y.; Fujishima, A. In Photocatalytic Purification and Treatment of Water and Air; Ollis, D. F . Al-Ekabi, H., Eds.; Elsevier: New York, 1994; pp 651658. (9) Jaeger, C. D.; Bard, A. J. J . Phys. Chem. 1979, 83, 3146. (10) Cai, R.-X.; Hashimoto, K.; Fujishima, A,; Kubota, Y. J . Electroanal. Chem. 1992, 326, 345. (11) Harvour, J . R.; Tromp, J.; Hair, M. L. Can. J . Chem. 1985, 63, 204.
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(18) Gregg,B. A,; Heller, A. J . Phys. Chem. 1991, 95, 5970. (19) Heller, A., J. Phys. Chem. 1992, 96, 3579. (20) Pishko, M.; Michael, A. C.; Heller, A. Anal. Chem. 1991,63,2268. (21) Vreeke, M.; Maiden, R.; Heller, A., Anal. Chem. 1992, 96, 3579. (22) Kikuchi, E.; Itoh, K.; Fujishima, A. Nihon Kagaku Kaishi 1987, 11, 1970. (23) Bard, A. J.; Fan, F.-R. F.; Pierce, D. T.; Unwin, P. R.; Wipf, D. 0,;sou, F. science1991, 254, 69.
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