Single-Molecule Fluorescence Imaging of TiO2 Photocatalytic

pubs.acs.org/Langmuir © 2009 American Chemical Society

Single-Molecule Fluorescence Imaging of TiO2 Photocatalytic Reactions Takashi Tachikawa and Tetsuro Majima* The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan Received March 4, 2009. Revised Manuscript Received April 2, 2009 Heterogeneous photocatalysts have both potential and demonstrated applications for use in the water-splitting reaction that produces hydrogen, the degradation of organic pollutants, the surface wettability conversion, etc. In this feature article, we have focused on the in-site observation of various reactive oxygen species (ROS), such as singlet oxygen (1O2) and the hydroxyl radical (•OH), generated by the photoexcitation of TiO2 nanomaterials using singlemolecule fluorescence spectroscopy. The spatially resolved photoluminescence (PL) imaging techniques enable us to determine the location of the (photo)catalytically active sites that are related to the heterogeneously distributed defects on the surface. We also present the results that revealed the formation and reaction dynamics of the photogenerated charge carriers in individual TiO2 nanoparticles. Furthermore, we introduce the single-molecule single-mismatch detection of the nucleotide sequence upon the photoexcitation of a novel nanoconjugate consisting of TiO2 and DNA on the basis of the mechanistic aspects. Notably, the present conjugates can recognize the difference in a single nucleotide. Consequently, this article provides a significant opportunity to understand the temporal and spatial distributions of ROS generated during the photoirradiation of TiO2 nanomaterials and directly explore the microscopic world in many fields ranging from fundamental physics and chemistry to practical applications.

1. Introduction Metal oxide photocatalysts, such as TiO2 and ZnO, have been extensively studied and used for the water-splitting reaction that produces hydrogen, the degradation of organic pollutants, the surface wettability conversion, etc.1-5 It has been reported that various reactive oxygen species (ROS), such as superoxide (O2•-), singlet oxygen (1O2), the hydroxyl radical (•OH), the hydroperoxyl radical (HO2•), and hydrogen peroxide (H2O2), are generated on the TiO2 surface and react with organic or inorganic compounds in the gas and liquid phases. The proposed photocatalytic reaction schemes for the generation of ROS are summarized in Scheme 1. The photoinduced reactions based on the semiconductor property of metal oxides are basically initiated by the band gap excitation (in the case of anatase TiO2, ultraviolet (UV) light irradiation (λ < 390 nm) corresponding to its band gap of 3.2 eV) to generate the valence band (VB) holes (h+) and conduction band (CB) electrons (e-), which are immediately captured on a picosecond time scale at various trap sites in the bulk or on the surface. In the TiO2-assisted oxidation processes of organic compounds, there are two important processes, i.e., the direct oxidation on the TiO2 surface by free or trapped h+ and the indirect oxidation by the ROS, which are mainly formed via the reduction of O2 by free or trapped e-. The direct oxidation process has been well characterized by the transient absorption measurements of radical cations of adsorbed substrates (Sads•+), which are formed by the reaction between the substrates adsorbed *Corresponding author: Tel: +81-6-6879-8495; Fax: +81-6-6879-8499; E-mail: [email protected]. (1) Fujishima, A.; Rao, T. N.; Tryk, D. A. J. Photochem. Photobiol., C 2000, 1, 1–21. (2) Hoffmann, M. R.; Martin, S. T.; Choi, W.; Bahnemann, D. W. Chem. Rev. 1995, 95, 69–96. (3) Thompson, T. L.; Yates, J. T.Jr. Chem. Rev. 2006, 106, 4428–4453. (4) Chen, X.; Mao, S. S. Chem. Rev. 2007, 107, 2891–2959. (5) Tachikawa, T.; Fujitsuka, M.; Majima, T. J. Phys. Chem. C 2007, 111, 5259–5275.

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on the TiO2 surface and h+.5 On the other hand, in the indirect reaction process, the generation of a variety of ROS during the TiO2 photocatalytic reactions has been investigated using various methods, such as electron spin resonance (ESR) spectroscopy,6 near-infrared spectroscopy,7 laser-induced fluorescence spectroscopy,8 and the use of fluorescent probes.9,10 The indirect oxidation caused by these ROS diffusing in the medium should be the dominant process at a site distant from the TiO2 surface, where direct oxidation cannot occur. To design an efficient photocatalyst for the above-mentioned applications, it is important to reveal and understand the mechanism of the photocatalytic reactions, in particular, the oxidation of organic compounds. However, ROS present some characteristics that make them difficult to be detected and identified due to the wide ranges of lifetimes and reactivities under various conditions. For example, as shown in Figure 1, the diffusion distances of •OH (dOH) in water and air containing acetaldehyde (4.5 μM, 100 ppm) are calculated to be 1.1 and 37 μm, respectively. Therefore, it is essential to develop methodologies capable of overcoming these types of obstacles. Although the fluorescence methodology associated with the use of suitable probes is an excellent approach to measure ROS because of its high sensitivity, simplicity in data collection, and high spatial resolution of its microscopic imaging, it is extremely difficult or impossible to elucidate the influence of the structural disorders, such as defects and size and shape nonuniformities, on the spatial distribution of (photo)catalytically active sites using conventional ensemble measurements. Thus, future techniques for examining a single piece of material are strongly required for elucidating the details of the reaction mechanisms. (6) Anpo, M.; Shima, T.; Kubokawa, Y. Chem. Lett. 1985, 1799–1802. (7) Daimon, T.; Nosaka, Y. J. Phys. Chem. C 2007, 111, 4420–4424. (8) Murakami, Y.; Kenji, E.; Nosaka, A. Y.; Nosaka, Y. J. Phys. Chem. B 2006, 110, 16808–16811. (9) Ishibashi, K.; Fujishima, A.; Watanabe, T.; Hashimoto, K. J. Photochem. Photobiol., A 2000, 134, 139–142. (10) Hirakawa, T.; Nosaka, Y. Langmuir 2002, 18, 3247–3254.

Published on Web 04/29/2009

DOI: 10.1021/la900790f

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Invited Feature Article

Tachikawa and Majima

Scheme 1. Photocatalytic Reaction Schemes for the Generation of ROS

Figure 1. Spatial distribution of ROS during TiO2 photocatalytic

Single-molecule fluorescence spectroscopy is emerging as an important tool for studying the photophysical and photochemical processes of all types of molecular systems from simple dye molecules to fluorescent proteins.15,16 Until now, the requirements for observing very weak emissions from a single fluorophore have required the development of advanced detection methods,17 such as total internal reflection fluorescence microscopy (TIRFM, see Figure 2) and confocal fluorescence microscopy. The former technique is advantageous in visualizing the fluorescence from single molecules immobilized or located at the interface (e.g., a glass surface) with a very low background noise using an evanescent field and thus has been applied to the investigation of the temporal dynamics of biomolecules (e.g., DNA, proteins, and enzymes) labeled with dyes, which provides information that is useful for revealing various biological functions at the molecular level.18 The latter, confocal microscopy, is widely used for the detection of a molecule freely diffusing in solution, often referred to as fluorescence correlation spectroscopy (FCS),19 which enables us to investigate and clarify the binding interactions, such as protein-ligand binding and DNA hybridization, by measuring the correlation time of diffusing molecules into the focal volume. These techniques for singlemolecule fluorescence detection have advantages superior to the conventional ones that relies on the bulk sample, providing us with opportunities such as the ultimate high sensitivity, the possible observations of the properties hidden in ensemble measurements (subpopulation existing in the sample), and eliminating the need for synchronization. Therefore, the single-molecule (single-particle) fluorescence spectroscopy has been applied to elucidate the inherent features of heterogeneous catalyzes, such as the dynamics of reagent molecules in mesoporous silica,20 and the mechanism of the hydrolysis or redox reaction occurring on the surface of the layered double hydroxide (LDH) crystal21 and gold nanoparticles.22 (11) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref. Data 1988, 17, 513–886. (12) Massman, W. J. Atmos. Environ. 1998, 32, 1111–1127. (13) Schuchmann, M. N.; Von Sonntag, C. J. Am. Chem. Soc. 1988, 110, 5698–5701. (14) Atkinson, R. Chem. Rev. 1986, 86, 69–201. (15) Xie, X. S.; Trautman, J. K. Annu. Rev. Phys. Chem. 1998, 49, 441–480. (16) Moerner, W. E.; Orrit, M. Science 1999, 283, 1670–1676. (17) Moerner, W. E.; Fromm, D. P. Rev. Sci. Instrum. 2003, 74, 3597–3619. (18) Cornish, P. V.; Ha, T. ACS Chem. Biol. 2007, 2, 53–61. (19) Rigler, R.; Elson, E. S. Fluorescence Correlation Spectroscopy: Theory and Applications; Springer: Berlin, 2001. (20) Zuerner, A.; Kirstein, J.; Doeblinger, M.; Braeuchle, C.; Bein, T. Nature 2007, 450, 705–708. (21) Roeffaers, M. B. J.; Sels, B. F.; Uji-i, H.; De Schryver, F. C.; Jacobs, P. A.; De Vos, D. E.; Hofkens, J. Nature 2006, 439, 572–575. (22) Xu, W.; Kong, J. S.; Yeh, Y.-T. E.; Chen, P. Nat. Mater. 2008, 7, 992–996.

7792 DOI: 10.1021/la900790f

reactions. The direct oxidation by the valence hole (hVB+) and the indirect oxidation by ROS diffused from the surface are shown. For example, the diffusion distances of •OH (dOH) in water and air were calculated to be 1.1 and 37 μm, respectively, from the Brownian motion expression in three dimensions, dOH = (6Dt)1/2, where D is the diffusion coefficient of •OH (2.3 10-9 and 2.2 10-5 m2 s-1, respectively)11,12 and t is the lifetime of •OH estimated from the reaction rate between •OH and acetaldehyde (3.6 109 and 9.8 109 M-1 s-1, respectively).13,14 In this case, both the concentrations of acetaldehyde in water and air are assumed to be 4.5 μM (corresponding to 100 ppm in air).

Recently, we have successfully applied the single-molecule fluorescence techniques with TIRFM to reveal the temporal and spatial distributions of ROS during the TiO2 photocatalytic reactions.23 In this feature article, we first focus on the development of the methodology of the single-molecule detection of ROS with specific fluorescent probes and discussed the temporal and spatial contributions of ROS generated by the photoexcitation of TiO2 nanoparticles or nanotubes (sections 2-5). Next, in section 6, we present our recent studies on the defect-mediated photoluminescence (PL) of individual TiO2 nanoparticles to clarify the transport and reaction dynamics of photogenerated charge carriers in the materials. In section 7, in order to motivate the application of TiO2 nanoparticles in the fields of nanobiotechnology and nanomedicine, we describe the single-molecule single-mismatch detection of the nucleotide sequence upon the photoexcitation of a novel nanoconjugate consisting of TiO2 and DNA on the basis of the mechanistic aspects. Finally, we summarize our contributions and suggest future directions.

2. Remote Oxidation of Single Fluorescence Dye Molecules Induced by Photoirradiation of TiO2 Film The TiO2 photocatalytic reaction has been utilized to oxidize organic and inorganic substrates, such as the saturated alkyl chain monolayer, polymer, and silicon carbide, at a distant site with an intervening gap (so-called “remote TiO2 photocatalytic oxidation”). According to the literature,24,25 it is strongly believed that the principal airborne ROS causing the remote oxidation at the distant position should be •OH molecules, which are produced by the proposed mechanisms including the photolysis of H2O2 by UV irradiation in the vicinity of the oxidizing substrate surface. Recently, Murakami et al. directly detected •OH diffused in the gas phase from the photocatalytic TiO2 surfaces using the in situ laser-induced fluorescence technique.8 To answer whether or not the •OH is the sole reactive species, we have proposed a new strategy to detect the airborne ROS diffused from the surface of TiO2 nanoparticles at the single-molecule level.26 (23) Tachikawa, T.; Majima, T. J. Fluoresc. 2007, 17, 727–738. (24) Kubo, W.; Tatsuma, T.; Fujishima, A.; Kobayashi, H. J. Phys. Chem. B 2004, 108, 3005–3009. (25) Kubo, W.; Tatsuma, T. J. Am. Chem. Soc. 2006, 128, 16034–16035. (26) Naito, K.; Tachikawa, T.; Fujitsuka, M.; Majima, T. J. Phys. Chem. B 2005, 109, 23138–23140.

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Figure 2. (A) Illustration of the experimental setup for the total internal reflection fluorescence microscopy (TIRFM) used in part of the present studies. The movement of the incident laser light (green) can control the angle of incident at the interface, enabling the switching from the epi-fluorescence to TIRF excitation. (B) The principle of evanescent wave excitation for single-molecule fluorescence experiments (λex = 532 nm). Only molecules near the interface (within the penetration depth d ∼ 200 nm) can be efficiently excited and then emitting the detectable fluorescence.

The experimental setup for the detection of ROS with a singlemolecule fluorescence probe (Alexa Fluor 532 dye) is illustrated in Figure 2A. The TiO2 aqueous sol was coated on a slide glass by spin coating, and the resulting TiO2 film was calcined to obtain an optically opaque TiO2-coated glass plate. The intervening gap was controlled using polyimide films. The TiO2 coating was irradiated with a 100 W Hg lamp that passed through a bandpass filter (310-370 nm), an objective lens, and a dye-modified cover glass. Figure 3A shows the fluorescence images observed during the 532 nm excitation of single dye molecules before and after the UV irradiation of the TiO2 film with the intervening gap of 12.5 μm in ambient air. It was found that the number of single fluorescence dyes (N) clearly decreased with the increasing UV irradiation time, and the spatial distribution of ROS reached about 100 100 μm2 at the surface of the cover glass (Figure 3A). In addition, as shown in Figure 3B, the bleaching rates of the dyes significantly decreased with the increasing gap.27 To identify the origin of the ROS, 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 nanocrystalline TiO2 film immersed in 2-propanol for 10 min. As shown in Figure 3C, a significant decrease in the bleaching rate was observed when compared with that obtained for the bare TiO2. The possible explanation is that 2-propanol scavenges holes and/or •OH generated at the TiO2 surface. Most importantly, it was found that a relatively low bleaching rate of single dye was obtained for the H2O2 (27) In the absence of a self-assembled monolayer of N-[3-trimethoxysilylpropyl]ethylenediamine (TSE) as a linker between the cover glass and the dye molecule, a significant decrease in the bleaching rate of dyes, which are spread over the cover glass using a spin-coater, was observed. These results strongly support the fact that the degradation of dyes is caused by the bimolecular reaction with ROS, not by some artifacts such as the UV light scattering.

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aqueous solution (30 wt %)-coated glass, when compared to the TiO2-coated glass. These results infer that •OH is not the sole reactive species. To confirm the oxidation process by ROS, we measured the single-molecule fluorescence spectra of dye molecules before and after the UV irradiation. It was found that the histogram of the peak wavelength of the spectra observed after UV irradiation indicates two maxima around 561 and 544 nm. The former, which is almost identical to that (562 nm) obtained before the UV irradiation, should be assigned to the unoxidized dyes. These experimental results strongly support multistep decomposition processes of the dyes, i.e., further oxidation of the intermediate (oxidized) species is included in the remote oxidation processes of the dyes.

3. Selective Detection of Single Airborne Singlet Oxygen Molecules As a next step, we tried to selectively detect an airborne 1O2 molecule that diffused from the surface of the TiO2 nanoparticles using TIRFM.28 Although 1O2 molecules are believed to be generated during the TiO2 photocatalytic reactions (Scheme 1), the formation mechanism has been proposed as follows:7 TiO2 þ hν f TiO2 ðh þ Þ þ TiO2 ðe - Þ

ð1Þ

O2 þ TiO2 ðe - Þ f O2 • -

ð2Þ

O2 • - þ h þ f 1 O2 ðor 3 O2 Þ

ð3Þ

3

(28) Naito, K.; Tachikawa, T.; Cui, S.-C.; Sugimoto, A.; Fujitsuka, M. Majima, T. J. Am. Chem. Soc. 2006, 128, 16430–16431.

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Figure 3. (A) Fluorescence images observed during the 532 nm excitation of single dyes immobilized on the cover glass before and after UV irradiation (all images are 30 μm 30 μm). The cover glass coated with the fluorescent probes was attached to the TiO2 film-coated slide glass with an intervening gap. The intervening gap between the fluorescent probe-coated cover glass and the TiO2 film-coated slide glass was controlled using polyimide films. See Figure 2A for the experimental setup. (B) Time dependence of the N/N0 values in the gaps of 12.5 (black), 50 (red), and 125 μm (blue). The solid lines are visual guides. (C) Time dependences of the N/N0 values in the 12.5 μm gap observed for the uncoated (black), the TiO2-coated (red), the TiO2-coated slide glasses immersed in 2-propanol (green), and the uncoated slide glass spincoated with 30 wt % H2O2 (blue). For the H2O2 aqueous solution-coated glass, the space between the glasses was filled with air saturated with the vapor of the residual solution (vapor pressure of the solution is ca. 20 mmHg at room temperature). The solid lines are visual guides. For additional results, see ref 26.

Otherwise, O2 is generated by the energy exchange between the excited TiO2 and 3O2 molecules:29 TiO2 þ hν f TiO2

ð4Þ

TiO2 þ 3 O2 f TiO2 þ 1 O2

ð5Þ

In the remote TiO2 photocatalytic oxidation, 1O2 generated on the TiO2 surface is considered to barely diffuse into the gas phase forming airborne 1O2,7 but there is no direct experimental evidence. Our strategy to detect 1O2 molecules at the single-molecule level is summarized in Figure 4A. The terrylenediimide derivative (TDI) was used as an 1O2 nanosensor in place of Alexa dye described in section 2. According to a pioneering study,30 a single TDI molecule should be oxidized by a single 1O2 molecule to form a less fluorescent endoperoxide and successively a strongly fluorescent diepoxide with a spectral blue shift that is easily detected upon 532 nm laser excitation. Figure 4B depicts the single-molecule fluorescence images observed before (a) and after (b) the UV irradiation of the TiO2 film for 5 min. Before the UV irradiation, only a few fluorescent spots were observed due to the weak fluorescence of the TDI excited at 532 nm. Because of the lack of sensitivity against the weak fluorescence, most of the TDIs cannot be recognized in image a. Interestingly, after UV irradiation, bright fluorescent spots emerged around the UV-irradiated region as described by the white circles. Based on the reaction scheme, these fluorescent spots would arise from the TDI diepoxide, which is formed by the cycloaddition (29) Janczyk, A.; Krakowska, E.; Stochel, G.; Macyk, W. J. Am. Chem. Soc. 2006, 128, 15574–15575. (30) Christ, T.; Kulzer, F.; Bordat, P.; Basche, T. Angew. Chem., Int. Ed. 2001, 40, 4192–4195.

7794 DOI: 10.1021/la900790f

reaction between the TDI and airborne 1O2 generated during the TiO2 photocatalytic reactions. To clarify the formation of the TDI diepoxide, the single-molecule fluorescence spectra were measured for each spot before and after the UV irradiation as shown in panel c of Figure 4B. Compared to that before the UV irradiation, it was found that the spectrum after UV irradiation is blue-shifted by ∼70 nm and has a strong fluorescence intensity, suggesting that the bright fluorescent spots are attributed to the TDI diepoxide. Under this experimental condition, 40% of the TDI molecules show complete digital switching.31 The fact that the TDI diepoxide shows a strong fluorescence intensity, when compared with that of the TDI, is explained as the increase in the absorbed photon due to the blue shift of the absorption spectrum. These speculations were well supported by quantum calculations of the optical transitions. Using this 1O2 nanosensor, the spatial and temporal distributions of the airborne 1O2 molecules diffused from the TiO2 film have been investigated. Interestingly, several dozen 1O2 molecules are detected even at the intervening gap of 2 mm as shown in Figure 4C. To the best of our knowledge, this is the first example of the single-molecule detection of molecules traveling such a long distance in ambient air. The quantitative analysis enables us to estimate the generation efficiency of the airborne 1O2 molecules during the TiO2 photocatalytic reactions. Using the detected number of 1O2 molecules and the number of photons absorbed by the TiO2 film, the generation efficiency of the airborne 1 O2 molecules diffused from the TiO2 film was determined to be about 10-8. This value is much lower than the quantum yield (0.12-0.38) for the 1O2 generation measured for the TiO2 photocatalysis using near-infrared phosphorescence spectroscopy.7 (31) It should be noted that no further spectral blue shift due to multiple attacks of 1O2 was observed in the present wavelength range. Therefore, the bright spots in the fluorescence images can be regarded as a signal of single 1O2 molecules. This enables us to count the 1O2 molecules, although a part of 1O2 molecules might be deactivated in the PMMA film.

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Figure 4. (A) Single-molecule detection of the airborne 1O2 with TDI. (B) Fluorescence images of single TDIs spin-coated on the PMMAcoated cover glass before (a) and after (b) UV irradiation for 5 min (scale bars are 10 μm). The intervening gap is 12.5 μm. The bright spots correspond to the blue-shifted, TDI diepoxide. The UV irradiation area is inside the white circle in the images. The absence of dyes at the center of the image after UV irradiation is due to the bleaching of TDI caused by the direct UV irradiation. (C) The spatial and temporal distributions of 1O2 molecules diffused from the surface of the TiO2 film. The observation region is 70 70 μm2. These results are reported in ref 28.

This prominent discrepancy indicates that only a small number of generated 1O2 molecules can be desorbed from the surface and diffuse into air, although almost all molecules should be deactivated without their release from the surface.

4. In Situ Observation of the Spatial and Temporal Distribution of Reactive Oxygen Species In this study, we have developed the methodology for the selective single-molecule detection of the airborne ROS diffused from the pure and nitrogen (N)-doped TiO2 nanoparticles under UV- or visible-light irradiation.32 The visible-light-responsive TiO2 doped with nonmetal atoms, such as N, sulfur (S), and carbon (C), have been synthesized and studied to produce photocatalysts working under visible light, which is a significant part of sunlight.33-36 Furthermore, we performed the real-time single-molecule imaging of the spatial and temporal distributions of the •OH molecules at the solid-air interfaces including glass substrate, surface-adsorbed water, and air. For the selective single-molecule detection of •OH, we used 0 3 -(p-hydroxylphenyl)fluorescein (HPF). HPF is a fluorescent probe for the selective detection of •OH that was developed by Nagano et al.,37 which selectively reacts with a single •OH molecule to form a strongly emissive fluorescein molecule, but (32) Naito, K.; Tachikawa, T.; Fujitsuka, M.; Majima, T. J. Phys. Chem. C 2008, 112, 1048–1059. (33) Asahi, R.; Morikawa, T.; Ohwaki, T.; Aoki, K.; Taga, Y. Science 2001, 293, 269–271. (34) Tachikawa, T.; Tojo, S.; Kawai, K.; Endo, M.; Fujitsuka, M.; Ohno, T.; Nishijima, K.; Miyamoto, Z.; Majima, T. J. Phys. Chem. B 2004, 108, 19299–19306. (35) Tachikawa, T.; Takai, Y.; Tojo, S.; Fujitsuka, M.; Irie, H.; Hashimoto, K.; Majima, T. J. Phys. Chem. B 2006, 110, 13158–13165. (36) Tojo, S.; Tachikawa, T.; Fujitsuka, M.; Majima, T. J. Phys. Chem. C 2008, 112, 14948–14954. (37) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T. J. Biol. Chem. 2003, 278, 3170–3175.

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not with other ROS, such as O2•-, 1O2, and H2O2, generated during the TiO2 photocatalytic reactions. This reaction scheme is summarized in Figure 5A. During the simultaneous excitation of TiO2 and fluorescent dyes, we succeeded in detecting single airborne •OH molecules in real time. The simultaneous excitation of the fluorescent probe on a cover glass and the TiO2 film requires that the bimolecular reaction between the excited fluorescent probe and the ROS is negligible. It has been observed that both the HPF and TDI in the ground state do not react with H2O2, while the excited TDI reacts with H2O2 to form the fluorescent species. A typical example of the real-time single-molecule detection of • OH diffused from the pure TiO2 film under UV irradiation at the intervening gap of 12.5 μm is shown in Figure 5B. The vertical axis is the number of the detected fluorescein (Fl) molecules per 33 ms. Thus, this corresponds to the count rate of the •OH molecules. The inset clearly indicates that the integrated fluorescein molecules increased with the increasing UV irradiation time, in which the saturated value depends on the number of HPF molecules coated on the cover glass. It is worth emphasizing here that the rate constant derived from the evolution of the integrated fluorescein molecules is important for estimating the concentration of airborne •OH molecules. Assuming that the bimolecular reaction between •OH and HPF obeys pseudo-first-order kinetics, the reaction rate constant (kOH) was estimated to be 0.16 s-1 by a single-exponential function. It should be noted that the kinetics of the formation of •OH for the pure and N-doped TiO2 films are almost the same. This result agrees with the fact that the photocatalytic activity of the N-doped TiO2 under UV irradiation is comparable to that of the pure TiO2 in the ensemble measurements.35 On the other hand, no significant •OH molecules were detected for both TiO2 films, although a significant number of 1O2 molecules were detected with TDI only for the N-doped TiO2 under visible-light irradiation. These results suggest that the photon energy in the visible light (wavelength: 435 nm) is DOI: 10.1021/la900790f

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Figure 5. (A) Single-molecule detection of •OH molecules with HPF. (B) Real-time single-molecule detection of the airborne •OH diffused

from the surface of pure TiO2 with the simultaneous 365 nm irradiation. The intervening gap is 12.5 μm. The vertical axis is the number of fluorescein molecules that emerged per bin time (33 ms), which corresponds to the count rate of the •OH molecules. The 365 nm excitation of the TiO2 film was started at t = 14 s (top). The airborne •OH molecules were detected immediately after the 365 nm excitation. Integrated •OH molecules increased with the increasing UV-irradiation time (red line). The saturated value of the integrated •OH molecules (ca. 2700 molecules) is limited by the number of HPF coated on the cover glass. The blue line indicates the single-exponential fit (t > 14 s), of which the decay rate corresponds to the pseudo-first-order rate constant (kOH) between the HPF and •OH molecules. Inset shows the semilog plot of the dependence of the intervening gap. The lifetime of the airborne •OH in ambient air is estimated to be ∼170 μs from the slope of the straight line. (C) (top) Schematic representation of the observation of the lateral diffusion of •OH molecules from the UV-irradiated pure TiO2 film. The fluorescence image of the HPF-coated cover glass captured during the UV irradiation is shown. Immediately after the UV irradiation, •OH molecules spread on the cover glass within a couple of seconds. (bottom) Plot of the mean-square displacement Ær2æ vs the arrival R time of the •OH molecules. Fitting lines using the mean-square displacement in one dimension, Ær2æ = 2Dsurf OHt , are shown by the solid line (Brownian motion model, R = 1) and dotted line (superdiffusive model, R = 1.5). The diffusion coefficients of the •OH molecules on the cover -10 glass (Dsurf and 7.0 10-10 m2 s-1, respectively. For additional results including movies, OH) calculated from the fitting lines are 7.5 10 see ref 32.

insufficient to cause the photolysis of H2O2 molecules to form • OH molecules. The dependence of kOH on the intervening gap under UV irradiation is shown in the inset of Figure 5B. As expected, the kOH decreased with the intervening gap. From the data regarding the influences of the intervening gap, the spatial distribution was examined by postulating the Gaussian distribution kOH ¼ A expð -ðkq =2DOH Þd 2 Þ

ð6Þ

which was applied by Midden et al. to describe the concentration of the airborne 1O2 molecules generated from the photoirradiated sensitizers coated on slica gels.38 In this equation, kOH is the pseudo-first-order rate constant of the reaction between the HPF and •OH molecules at the intervening gap of d, A is the rate constant at the TiO2 surface, kq is the decay rate of •OH in ambient air, and DOH is the diffusion coefficient of the •OH molecule in ambient air (2.2 10-5 m2 s-1).12 A semilog plot of the dependence of the intervening gap according to eq 6 is shown in the inset. Using kq, which can be estimated from the slope of the straight line, the lifetime of •OH in ambient air is roughly estimated to be 170 μs. This is relatively lower than that of •OH in ambient air previously reported (ca. 20 ms).39 This would be explained by the escape of the (38) Midden, W. R.; Wang, S. Y. J. Am. Chem. Soc. 1983, 105, 4129–35. (39) Sadanaga, Y.; Yoshino, A.; Watanabe, K.; Yoshioka, A.; Wakazono, Y.; Kanaya, Y.; Kajii, Y. Rev. Sci. Instrum. 2004, 75, 2648–2655.

7796 DOI: 10.1021/la900790f

airborne •OH molecules from the detection region by the spatial diffusion and the scavenging of •OH by H2O2 or other airborne species in the gas phase, which are accompanied by the formation of •OH during the TiO2 photocatalytic reactions. Furthermore, we have demonstrated the single-molecule fluorescence imaging of the diffusing •OH molecules at the glass-air interface. Figure 5C (upper) shows the fluorescence image of the HPF-coated cover glass captured during the UV irradiation. The pure TiO2 film was placed on the edge of a cover glass and irradiated with UV light to form • OH molecules. Interestingly, the single-molecule fluorescence spots emerged from the edge of the cover glass irradiated with UV light and spread on the glass surface within a couple of seconds. We have determined the DOH on the cover glass (Dsurf OH) from the apparent lateral diffusion of the generated fluorescein molecules that is produced by the reaction with •OH molecules. Because of the nonuniform illumination and the simplification of the analysis, we assumed the one-dimensional diffusion of • OH molecules on the cover glass, although •OH molecules generated on the UV-irradiated TiO2 film would two-dimensionally diffuse. To roughly estimate Dsurf OH, the mean-square displacement Ær2æ vs the arrival time of •OH molecules was plotted, as shown in Figure 5C (bottom). By fitting the data using the mean-square displacement in one dimension, Ær2æ = 2Dsurf OHt -10 m2 s-1. (solid line), we calculated Dsurf OH to be 7.5 10 The estimated Dsurf OH is somewhat consistent with the diffusion coefficient of water adsorbed in the near-monolayer films on a Langmuir 2009, 25(14), 7791–7802


silica surface ((3-6) 10-10 m2 s-1 at 298 K).40,41 This fact presumably suggests that the •OH molecules are spread over the cover glass through the adsorbed water layer because the relative humidity in this measurement was maintained at about 20-30%, which corresponds to that in the formation of the near-monolayer water. A plausible mechanism of the anomalous diffusion behavior of • OH molecules on the cover glass would be explained by the involvement of a number of adsorbed water domains or network formed on the glass and mica, as already reported.42 In addition, the accommodation and the surface activity of the airborne •OH molecules at the air-water interface were previously predicted by Tobias et al.43 Consequently, we hypothesized that the airborne • OH molecules generated from the TiO2 film might laterally diffuse on the cover glass across a number of air-water interfaces of the domains or networks, accompanied by diffusion into the air phase (air-mediated effective surface diffusion). If the hypothesis of this mechanism is correct, the diffusion behavior of •OH molecules at the interface no longer obeys the Brownian motion characterized by a mean-squared displacement R 44 that linearly increases with time, Ær2æ = 2Dsurf OHt , where R = 1. On the other hand, such an air-mediated effective surface diffusion through the adsorbed water domains is considered to obey the superdiffusive behavior (the so-called Levy walk), in which R = 1.5.45,46 The fitting line using the superdiffusive model is shown in Figure 5C (red line). The observed data can be well fitted by the superdiffusive model compared to the Brownian motion (black line). The Dsurf OH calculated from the fitting line of this model is 7.0 10-10 m2 s-1. These results indicate that the anomalous diffusion behavior of •OH molecules on the cover glass should involve the heterogeneous diffusion processes between the adsorbed water domains or networks and the air phase. This is the first real-time observation of the diffusion of •OH molecules at the heterogeneous interface under a fluorescence microscope. Although the reliable evidence to fully explain the mechanism has not yet been obtained in the present study, our findings in this single-molecule study provide new insights into the generation, diffusion, and reaction processes of the airborne ROS at the heterogeneous interfaces.

5. Single-Molecule Observation of Photocatalytic Reaction in TiO2 Nanotubes Recently, mesoporous TiO2 materials such as nanotubes, hollow spheres, and opals have been synthesized with the objective of designing an efficient photocatalyst having a high specific surface area and high molecular selectivity.47-50 In these applications, the performance of reagent molecules should be directly determined by their accessibility to the active sites for adsorption onto and reaction with the surfaces of the materials. However, the (40) Morariu, V. V.; Mills, R. Z. Phys. Chem. (Munich) 1972, 79, 1–9. (41) Clark, J. W.; Hall, P. G.; Pidduck, A. J.; Wright, C. J. J. Chem. Soc., Faraday Trans. 1 1985, 81, 2067–2082. (42) Xu, L.; Lio, A.; Hu, J.; Ogletree, D. F.; Salmeron, M. J. Phys. Chem. B 1998, 102, 540–548. (43) Roeselova, M.; Vieceli, J.; Dang, L. X.; Garrett, B. C.; Tobias, D. J. J. Am. Chem. Soc. 2004, 126, 16308–16309. (44) Shlesinger, M. F.; Zaslavsky, G. M.; Klafter, J. Nature 1993, 363, 31–37. (45) Bychuk, O. V.; O’Shaughnessy, B. Langmuir 1994, 10, 3260–3267. (46) Bychuk, O. V.; O’Shaughnessy, B. Phys. Rev. Lett. 1995, 74, 1795–1798. (47) Lakshmi, B. B.; Dorhout, P. K.; Martin, C. R. Chem. Mater. 1997, 9, 857–862. (48) Shiraishi, Y.; Saito, N.; Hirai, T. J. Am. Chem. Soc. 2005, 127, 12820–12822. (49) Li, H.; Bian, Z.; Zhu, J.; Zhang, D.; Li, G.; Huo, Y.; Li, H.; Lu, Y. J. Am. Chem. Soc. 2007, 129, 8406–8407. (50) Pan, J. H.; Zhang, X.; Du, A. J.; Sun, D. D.; Leckie, J. O. J. Am. Chem. Soc. 2008, 130, 11256–11257.

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structural disorders and agglomerates present in bulk samples often hinder efficient molecular transport. Furthermore, because conventional measurement methods cannot be used to observe and distinguish between (photo)catalytic reactions occurring on active sites distributed over the catalyst, the inherent properties can only be obtained as the ensemble mean of the heterogeneities. In this section, we describe the methodology for evaluating the photocatalytic activity of individual porous TiO2 nanotubes,51 which have a porous structure containing a straight macropore (pore size: 100-150 nm) and mesopores between the anatase nanoparticles (pore size: 5-10 nm), by the single-molecule counting of •OH using a specific fluorescent probe, aminophenyl fluorescein (APF) (Figure 6A).37 The time- and space-resolved observation of emissive fluorescein (Fl) generated by the photocatalytic reaction clearly reveals the importance of the transport behavior of reagents through the porous structures on the photocatalytic activity and the existence of the spatial heterogeneity of reactive sites even in an isolated TiO2 nanotube. Figure 6B shows the results of the single-molecule counting of • OH generated in a single TiO2 nanotube under static conditions (i.e., without sample flow). The fluorescence intensity significantly increased immediately after UV irradiation (t > 60 s) with a 500 nM APF phosphate buffer solution (pH 7.4) (black). In contrast, in the presence of DMSO as an •OH quencher, the increase in fluorescence was suppressed (red). These results confirm that fluorescein molecules were produced by •OH and not by the auto-oxidation of APF caused by UV irradiation. Importantly, the fluorescence can be observed only within the time resolution (33 ms), suggesting that the fluorescein generated in the pores rapidly diffused out of the nanotube and into the bulk solution. Figure 6B also represents TiO2 nanotubes that exhibit a diffusion behavior inherent in the macropore (b) and mesopore (c). The figures show the transmission (left) and fluorescence images (right) captured at the same position, and the trajectories of the displacement of the spots are indicated by the arrows (inset, blue). The fluorescent spot in image b (right) is much broader than the size of the diffraction-limited spot (ca. 200 nm). This result infers that the fluorescein molecule is generated in the straight macropore in which the molecule can freely diffuse. On the other hand, the fluorescent spot shown in image c (right) represents contrasting results as follows: (1) the trajectory due to slow diffusion, (2) the line profile seen as the diffraction-limited spot, and (3) maintenance of the fluorescence intensity for a duration longer than the time resolution of the data acquisition. It is also noteworthy that the diffusing molecule mainly exists in a region within an area of ca. 40 40 nm2, which is of the same order as the wall thickness of the nanotube. The possible adsorption of fluorescein on a TiO2 particle can be ruled out because of the following reasons: movement of the molecule, fluorescence quenching due to electron transfer, and electrostatic repulsion between the negatively charged fluorescein and the TiO2 particle in the phosphate buffer solution.52 Thus, the fluorescein molecule shown in image c must be present in the mesopores between the nanoparticles. From the results mentioned above, the photocatalytic activity inherent in the porous structures can be separately evaluated. Panel a of Figure 6C shows the histograms of the counting rates of single fluorescein molecules, RFl, measured at the macropore and mesopore, indicating that the average values of RFl in these pores (51) Naito, K.; Tachikawa, T.; Fujitsuka, M.; Majima, T. J. Am. Chem. Soc. 2009, 131, 934–936. (52) Li, X.; Chen, C.; Zhao, J. Langmuir 2001, 17, 4118–4122.

DOI: 10.1021/la900790f

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Figure 6. (A) Schematic representation of photocatalytic reaction occurring on a single nanotube (a). The porous TiO2 nanotube on the cover glass is simultaneously irradiated with UV light (wavelength: 365 nm) and evanescent light produced by a CW Ar+ laser (wavelength: 488 nm) to excite the nanotube and fluorescein, respectively. TEM images of TiO2 nanotubes synthesized by the sol-gel template method, which have porous structures containing a straight macropore (pore size: 100-150 nm) (b) and mesopores between the anatase nanoparticles (pore size: 5-10 nm) (c). (B) Time trajectories of the fluorescence intensity of the entire TiO2 nanotube in the absence (black) and presence (red) of 100 mM DMSO as an •OH quencher. The black line is the threshold level. Transmission image (left) and fluorescence image (right) obtained for single TiO2 nanotubes (b and c). Inset shows the peak trajectory of single-molecule fluorescence generated in the macropore (b) and mesopore (c) of the TiO2 nanotube. (C) Histograms of the counting rates of single fluorescein molecules, RFl, in the macropore (red) and mesopore (blue) of the TiO2 nanotube (a). Line profile of the integrated fluorescence intensity along the long axis shown by the dotted line in the inset (blue) and the histogram of the number of fluorescein molecules generated at that position, which is determined from the peak of the Gaussian distribution (red) (b). The fluorescence intensity should be directly correlated with the photocatalytically active sites. The integrated fluorescence intensity of the TiO2 nanotube over 120 s during the photocatalytic reaction (inset of panel c). These results are reported in ref 51.

are 0.85 and 0.065 molecules μm-1 s-1, respectively. RFl of the mesopore is 1 order of magnitude lower than that of the macropore, despite the possible advantage that its small volume facilitates the accumulation of •OH. This finding strongly indicates the effect of the kinetics of the transport of reagents on the photocatalytic activity, which is not evident in the bulk measurement. Furthermore, we have attempted to study the spatial heterogeneity of active sites on individual TiO2 nanotubes. To avoid any potential artifacts due to the enhanced light scattering arising from the existence of cracks or branches on the nanotube, the histogram of the number of fluorescein molecules counted along the dotted line in the image shown in the inset was carefully examined (red). In this case, the precise position at which fluorescein is generated is determined from the Gaussian fits to the fluorescence intensity profiles of fluorescein that immediately disappeared by fast diffusion. For the sake of comparison, the line profile of the integrated fluorescence intensity is also shown (blue). 7798 DOI: 10.1021/la900790f

The fact that the peaks in both profiles approximately correspond to each other reveals the heterogeneous distribution of the active sites even in the isolated nanotube. The origin of the observed heterogeneity has not yet been fully explained; however, it is possibly attributed to the intrinsic distribution of surface defects, such as oxygen vacancies that are present in the respective nanotubes, which should mediate the electron transfer from the conduction band to O2, thus leading to the generation of H2O2 as the precursor of •OH.53 In summary, we have investigated the photocatalytic activity of individual porous TiO2 nanotubes by the single-molecule counting of •OH using a specific fluorescent probe. The time- and (53) •OH is trapped on the TiO2 surface and is converted to a less-active surface OH with a nearly diffusion-controlled rate, indicating that the •OH generated in the macropore and mesopore scarcely diffuses out of the pores, and hence, the equilibrium concentration of •OH in the nanotube under UV irradiation depends on the local concentration of the precursor H2O2 molecules in the pores.

•

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space-resolved observation of the emissive fluorescein generated by the photocatalytic reaction clearly reveals that the transport of reagents inherent in the porous structures is closely related to the photocatalytic activity. Furthermore, we discovered the spatial heterogeneity of reactive sites in an isolated TiO2 nanotube for the first time. Experiments on a single nanotube provide information that is useful for elucidating the reaction mechanism of the heterogeneous (photo)catalyst and for designing advanced porous materials.

6. Defect-Mediated PL Dynamics of Eu3+-Doped TiO2 Nanoparticles Rare-earth (RE)-doped materials are finding use in a wide variety of applications in optics as gain media for amplifiers and lasers and as biolabels, white-light emitters, and fullcolor phosphors for displays.54-56 Since direct excitation of the parity-forbidden intra-f-shell RE ion crystal-field transitions is inefficient, it is anticipated that the luminescence of RE ions incorporated in the wide band gap semiconductor lattice (e.g., ZnO and TiO2) could be efficiently sensitized by exciton recombination in the host.57-60 Although the various types of defect states have been considered to play an important role in energy transfer between TiO2 and the activator Eu3+ ions, the mechanism of the energy transfer process from the defect energy levels of the host to dopants has not yet been clarified due to several difficulties, such as the nonhomogeneous distribution of ions in the material. In this study, we investigated the PL dynamics of undoped TiO2 and TiO2:Eu3+ (0.5 atom %) nanoparticles (or their aggregates) using single-particle PL spectroscopy.61 The photostimulated formation of emissive defects at the TiO2 surface and the defect-mediated PL of the doped Eu3+ were examined at the single-particle or aggregate level. As shown in Figure 7A, individual TiO2 nanoparticles or aggregates show a very weak emission in the visible region, which mostly comes from scattered light during the 405 nm laser irradiation in ambient air (left). However, a dramatic increase in the emission intensity was observed by changing the atmosphere from air to Ar during the photoirradiation (right). The spectral measurements suggest the growth of a broad PL band in the visible region (500-750 nm), which can be mainly assigned to the oxygen-vacancy-related defects (color centers) (for example, see the blue line in Figure 7C).62,63 According to the literature,64 the main feature of the kinetics under visible-light irradiation is the dependence of the absorbance of the sample on the number of color centers (N). Assuming that the rates of formation (k+) and deactivation (k-) of the color (54) Chen, J.; Patil, S.; Seal, S.; McGinnis, J. F. Nat. Nanotechnol. 2006, 1, 142–150. (55) Di Maio, J. R.; Sabatier, C.; Kokuoz, B.; Ballato, J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 1809–1813. (56) Wang, F.; Liu, X. J. Am. Chem. Soc. 2008, 130, 5642–5643. (57) Conde-Gallardo, A.; Garcia-Rocha, M.; Hernandez-Calderon, I.; Palomino-Merino, R. Appl. Phys. Lett. 2001, 78, 3436–3438. (58) Frindell, K. L.; Bartl, M. H.; Robinson, M. R.; Bazan, G. C.; Popitsch, A.; Stucky, G. D. J. Solid State Chem. 2003, 172, 81–88. (59) Jia, C. W.; Xie, E. Q.; Zhao, J. G.; Sun, Z. W.; Peng, A. H. J. Appl. Phys. 2006, 100, 023529/1–023529/5. (60) Li, J.-G.; Wang, X.; Watanabe, K.; Ishigaki, T. J. Phys. Chem. B 2006, 110, 1121–1127. (61) Tachikawa, T.; Ishigaki, T.; Li, J.-G.; Fujitsuka, M.; Majima, T. Angew. Chem., Int. Ed. 2008, 47, 5348–5352. (62) Serpone, N.; Lawless, D.; Khairutdinov, R. J. Phys. Chem. 1995, 99, 16646–16654. (63) Serpone, N. J. Phys. Chem. C 2006, 110, 24287–24293. (64) Kuznetsov, V. N.; Serpone, N. J. Phys. Chem. C 2007, 111, 15277–15288.

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centers are directly proportional to N, then one obtains NðtÞ ¼

kkþ

-

kkþ

1

- N10

expð -k þ tÞ

ð7Þ

where N0 is the number of color centers that exist prior to irradiation. The observed trajectories of the PL intensity were well reproduced by eq 7 (Figure 7B). Although it is very difficult to estimate the absolute value, the N0 values significantly increased by about 20 times after the photoactivation. Assuming that the absorption cross section and PL quantum yield of color centers are constant, the k+ value is determined to be 0.05 ( 0.01 s-1 for both processes, while the k- values are 0.5 ( 0.2 and 15 ( 5 s-1 for the photoactivation and deactivation processes, respectively. This remarkable difference in k- should be due to the different oxygen concentrations in the gas phase. Figure 7C shows the time evolution of the PL spectra observed for a single TiO2:Eu3+ nanoparticle (or aggregate) under the Ar atmosphere. Only the PL bands due to Eu3+ were observed immediately after the laser irradiation, and then, the PL band from the defects appeared in the visible region (500-750 nm) and increased over time. It should be noted that the PL bands attributed to the 5D0 f 7F1 and 5D0 f 7F2 transitions increased and decreased with the irradiation time, respectively, and this change seems to be synchronized with the photoactivation event. This contains information difficult or impossible to obtain from ensemble experiments since each particle or aggregate behaves differently. According to the fact that the Eu3+ ions located at the surface have a lower R value, where R is the relative intensity (area) ratio of 5D0 f 7F2 to 5D0 f 7F1, than that in the interior region of the TiO2 host in ambient air,65 it is inferred that the emission from the Eu3+ ions located at the surface is significantly enhanced by the continuous photoirradiation. On the basis of the above results and discussion, we have proposed the PL mechanisms as summarized in Figure 7D. First, visible color centers, most probably, trapped e- in the vacancy defect sites, are generated by the intrinsic and/or extrinsic excitations of TiO2 nanoparticles under the 405 nm laser irradiation. Both free and trapped e- can then recombine with the photogenerated h+ to produce the PL in the UV and visible regions, respectively. In our experimental setup, only the latter could be detected. The quenching of e- by O2 molecules consequently results in the decreased PL. Free excitons should excite both the interior and surface-located Eu3+ ions, while the recombination of trapped charges at the surface would only excite the surfacelocated Eu3+ ions. This interpretation is supported by the fact that the photoactivation of the color centers in the TiO2 host, i.e., the formation of trapped charges at the defect sites, is accompanied by a significant decrease in R. Our method will allow us to gain a further insight into the mechanisms of charge and energy transfer within hybrid inorganic nanomaterials.

7. Photoinduced Cleavage of Single TiO2/DNA Nanoconjugates Bioconjugated nanomaterials that are composites between metal or semiconductor nanomaterials and biomolecules, such as DNA and proteins, have the potential to produce various unique and new functions in a variety of areas including (65) When the TiO2 surface was modified with octadecyltrimethoxysilane, the distribution of R values shifted to higher values (from 4.8 to 6.3) and became narrower relative to the PVA-coated sample. From these findings, it is considered that the Eu3+ ions located at the surface have a lower R value than those in the interior region of the TiO2 host in ambient air.

DOI: 10.1021/la900790f

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Figure 7. (A) Typical PL images observed during the 405 nm laser excitation for single TiO2 nanoparticles (or aggregates) under air (left) and Ar (right) atmospheres (scale bars are 10 μm). (B) Trajectories of PL intensities under Ar (red) and air (blue) atmospheres (bin time is 33 ms). The photoactivation is accompanied by numerous “photon bursts” until saturation of the PL intensity occurred (and vice versa). Solid lines indicate the kinetic traces calculated using eq 7. The k+ value was determined to be 0.05 ( 0.01 s-1 for both processes, while the k- values are 0.5 ( 0.2 and 15 ( 5 s-1 for the photoactivation and deactivation processes, respectively. (C) Time evolution of the PL spectra obtained during the 405 nm laser excitation for a single TiO2:Eu3+ nanoparticle (or aggregate) under an Ar atmosphere. The PL bands around 590 and 615 nm are attributable to transitions from the 5D0 level to the 7F1 and 7F2 levels of Eu3+, respectively. (D) Schematic illustration of the energy transfer from the TiO2 host to the doped Eu3+ ions. The Eu3+ ions located on the surface of the TiO2 host are sensitized efficiently by charge recombination of the trapped carriers. For additional results including movies, see ref 61.

biosensors, drug and gene delivery, etc.66-69 While the conjugations can be significant, several problems cannot be ignored, i.e., the undesirable reactions and byproducts, the possible toxicity or nonbiocompatibility of the used materials, and the higher cost of controlled-release systems. Most significant, the adsorption and chemical reaction dynamics presumably associated with the spatial heterogeneities of the surface and the local environments make it rather difficult for ensemble-averaged measurements to evaluate their functions.70 In this study, to overcome the above-mentioned problems, we investigated the photocatalytic reactions of a novel nanoconjugation consisting of TiO2 nanoparticles and DNA at the singlemolecule level.71 As illustrated in Figure 8A, we selected the catechol (CA) moiety as a key feature of the synthetic DNA for the fabrication of nanoconjugates with TiO2 nanoparticles (diameter: 3-4 nm).72 Here, a dopamine molecule was introduced into the 5-methylcytosine (CMe) group of the DNA by postmodification. Also, we applied the photocatalytic oxidation of the CA moiety, which produces the nonadsorbed products, such as quinones, to release the modified DNA into solution. In order to detect the photoinduced cleavage of single nanoconjugates upon UV irradiation, the DNA strand was modified with a strongly (66) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042–6108. (67) Paunesku, T.; Rajh, T.; Wiederrecht, G.; Maser, J.; Vogt, S.; Stojicevic, N.; Protic, M.; Lai, B.; Oryhon, J.; Thurnauer, M.; Woloschak, G. Nat. Mater. 2003, 2, 343–346. (68) Dimitrijevic, N. M.; Saponjic, Z. V.; Rabatic, B. M.; Rajh, T. J. Am. Chem. Soc. 2005, 127, 1344–1345. (69) Clarke, S. J.; Hollmann, C. A.; Zhang, Z.; Suffern, D.; Bradforth, S. E.; Dimitrijevic, N. M.; Minarik, W. G.; Nadeau, J. L. Nat. Mater. 2006, 5, 409–417. (70) Tachikawa, T.; Takai, Y.; Tojo, S.; Fujitsuka, M.; Majima, T. Langmuir 2006, 22, 893–896. (71) Tachikawa, T.; Asanoi, Y.; Kawai, K.; Tojo, S.; Sugimoto, A.; Fujitsuka, M.; Majima, T. Chem.;Eur. J. 2008, 14, 1492–1498. (72) Rajh, T.; Chen, L. X.; Lukas, K.; Liu, T.; Thurnauer, M. C.; Tiede, D. M. J. Phys. Chem. B 2002, 106, 10543–10552.

7800 DOI: 10.1021/la900790f

luminescent semiconductor quantum dot (QD) (Invitrogen, Qdot 605 Streptavidin Conjugate) as a probe via a streptavidin-biotin interaction. It is expected that the moving away of the QDmodified DNA from the evanescent field or focus area, which is caused by the photocatalytic oxidation of the CA moiety to produce quinones, should quench the luminescence. Figure 8B shows the UV irradiation time dependence of the N/N0 values obtained for the fully matched and mismatched DNA duplexes, where N0 and N denote the number of luminescent spots before and after the UV irradiation, respectively. Before the UV irradiation, a large number of luminescent spots were observed for the fully matched DNA, while only a few luminescent spots were observed after a 30 s UV irradiation (black). On the other hand, the decrease in N was significantly inhibited by a single A:C mismatch in the DNA sequences, inferring that the migration of holes, which are injected from the photoexcited TiO2 into the DNA, through the DNA bases plays an important role in the decreased N. It should be noted that the yield of the cleaved TiO2/DNA nanoconjugates was changed by over 30% with only a single base mismatch, and this variation is much greater than that (ca. 10%) obtained at the bulk level for the TiO2/DNA suspensions. This difference is possibly due to a nonspecific adsorption on the TiO2 surface and the possibility for changing the adsorption behaviors, i.e., physisorbed and chemisorbed species. The characteristic lifetimes of the nanoconjugates during UV irradiation were tentatively determined to be 4 and 11 s for the fully matched and mismatched DNA duplexes, respectively, by single-exponential fits, although the decay profile should display a nonexponential nature that may be a consequence of the complexity and nonhomogeneity of the reaction dynamics. When considering the TiO2 photocatalytic reaction mechanisms, the influences of the photogenerated ROS cannot be ignored (see Scheme 1). As is well-known, oxidative DNA damage is Langmuir 2009, 25(14), 7791–7802



Figure 8. (A) Optical detection of single TiO2/DNA nanoconjugates. The luminescence from the photoexcited QD modified at the DNA should be quenched due to the dissociation from the surface over the evanescent field or focus area, which is caused by the photocatalytic oxidation of the catechol (CA) moiety to produce the nonadsorbed products, such as quinines. (B) UV irradiation time dependence of the N/N0 values obtained for the fully matched and mismatched DNA duplexes. N0 and N denote the number of luminescent spots before (over 300 spots) and after the UV irradiation, respectively. A single mismatch (A:C mismatch) is shown in pink. The characteristic lifetimes of the nanoconjugates under UV irradiation were determined to be 4 and 11 s for the fully matched and mismatched DNA, respectively, by single-exponential fits. (C) N/N0 values obtained from the single-particle PL measurements for the fully matched DNA in the absence and presence of NaN3 (100 mM), DMSO (100 mM), SOD (5 nM), and the mismatched DNA. The UV irradiation time is 30 s. (D) Proposed photocatalytic reaction mechanisms of the TiO2/DNA nanoconjugate. The minus and plus signs denote an electron and hole, respectively. HT and CR denote the hole transfer and charge recombination reactions, respectively. SS denotes the surface states. For additional results, see ref 71.

initiated by a reaction with the ROS, such as 1O2, by hydrogen atom abstraction from the deoxyriboses to form intermediate radicals, and by the loss of electrons from the aromatic bases that form radical cations. To identify the ROS, several of the following experiments have been performed: (1) in the presence of NaN3, (2) in the presence of DMSO (•OH quencher), and (3) in the presence of superoxide dismutase (SOD) (O2•- quencher). In all the experiments, the decrease in N upon UV irradiation was suppressed (Figure 8C). In particular, in the case of NaN3, which is an effective scavenger of • OH, O2•-, and 1O2, as well as the photogenerated holes, the decrease in N was completely inhibited. On the other hand, a relatively weak inhibition was observed for the other scavengers. Our experimental results clearly suggest that several free ROS in solution are involved in the photocatalytic oxidation processes of DNA. The proposed photocatalytic reaction mechanism of the TiO2/ DNA nanoconjugate is summarized in Figure 8D. Our results clearly suggest that both the photogenerated holes in TiO2 and the free ROS in solution are involved in the oxidation processes of the CA moiety and/or DNA itself. According to the literature,73 the forward and return hole transfer rates from G•+ to GG separated by a single A:T base pair (GAGG sequence) were reported to be 6.0 and 1.7 107 s-1, respectively. It should be noted that the hole transfer rate dramatically decreased due to the longer bridge consisting of two A:T base pairs separating the proximal G and the distal GG. The determined forward and return hole transfer (73) Senthilkumar, K.; Grozema, F. C.; Guerra, C. F.; Bickelhaupt, F. M.; Lewis, F. D.; Berlin, Y. A.; Ratner, M. A.; Siebbeles, L. D. A. J. Am. Chem. Soc. 2005, 127, 14894–14903.

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rates from G•+ to GG separated by two A:T base pairs (GAAGG sequence) were 4.8 105 and 2.4 104 s-1, respectively. On the other hand, the charge recombination dynamics between the surface-bound radical cations, such as CA•+, and the electrons in TiO2 showed a wide distribution from 105 to 1012 s-1 due to the heterogeneous electron trapping and detrapping processes. These kinetic data would partially explain why we could observe a significant mismatch effect on the cleavage of the nanoconjugates upon UV irradiation, such that the formation of intermediate radical cations near the surface increases the charge recombination and thus lowers the quantum yield for degradation. Thus, it was considered that the spatial separation between the photogenerated charge carriers by the G-hopping of holes enhanced the oxidation efficiency of CA, which acts as a deep hole trap, because the electrons in TiO2 are allowed to competitively react with oxygen molecules at the interface. In other words, for the fully matched DNA duplex, the photogenerated electrons in TiO2 would be eventually consumed by oxygen molecules before recombining with G•+, resulting in the efficient oxidation of the CA moiety. On the other hand, for the mismatched DNA duplex, the photogenerated holes should localize near the surface of the TiO2 nanoparticles, resulting in the efficient charge recombination with electrons in TiO2. In summary, we have successfully observed the photocatalytic cleavage of TiO2/DNA nanoconjugates upon UV irradiation at the single-molecule level. Notably, the present conjugates can recognize a difference in a single nucleotide. This means that the present conjugate has the potential for applications such as novel biosensing and photoinduced drug release systems. DOI: 10.1021/la900790f

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8. Conclusion and Perspectives We have investigated the temporal and spatial distribution of ROS generated during the photoirradiation of TiO2 nanoparticles using single-molecule fluorescence spectroscopy. The methodologies for the single-molecule detection of ROS, such as 1 O2 and •OH, have been introduced together with examples. In 2007, we published a feature article entitled “Mechanistic Insight into the TiO2 Photocatalytic Reactions: Design of New Photocatalysts” in J. Phys. Chem. C.5 In this article, we mentioned that the single-molecule nanosensors for the detection of airborne ROS will be applicable for the multiple detection of the ROS generated during the photocatalytic reactions. At the present time, although it is still difficult to simultaneously detect two different kinds of ROS, we have developed our methodology and have adopted the following core conclusions: (1) The contribution of the excited-state of single fluorescent probes in the real-time detection of ROS should be considered. (2) Upon UV excitation, 1O2 and •OH were detected from both the pure and N-doped TiO2 samples, while 1O2 was exclusively detected only from the N-doped TiO2 upon visible excitation. (3) The bimolecular reaction rate constant between •OH and the substrate (kOH), which obeys pseudo-first-order kinetics, can be regarded as a steady-state concentration of the airborne •OH molecules. (4) The diffusion of •OH molecules through the air-water interface on a glass surface was directly observed and interpreted in terms of the superdiffusive model. It is worth emphasizing that the diffusion distances of airborne 1O2 (>1000 μm) and •OH (ca. 100 μm) in ambient air are significantly different. This implies the possibility of selective oxidation of substrates by changing the intervening gap between the substrates and the TiO2 film. Current efforts are also directed toward developing and applying this method for studying the (photo)catalytic reactions in various heterogeneous systems such as solid catalysts and living cells. In section 5, we have studied the photocatalytic activity of individual porous TiO2 nanotubes by the single-molecule counting of •OH using a specific fluorescent probe. The time- and space-resolved observation of emissive fluorescein generated by the photocatalytic reaction clearly reveals that the transport of reagents inherent in the porous structures is closely related to the photocatalytic activity. Furthermore, we discovered the spatial heterogeneity of reactive sites in an isolated TiO2 nanotube for the first time. There can be no doubt that the diffusion behavior of probe molecules or analytes in heterogeneous structures of TiO2 materials is influenced by the structural disorders, such as the existence of agglomerates, cracks, or branches, and the distribution of the particle size and pore diameter, which cannot be addressed by the wide-field fluorescence imaging. To assess the structure of nanotubes at the nanometer scale, a combination of fluorescence imaging and TEM or AFM analysis on a single nanotube is required, as already performed by other groups.20 In section 6, we have clarified the defect-mediated PL dynamics of pure and Eu3+-doped TiO2 nanoparticles at the single-particle or single-aggregate level. The quantitative examination of the spectral and kinetic characteristics revealed that the PL bands originating from defects in the bulk and/or on the surface appeared in the visible region with numerous photon bursts by the photoirradiation with a 405 nm laser in an inert gas atmosphere. The spatially resolved PL imaging techniques thus enabled us to ascertain the location of the active sites and to characterize the transport behavior of charge carriers in the 7802 DOI: 10.1021/la900790f


TiO2 nanostructures with various morphologies, e.g., nanotubes, nanowires, nanosheets, etc. Such information is useful not only for exploration but also for the development of TiO2-based solar cells and photocatalysts and provides us insights not available from other methods. Furthermore, to clarify the relationship between the distributions of the photocatalytically active sites and surface defects, the direct monitoring of the defect-mediated PL and ROS on a single TiO2 nanostructure should be attempted. The feasibility of studying heterogeneous reactions at the single-molecule level permits us to devise completely new experimental schemes. In section 7, we fabricated TiO2/DNA nanoconjugates using the catechol moiety as a binding functional group and demonstrated that this novel conjugate can recognize a difference in a single nucleotide upon UV irradiation. The photocatalytic degradation mechanism of catechols at the TiO2 surface was interpreted in terms of the interfacial charge recombination reaction of their radical cations with the conduction band electrons. It should be noted that the interfacial electrontransfer processes are closely associated with the spatial heterogeneities of the nanoscale local environments and the nonhomogeneous vibronic coupling between the adsorbed molecules and the rough surfaces of the semiconductors.74-79 Forthcoming studies will focus on the mechanistic details to resolve the above-mentioned issues. Further developments of single-molecule fluorescence spectroscopy will extend the range of applications by adding new tools. Recently, advanced “nanoscopy” techniques, such as stimulated emission depletion (STED) microscopy80 and photoactivation localization microscopy (PALM),81 achieved optical imaging at high spatial resolution beyond the diffraction limit (below a few tens of nanometers). This feature should be highly advantageous when exploring the active sites heterogeneously distributed over the materials. It is also possible to apply the external fields or forces (e.g., pressure, current, voltage, and magnetic fields) to the system for monitoring the structural or functional response in real time.82-84 In conclusion, we believe that single-molecule (singleparticle) experiments can provide novel information for elucidating the reaction mechanism of heterogeneous (photo)catalysts and for designing or identifying new materials for applications in a variety of areas. Acknowledgment. We are grateful to a number of colleagues, especially Dr. Kazuya Naito and Prof. Mamoru Fujitsuka, and co-workers for their experimental support, invaluable suggestions, and discussions. 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 the Japanese Government. (74) Lu, H. P.; Xie, X. S. J. Phys. Chem. B 1997, 101, 2753–2757. (75) Biju, V.; Micic, M.; Hu, D.; Lu, H. P. J. Am. Chem. Soc. 2004, 126, 9374–9381. (76) Wang, Y.; Wang, X.; Ghosh, S. K.; Lu, H. P. J. Am. Chem. Soc. 2009, 131, 1479–1487. (77) Tachikawa, T.; Cui, S.-C.; Tojo, S.; Fujitsuka, M.; Majima, T. Chem. Phys. Lett. 2007, 443, 313–318. (78) Cui, S.-C.; Tachikawa, T.; Fujitsuka, M.; Majima, T. J. Phys. Chem. C 2008, 112, 19625–19634. (79) Issac, A.; Jin, S.; Lian, T. J. Am. Chem. Soc. 2008, 130, 11280–11281. (80) Hell, S. W. Science 2007, 316, 1153–1158. (81) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Science 2006, 313, 1642–1645. (82) Palacios, R. E.; Fan, F.-R. F.; Grey, J. K.; Suk, J.; Bard, A. J.; Barbara, P. F. Nat. Mater. 2007, 6, 680–685. (83) Becker, K.; Lupton, J. M.; Muller, J.; Rogach, A. L.; Talapin, D. V.; Weller, H.; Feldmann, J. Nat. Mater. 2006, 5, 777–781. (84) Protasenko, V.; Gordeyev, S.; Kuno, M. J. Am. Chem. Soc. 2007, 129, 13160–13171.

Langmuir 2009, 25(14), 7791–7802

Single-Molecule Fluorescence Imaging of TiO2 Photocatalytic

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