Single-Molecule Fluorescence Imaging of the Remote TiO2

The remote TiO2 photocatalytic oxidation reaction of single dyes has been investigated by the single-molecule fluorescent imaging technique. The prese...
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23138

2005, 109, 23138-23140 Published on Web 11/17/2005

Single-Molecule Fluorescence Imaging of the Remote TiO2 Photocatalytic Oxidation Kazuya Naito, Takashi Tachikawa, Mamoru Fujitsuka, and Tetsuro Majima* The Institute of Scientific and Industrial Research (SANKEN), Osaka UniVersity, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan ReceiVed: October 1, 2005; In Final Form: NoVember 7, 2005

The remote TiO2 photocatalytic oxidation reaction of single dyes has been investigated by the single-molecule fluorescent imaging technique. The present results suggest that the active oxygen species (Ox) is most probably the •OH radical, which is generated from the photodecomposition of H2O2 by UV light. The analyses of the number, intensity, and spectrum of individual fluorescence spots at the single-molecule level also indicate that unoxidized and oxidized dyes exist during the bleaching processes of single dyes.

TiO2 photocatalysts have been extensively studied and used for the water-splitting reaction that produces hydrogen, the degradation of organic pollutants, the surface wettability conversion, and so on.1-7 Recently, Tatsuma et al. reported the remote oxidation of organic and inorganic materials using the TiO2 photocatalyst.2 They explained this phenomenon in terms of a double excitation scheme in which the photodecomposition of gaseous H2O2 by UV light to give the hydroxyl radical (•OH) is the key process.2d Park and Choi also suggested that airborne active oxygen species (Ox) is mostly •OH, although they doubted the double excitation mechanism.3b It has also been reported that the superoxide anion (O2•-),6 singlet oxygen (1O2),7 and hydrogen peroxide (H2O2)2d are generated on the TiO2 surface in the gas and liquid phases. Indeed, it is difficult to identify Ox and clarify the reaction mechanism, because the concentration of Ox is too low to detect at the surface using conventional steady-state spectroscopies. Single-molecule fluorescence studies can reveal the detailed kinetics of physical processes and chemical reactions that are often hidden in ensemble measurements.8,9 In particular, total internal reflection fluorescence microscopy (TIRFM) is an elegant optical technique that is used to observe single molecule fluorescence at surfaces and interfaces.9 Herein, we have successfully applied this technique to clarify the oxidation process of single dyes during the remote TiO2 photocatalytic reactions. To the best of our knowledge, this is the first example of the TiO2 photocatalytic oxidation reaction of single dye molecules. The experimental setup is based on using an Olympus IX71 TIRF microscope. Light emitted from a continuous wave (CW) Nd:YAG laser (532 nm, 50 mW) or a CW Ar ion laser (488 nm, 10 mW) passing through an objective lens (Olympus, PlanApo, 1.45 NA, 100×) was totally reflected at the coverslip-air interface to obtain an evanescent field which can excite a dye molecule (see Supporting Information for details). The fluorescence from single dyes (Alexa Fluor 532, Molecular Probes) immobilized onto a glass coverslip via N-[3-trimethoxysilylpropyl]ethylenediamine (TSE) was collected using an oil-immersion microscope objective. * Corresponding author. Tel: +81-6-6879-8495. Fax: +81-6-6879-8499. E-mail address: [email protected].

10.1021/jp0556005 CCC: $30.25

The TiO2 aqueous sol (Ishihara Sangyo, STS-21) was diluted with MiliQ water (80 vol %), sonicated overnight, and coated on a slide glass by spin coating at 2000 rpm for 15 s. The resulting TiO2 film was calcined at 400 °C for 1 h to obtain an optically opaque TiO2-coated glass plate. The film thickness was determined to be 1 µm by a Veeco Instruments Dektak3 surface profiler. The intervening gap was controlled using polyimide films (Nilaco, thickness, 12.5-125 µm). The TiO2 coating was irradiated with a 100 W Hg lamp (0.2 mW cm-2) that passed through a band-pass filter (330-385 nm), an objective lens, and a dye-modified coverslip (Figure 1). Figure 2A shows the fluorescence images observed during the 532-nm excitation of the single dyes before and after the UV irradiation of the TiO2 film with the intervening gap of 12.5 µm in ambient air. The number of single fluorescence dyes (N) clearly decreased with the increasing UV irradiation time. It was found that the spatial distribution of Ox reaches about 100 × 100 µm2 at the surface of the coverslip. In addition, as shown in Figure 2B, the bleaching rates of the dyes significantly decreased with the increasing gap. This tendency is quite similar to that reported elsewhere.2 It should also be noted that the bleaching of dyes was achieved by the very weak UV light. In the absence of a self-assembled monolayer of TSE, the decrease in the bleaching rate of dyes, which are spread over the cover glass using a spin coater, was observed (see Supporting Information). In addition, no bleaching was observed over a much larger area than the focal area of the incident UV light, even after a 1 h UV irradiation time using the Spectralon diffuse reflectance standard (Labsphere) instead of the TiO2-coated slide glass. These results strongly support the fact that the degradation of dyes is caused by the bimolecular reaction with Ox, not by some artifacts such as the UV light scattering. Various Ox species, such as •OH, O2•-, and 1O2, would be generated during the TiO2 photocatalytic reactions. For example, •OH is generated as described by eqs 1 and 2,1

TiO2 + hν f h+ + e-

(1)

h+ + H2O (or OH-) f •OH + H+ (or •OH)

(2)

where h+ and e- are holes and electrons, respectively, generated © 2005 American Chemical Society

Letters

J. Phys. Chem. B, Vol. 109, No. 49, 2005 23139

Figure 1. Schematic illustration of the experimental setup.

Figure 3. (A) Histogram of the fluorescence intensity observed during the 532-nm excitation of the single dyes immobilized on the cover glass in the 12.5 µm gap before (a) and after (b) UV irradiation for 15 min. Solid and dotted lines indicate Gaussian distributions fitted with the histograms for one and two dye chromophores, respectively. Inset: UV irradiation time dependence of the center of the distribution. (B) Typical trajectories of the fluorescence spots obtained before (a) and after (b) UV irradiation for 15 min. (C) Typical single-molecule fluorescence spectra observed during the 488-nm excitation of the single dyes immobilized on the cover glass in the 12.5 µm gap before (a) and after (b) UV irradiation for 15 min. The spectra are cut below 510 nm by a long-pass filter on the blue edge. (D) Histogram of the peak wavenumber of the fluorescence spectra observed during the 488-nm excitation for the single dyes before (a) and after (b) UV irradiation for 15 min. Solid lines indicate Gaussian distributions fitted with the histograms. Figure 2. (A) Fluorescence images observed during the 532-nm excitation of single dyes immobilized on the coverslip before and after UV irradiation (scale bars are 30 µm). The UV irradiation area is the inside of the white circle in the middle image. The bleaching of dyes at the center of the images is due to the UV irradiation. (B) Time dependence of the N/N0 values in the gaps of 12.5 (a), 50 (b), and 125 (c) µm. The solid lines are visual guides. (C) Time dependences of the N/N0 values in the 12.5 µm gap observed for the uncoated (a), the TiO2coated (b), the TiO2-coated slide glasses immersed in 2-propanol (c), and the uncoated slide glass spin-coated with 30 wt % H2O2 (d). The solid lines are visual guides.

at the TiO2 surface excited by UV light. H2O2 can also be generated from •OH as follows:

2•OH f H2O2

(3)

To identify the origin of Ox, we examined the influence of hole scavengers, such as 2-propanol, on the bleaching process of the dyes. The fluorescence images were obtained after UV irradiation of the TiO2 film immersed in 2-propanol for 10 min. As shown in Figure 2C, a significant decrease in the bleaching rate was clearly observed when compared with that obtained for bare TiO2. The most likely explanation is that 2-propanol traps h+, which generates •OH from H2O or the resulting •OH at the TiO2 surface.10 We also investigated the possibility as to whether the photodecomposition of H2O2 is caused by the UV light that passed through the coverslip. The H2O2 has some absorption at 350-400 nm, while the coverslip has negligible absorption, suggesting that H2O2 could be photodecomposed to •OH by UV light.11 For this purpose, the slide glass spin-coated by the 30 wt % H2O2 solution is used instead of the TiO2-coated glass. As shown in Figure 2C, the observed bleaching of dyes would be evidence for the diffusion of •OH, which is generated from

the photodecomposition of H2O2.12 These experimental results indicate the involvement of the so-called double excitation mechanism during the bleaching process of dyes,2d although the bleaching of dyes was observed outside the UV irradiation area due to the diffusion of •OH because of the high sensitivity of our measurements. To date, the remote TiO2 photocatalytic oxidation has been successfully applied to various organic and inorganic substrates such as the saturated alkyl chain monolayer, polymer, carbon soot, copper, and silicon carbide,2-5 suggesting that only •OH is capable enough to oxidize such diverse materials. However, we cannot claim that •OH is the sole reactive species in the present system, because the bleaching of dyes due to 1O2 in a bulk aqueous solution was observed (see Supporting Information). It has been recently pointed out that 1O2 can be generated as a result of an energy exchange between the excited TiO2 and 3O2.2b The observed decrease in the bleaching rate in the presence of 2-propanol might be due to the fact that the adsorbed 2-propanol physically blocks the approach of 3O2. Considering that gas diffusion coefficients of O2 and •OH in air are about 0.2 cm2 s-1,13 the lifetimes of •OH and 1O2, which are reported to be several tens or hundreds of milliseconds,14 seem to be sufficient to migrate at least up to the dye-modified coverslip.15 Next, to clarify the bleaching processes of dyes on a singlemolecule level, the histogram of the fluorescence intensity for each single dye was examined. Figure 3A shows the fluorescence intensity histogram of individual spots, together with Gaussian functions fitted with the histograms. Before the UV irradiation, a dominant distribution centered at the fluorescence intensity of about 1.2 was accompanied by a broad bump centered at 2.3. This observation provides substantial evidence that the two maxima in the

23140 J. Phys. Chem. B, Vol. 109, No. 49, 2005 histogram are ascribable to one and two dye chromophores. Interestingly, the center of the distribution remarkably decreased by a 5 min UV irradiation and then reached almost the same value by subsequent UV irradiation. If Ox oxidizes dyes in a random order, and one-step staircase bleaching occurs on the fluorescent spot with a 1.2-intensity maximum, no change in the histogram should be observed. As shown in Figure 3B, the intermissive blinking, which is mainly due to the transition to the dark state, such as a triplet state,8 was typically observed for the trajectories of the fluorescence spots after UV irradiation, suggesting that the fluorescence properties, such as the fluorescence yield and lifetime, of dyes changed during the UV irradiation. Hence, we speculate that two states, such as the unoxidized and oxidized dyes, which indicate the fluorescence intensities of ∼1.2 and ∼0.6, respectively, exist during the bleaching processes of single dyes. To confirm the oxidation reaction of single dyes with Ox, we observed single-molecule fluorescence spectra of dyes before and after the UV irradiation. As shown in Figure 3C, it was found that the spectrum is blue shifted by approximately 1520 nm after the UV irradiation. This blue shift also leads to the irreversible signal drop of the fluorescence signal. A similar spectral change was observed during the bleaching processes of dyes in a bulk aqueous solution,16 suggesting that the observed blue shift is due to the remote oxidation of single dyes by Ox. It should also be noted that the histogram of the peak wavenumber of the spectra observed after UV irradiation indicates two maxima around 17820 and 18380 cm-1, which correspond to 561 and 544 nm, respectively (Figure 3D). The former, which is almost identical with that (17790 cm-1, 562 nm) obtained before the UV irradiation, should be assigned to the unoxidized dyes. These experimental results strongly support multistep decomposition processes of dyes, i.e., further oxidation of the intermediate (oxidized) species, should be included in the remote photocatalytic oxidation processes of dyes. In conclusion, we have demonstrated that single-molecule fluorescence imaging is a powerful tool to understand the TiO2 photocatalytic oxidation reaction of fluorescent molecules with airborne oxidants at the surface. The analyses of the number, intensity, and spectrum of individual fluorescence spots at the single-molecule level suggest that the bleaching process of dyes is accompanied by a change in the fluorescence properties during the TiO2 photocatalytic reactions. Acknowledgment. This work has been partly supported by a Grant-in-Aid for Scientific Research (Project 17105005, Priority Area (417), 21st Century COE Research, and others) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japanese Government. Supporting Information Available: The experimental setup and control experiments. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) For example, (a) Fujishima, A.; Rao, T. N.; Tryk, D. A. J. Photochem. Photobiol. C: Photochem. ReV. 2000, 1, 1-21. (b) Hoffmann, M. R.; Martin, S. T.; Choi, W.; Bahnemannt, D. W. Chem. ReV. 1995, 95, 69-96. (c) Mills, A.; Hunte, S. L. J. Photochem. Photobiol. A 1997, 108, 1-35. (2) (a) Tatsuma, T.; Tachibana, S.; Miwa, T.; Tryk, D. A.; Fujishima, A. J. Phys. Chem. B 1999, 103, 8033-8035. (b) Tatsuma, T.; Tachibana,

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