Real-Time Single-Molecule Imaging of the Spatial and Temporal

1048

J. Phys. Chem. C 2008, 112, 1048-1059

Real-Time Single-Molecule Imaging of the Spatial and Temporal Distribution of Reactive Oxygen Species with Fluorescent Probes: Applications to TiO2 Photocatalysts 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: August 7, 2007; In Final Form: October 24, 2007

The single-molecule detection of airborne reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydroxyl radical (•OH), diffused from the photoirradiated TiO2 surface, was successfully demonstrated using single-molecule fluorescence spectroscopy. Airborne single 1O2 and •OH molecules were selectively detected by the fluorescent probes, terrylenediimide (TDI) and 3′-(p-hydroxyphenyl) fluorescein (HPF), respectively. Generation of the airborne 1O2 and •OH from the TiO2 surface has been investigated under various conditions, such as the excitation wavelengths (UV or visible) and the types of TiO2 (pure or nitrogen (N)-doped). 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. Furthermore, the spatial and temporal distribution of the airborne •OH molecules diffused from the photoirradiated TiO2 surface was investigated by the real-time single-molecule imaging technique. The bimolecular reaction rate constant between •OH and HPF, which obeys pseudo first-order kinetics, can be regarded as a steady-state concentration of the airborne •OH molecules. Additionally, the anomalous diffusion of •OH molecules through an air-water interface (on glass surface, roughly 50 µm per 1 s) was directly observed and interpreted in terms of the superdiffusive model. Our finding in this single-molecule study provides new insights into the generation, diffusion, and reaction processes of the airborne ROS at the solid-air interfaces including TiO2 photocatalysts, air, and oxidizing substrates.

Introduction Single-molecule fluorescence spectroscopy has been used to detect single analyte molecules with specific fluorescent probes that bind to the analyte molecules or ions1,2 and recently utilized for quantitatively detecting a single oxygen (O2) molecule,3-6 which influences the fluorescence from a single-molecule such as the fluctuation of the fluorescence intensity arising from the triplet quenching.7,8 In addition, the reactive oxygen species (ROS), which can be formed by the reactions concerned with O2, such as the superoxide (O2•-), singlet oxygen (1O2), hydroxyl radical (•OH), and hydrogen peroxide (H2O2), plays crucial roles in many fields, such as atmospheric, biological, and semiconductor material sciences.9-15 Despite the importance of ROS, to the best of our knowledge, an example of the single-molecule detection of ROS has been really limited to only a few reports.16,17 Therefore, much would be gained if single ROS molecules in air, solution, and biological systems could be directly monitored with both time and spatial resolutions. ROS is also fundamental to the TiO2 photocatalytic reactions, which have been extensively studied and used for the watersplitting reaction that promotes hydrogen generation, the degradation of organic pollutants, the surface wettability conversion, etc.11,18-21 In 1999, Tatsuma et al. first reported that ROS formed on a photoirradiated TiO2 surface can be transported into the gas phase and can decompose organic and inorganic materials away from the TiO2 surface.22 This phenomenon is called remote oxidation and has been applied to the photocatalytic lithography of soot,23,24 self-assembled monolayers,25 a * Corresponding author. Tel: +81-6-6879-8495. Fax: +81-6-6879-8499. E-mail: [email protected].

silicon plate,26 and silicon carbide.27 The principal airborne ROS causing remote oxidation should be the •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.26,28 During this remote oxidation, all of the generation, diffusion, and reaction processes of the airborne ROS at the solid-air interfaces including TiO2 photocatalysts, air, and oxidizing substrates should play key roles in the reaction mechanisms. The ROS produced during the TiO2 photocatalytic reactions has been investigated in ensemble measurements using electron spin resonance (ESR),29-32 multiple internal reflection infrared,33,34 near-infrared,35,36 and laser-induced fluorescence spectroscopies,37,38 and the use of fluorescent probes.39-44 The sensitivity in the conventional experiments is, however, too low to directly detect a small amount of the airborne ROS. Therefore, it is of great importance to develop a methodology for the detection and identification of airborne ROS that diffuses from the photoirradiated TiO2 surface at the single-molecule level. Recently, we have successfully investigated the photooxidation process of a single fluorescent dye (Alexa fluor 532) caused by the airborne ROS that diffused from the photoirradiated TiO2 surface45 and specifically detected a single 1O2 molecule at the distance of >1000 µm from the TiO2 surface in ambient air using total internal reflection fluorescence microscopy (TIRFM).17 Therefore, we concluded that not only the •OH molecules but also the 1O2 molecules provide significant contributions to the remote oxidation mechanisms, because the 1O2 molecule is an important ROS in atmospheric, biological, and therapeutic processes.46,47 However, further investigations, such as the

10.1021/jp076335l CCC: $40.75 © 2008 American Chemical Society Published on Web 01/08/2008

Single-Molecule Detection of Reactive Oxygen Species

J. Phys. Chem. C, Vol. 112, No. 4, 2008 1049

SCHEME 1. Single-Molecule Detection of the Reactive Oxygen Species

formation condition of airborne ROS and the selective detection method, are required for a deeper understanding of the reaction dynamics at the heterogeneous interface. In the present study, we have developed the methodology for the selective single-molecule detection of the airborne ROS, such as 1O2 and •OH, diffused from the pure and nitrogen (N)doped TiO2 under UV- or visible-light irradiation and successfully have demonstrated the real-time single-molecule imaging of the spatial and temporal distribution of the •OH molecules diffused from the photoirradiated TiO2 into air and the heterogeneous interface. For the real-time single-molecule fluorescence imaging, we improved the custom-built fluorescence microscope so that we are able to observe a single-molecule fluorescence image under UV- and visible-light irradiations, which means the simultaneous excitation of the fluorescent probes and TiO2. The visible-light-responsive TiO2, such as N-doped TiO2, has been developed and studied to produce photocatalysts working under visible light, which is a significant part of sunlight.48-50 Exploring the airborne ROS formed under the UV- or visiblelight irradiation is fundamental for the application of the visiblelight-responsive TiO2. In addition, the water oxidation on the surface of the N-doped TiO2 under visible-light irradiation was considered to be induced by the nucleophilic attack of a H2O molecule on the surface-trapped hole at a bridged O site.34,51 However, whether or not the oxidation mechanisms contribute even a little to the formation of the airborne •OH molecules have not yet been investigated due to the lack of sensitivity of the conventional measurements. Herein, we discuss the contribution of the water oxidation to the formation of the airborne •OH molecules. For the selective single-molecule detection of the ROS, such as 1O2 and •OH, we used two fluorescent probes, that is, terrylenediimide (TDI) and 3′-(p-hydroxyphenyl) fluorescein (HPF), respectively. TDI coated on a poly(methylmethacrylate) (PMMA) thin film can selectively react with a single 1O2 molecule to form a less fluorescent endoperoxide and a strongly fluorescent diepoxide with a spectral blue-shift, which can be easily detected upon 532 nm laser excitation.17 This strategy to detect 1O2 molecules at the single-molecule level is summarized in Scheme 1A. The optimized structures of parent TDI, TDI endoperoxide, and TDI diepoxide formed in the cycloaddition reaction with 1O2 were previously investigated in our work.17 The dashed lines in Scheme 1A mean a benzene moiety in TDI

structure where 1O2 is attacked. A similar structural change and spectral shift have been reported for a single terrylene molecule doped in the p-terphenyl crystal52 and a multichromophoric rigid polyphenylenic dendrimer with spectrally different rylene chromophors.53 HPF is a fluorescent probe for the selective detection of •OH that was developed by Nagano et al.54 HPF selectively reacts with a single •OH molecule to form a strongly emissive fluorescein molecule but not with other ROS, such as O2•-, 1O2, and H2O2, generated during the TiO2 photocatalytic reactions. This reaction scheme is summarized in Scheme 1B. These selective reactions of TDI with 1O2 or HPF with •OH were observed at the single-molecule level with an evanescent field generated by a continuous wave (CW) laser (532 or 488 nm, respectively) totally reflected at the cover glass-air interface (Figure 1A). Experimental Section Sample Preparation for Single-Molecule Detection of ROS. The synthesis and characterization of pure and N-doped TiO2 powders and the preparation of the TiO2 films were described in detail in Supporting Information. TDI was synthesized using the procedures reported by Mu¨llen et al.55 HPF was purchased from Daiichi Pure Chemicals Co., Ltd. The CHCl3 and toluene were purchased from Wako Co., Ltd., and PMMA (Mw ) 15 000) was purchased from Aldrich Co., Ltd. Sodium dihydrogenphosphate anhydrate (NaH2PO4), and disodium hydrogenphosphate (Na2HPO4) for the preparation of the phosphate buffer were purchased from Nacalai Tesque, Inc. All reagents were used as supplied. The cover glasses were purchased from Matsunami Glass Ind., Ltd. and cleaned by sonication in a 20% detergent solution (As One, Cleanace) for 6 h, followed by repeated washing with warm running water for 30 min. Finally, the cover glasses were washed again with Milli-Q water. Samples for the single-molecule detection of 1O2 were prepared by spin-coating a toluene solution of PMMA (40 µL, 10 gl-1) on a clean cover glass at 3000 rpm for 15 s, followed by spin-coating a chloroform solution of TDI (40 µL, 3 nM) at 3000 rpm for 15 s. The sequence of the spin-coating in preparing the samples is very important. The PMMA film should be spincoated prior to the TDI. This underlaying PMMA has the following two roles: (i) protecting TDI from the self-sensitiza-

1050 J. Phys. Chem. C, Vol. 112, No. 4, 2008

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Figure 1. (A) Experimental setup for single-molecule detection of the airborne reactive oxygen species diffused from the TiO2 surface and the simultaneous excitation of the fluorescent probe (TDI or HPF) and the TiO2 film. (B) Single-molecule fluorescence image of the fluorescent probe coated on the cover glass. The fluorescent probe on the cover glass was observed with an evanescent field generated by a CW laser (532 or 488 nm) totally reflected at the cover glass-air interface. The diameter of the irradiated region is ca. 50 µm. (C) Irradiated region for the TiO2 film. Only the center region (diameter is ca. 10 µm) was irradiated with a mercury lamp through a 365 nm or 435 nm BP filter.

tion due to excess 3O2 and (ii) excluding the oxidation of TDI due to the radical species, such as •OH and •HO2, generated during the TiO2 photocatalytic reactions, because PMMA can react with the radicals56 but not extensively with 1O2.57 Samples for the single-molecule detection of •OH were prepared by spincoating a 40 µL phosphate buffer solution (pH 7.4, 0.1 M) of HPF (5 nM) on a clean cover glass at 3000 rpm for 15 s. 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 (Nilaco; thickness, 12.5-125 µm). A schematic representation of the sample is shown in Figure 1A. The fluorescent probes on the cover glass were observed with an evanescent field generated by a CW laser (532 nm (TDI) or 488 nm (HPF)) totally reflected at the cover glass-air interface. The diameter of the observed region was ca. 50 µm (Figure 1B). The TiO2 film-coated slide glass was irradiated only in the center region (the diameter was ca. 10 µm) with a mercury lamp through a 365 nm or 435 nm band-pass (BP) filter (Figure 1C). It is noted that the image in Figure 1C was obtained using no emission filters to remove the scattered light for visualization of the UV-visible-irradiated region. In single-molecule fluorescence detection, the scattered region in the center of Figure 1C is invisible by using suitable emission filters. Single-Molecule Fluorescence Measurements. The experimental setup is based on an Olympus IX71 inverted fluorescence microscope. A schematic illustration for the experimental setup is shown in Figure 1A. Light emitted from a CW Nd:YAG laser (532 nm, 50 mW; JDS Uniphase, 4611-050) for the TDI imaging or a CW Ar ion laser (488 nm, 50 mW; Melles Griot, IMA101010BOS) for the HPF imaging was reflected by a first dichroic mirror (Olympus, RDM450), which reflects the wavelength longer than 450 nm and is transparent for light shorter

than 450 nm, toward a second dicroic mirror (Olympus, DM570 for 532 nm excitation, DM505 for 488 nm excitation). A CW laser light passing through an objective lens (Olympus, UPlanSApo, 1.40 NA, 100×) after the reflection at a second dichroic mirror was also totally reflected at the cover glass-air interface to generate an evanescent field (penetration depth is ca. 100 nm from the interface), which can excite a fluorescent probe. The diameter of the observed region at the interface was ca. 50 µm (Figure 1B). For the excitation of the TiO2 film-coated slide glass, light emitted from a mercury lump (0.2 mW cm-2, Ushio, USH-102D) passing through a BP filter (Olympus, BP330385 for UV excitation, BP420-440 for visible excitation, shown in Figure S1) was passed through an object lens to irradiate the TiO2 film only in the center region (Figure 1C). The switching of the BP filters for excitation of the UV or visible light was manually manipulated. The switching time was ca. 100 ms. The fluorescence emission from single dye molecules on a cover glass was collected using an oil-immersion microscope objective, magnified by the built-in magnification changer 1.6× (thus, net magnification is 160×), and passed through the emission filters to remove the undesired scattered light (Olympus, BA575IF and SCF560 for 532 nm excitation, BA510IF and SCF500 for 488 nm excitation) and intensified by an image intensifier (Hamamatsu Photonics, C8600-03) coupled to a charge-coupled device camera (Hamamatsu Photonics, C307770). All the experimental data were obtained at room temperature. The images were recorded on a videocassette recorder at the video frame rate of 30 frames s-1. The pictures recorded on the videotape were converted into an electronic movie file using the ADVC 1394 video capture board (Canopus). A number of fluorescent spots were analyzed using the mean gray scale in the region of interest, which was performed using Scion Image software (http://www.scioncorp.com). A temporal change in the number of fluorescent spots versus the irradiation time



Figure 2. The influence of the excitation wavelength of UV- (A) and visible-light (B) on the detected number of 1O2 molecules using pure and N-doped TiO2. The influences upon UV irradiation are also shown in (B) for comparison (in this case, x-axis means UV-light irradiation time). ∆ Number of TDI diepoxides is the difference in the number of fluorescent spots before and after the UV- or visible-light irradiations.

in the real-time single-molecule imaging was analyzed using Gray Val32 software (Library, Japan). The superposition image of the temporal fluorescence images was created using ImageJ software (http://rsb.info.nih.gov/ij/). In the present study, the following two methods were employed to excite the TiO2 film: (i) the alternative excitation and (ii) the simultaneous excitation of the fluorescent probe on a cover glass and the TiO2 film on a slide glass. The latter method is required so that the bimolecular reaction between the excited fluorescent probe and the ROS is negligible. As discussed below, it has been observed that the excited TDI reacts with H2O2 to form the fluorescent species, while the TDI in the ground state does not. Therefore, the reaction between the excited TDI and H2O2 cannot be negligible although the reaction mechanism is inexplicit at the present stage. Consequently, we used only the alternative excitation method for the singlemolecule detection of 1O2. On the other hand, the reaction between the excited HPF and H2O2 is negligible, because no significant increase in the fluorescence intensity was observed in both the excited state and the ground state when the H2O2 gas was flowed above the HPF-coated cover glass. Therefore, we used both the alternative and simultaneous excitation methods for the single-molecule detection of •OH. Results and Discussion Single-Molecule Detection of Airborne 1O2 with the Alternative Excitation. In our previous study, we successfully demonstrated the spatial and temporal distribution of 1O2 molecules diffused from the surface of a TiO2 film irradiated by UV light.17 The results clearly indicated that TDI molecules on the PMMA film coated on a cover glass can detect the single 1O molecule at the distance of >1000 µm from the TiO surface 2 2 in ambient air. The formation mechanisms of 1O2 are described as the photocatalytic oxidation of O2•- back to 1O2 molecules, that is, reaction 1, the conduction electron, e-, and the valence hole, h+, are generated after UV irradiation of the TiO2, reaction 2, O2•- is produced by the reduction of O2 molecules in the ground state (3O2) molecules with e-, and reaction 3, O2•- is oxidized back to the 1O2 molecule by h+, resulting in 1O2 (and 3O ) on the TiO surface.35 2 2

TiO2 + hV f e- + h+

(1)

O2 + e- f 3O2•-

(2)

O2•- + h+ f 1O2 (and 3O2)

(3)

3 3

Otherwise, 1O2 is also generated by the energy transfer from the excited TiO2* to 3O2 molecules.22,58

TiO2 + hV f TiO2*

(4)

TiO2* + 3O2 f TiO2 + 1O2

(5)

The oxidation process of O2•- is considered to be dominant, when compared with that of the energy transfer mechanisms, although the experimental results supporting the energy transfer mechanisms during the formation of 1O2 have recently been reported.58 In the remote TiO2 photocatalytic oxidation, if 1O2 is generated on the TiO2 surface, it is expected to partially diffuse into the gas phase, thus forming airborne 1O2. If these mechanisms of the 1O2 formation are right, 1O2 molecules should be formed even during the visible excitation of the N-doped TiO2, based on the fact that the h+s formed on the N-sites have an oxidation potential (Eox(h+) ) +1.81 V versus normal hydrogen electrode (NHE))59 sufficient to oxidize O2•- back to 1O2 molecule (Ered(O2/O2•-) ) -0.33 V versus NHE).60 Additionally, the advantage of the visible excitation of the N-doped TiO2 is that 1O2 molecules are exclusively generated on the TiO2 surface without the formation of •OH molecules by the photolysis of H2O2, which might cause the bleaching of the TDI molecules, although almost all •OH molecules might be deactivated in the PMMA film. As shown in Figure 2, the influence of the excitation wavelength of the TiO2 film on the detected number of 1O2 molecules has been investigated using pure or N-doped TiO2. The alternative excitation was used in this data acquisition for the exclusion of the possible involvement of the undesired reaction between the excited TDI and H2O2 that diffused from TiO2 (see Experimental Section and the following section). After the UV irradiation for a few minutes, a large number of 1O2 molecules were detected for both the pure and N-doped TiO2. 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.61 The 1O molecules were reached at the numerical saturation (ca. 800 2 TDI diepoxide molecules) on the cover glass within a few


Naito et al.

Figure 3. The detected number of fluorescein molecules using (A) pure and (B) N-doped TiO2 under the alternative irradiation with UV or visible light. Fluorescein molecules (emissive product) appeared after the UV irradiation in both the pure and N-doped TiO2 films. The decrease in the fluorescein molecules during the 532 nm excitation (t > 19 s) is due to the photobleaching. The initial value of the burst corresponds to the number of •OH molecules diffused from the TiO2.

minutes and successively decreased to several dozen molecules. This decrease is probably due to the further oxidation by the multiple attacks of 1O2 on the TDI diepoxide molecules.62 After the visible-light irradiation, in contrast to the UV irradiation a significant number of fluorescent spots were detected only for the N-doped TiO2. This finding strongly indicates the formation of the airborne 1O2 under visible-light excitation and the photocatalytic activity of the N-doped TiO2.63 Because H2O2 can decompose to •OH by absorbing UV light shorter than 365 nm, the visible-light excitation cannot generate airborne •OH from the photolysis of H2O2 molecules formed by the reduction of oxygen.65 Thus, it was revalidated that the formation of TDI diepoxide is obviously ascribed to the 1O2 molecules that diffused from the TiO2 surface. These results, which can be expected from their formation mechanisms but not obtainable from the conventional measurements, strongly indicate the validity of the methodology in the single-molecule detection of ROS performed in this study. Using the detected number of 1O2 molecules and the number of photons absorbed by the TiO2 film, the generation efficiency of the airborne 1O2 molecules generated from the pure and N-doped TiO2 film were determined to be about 10-8, and 10-9, respectively. The detail for the determination of the generation efficiency was described in our previous work.17 These values are much lower than the quantum yield for the 1O2 generation measured for the TiO2 photocatalysis using near-infrared phosphorescence spectroscopy (0.0264 or 0.12-0.3836). This prominent discrepancy indicates that only small number of generated 1O2 molecules can be desorbed from the surface and diffuse into air, although almost all 1O2 molecules should be deactivated without the release from the surface. Considering that the quantum efficiencies of 1O2 molecule formed on the surface and into air are 10-1 and 10-8, respectively, only a 1O2 molecule in 107 of 1O2 molecules can diffuse from the surface of TiO2 nanoparticles.

Single-Molecule Detection of Airborne •OH with the Alternative Excitation. The single-molecule detection of airborne •OH diffused from the surface of pure and N-doped TiO2 films under UV- or visible-light irradiation with the alternative excitation (either HPF or TiO2 is excited at a time) was performed as shown in Figure 3. Visible and UV light were successively irradiated at the same position (started at t ) 8 and 17 s, respectively). The detected number of fluorescein molecules generated by the reaction between HPF and •OH denoted by a green line is considered to correspond to the number of •OH molecules (Scheme 1B). After the visible-light irradiation, as expected by the formation mechanism of •OH molecules, which is the photolysis of H2O2 molecules formed by the reduction of O2, no significant •OH molecules were detected for both TiO2 films, suggesting that the photon energy in the visible light is insufficient to cause the photolysis of H2O2 molecules to form •OH molecules.65 Additionally, this result infers that the water oxidation mechanism on the TiO2 surface is not involved in the formation of the airborne •OH molecules, although the water oxidation under visible-light irradiation has been reported to occur by a nucleophilic attack of a H2O molecule on the surface-trapped h+ at a bridged O site.51 To confirm the formation mechanism of airborne •OH molecules in detail, we investigated the influences of the concentration of oxygen on the detected number of airborne •OH molecules. The detected number of •OH molecules decreased with the decreasing concentration of oxygen (See Supporting Information for details).66 These results obtained from the single-molecule fluorescence detection also support the fact that the photolysis of H2O2 molecules is the plausible mechanism for the formation of •OH molecules in ambient air. After the UV irradiation, a large number of •OH molecules, which should be generated by the photolysis of H2O2, were observed (around t ) 19 s). The initial value of the curve



Figure 4. Influences of H2O2 vapor on the excited state of TDI. (A) H2O2 vapor was flowed during the excitation of the TDI (532 nm laser to excite TDI molecules is simultaneously irradiated with H2O2 flowing). (B) The fluorescence images of the oxidized TDI, which show all the fluorescent spots visible during the data acquisition (1 s), before (left) and after (right) the H2O2 flow. The number of the oxidized TDI molecules apparently increased due to the reaction between the excited TDI and H2O2 vapor. The time trajectories during 1 s clearly indicate the increase in the oxidized TDI after the flow and the blinking behavior (bottom). The blinking behavior of the oxidized TDI hides the signal-on arising from the 1 O2 molecules during the simultaneous excitation.

corresponds to the detected number of •OH molecules diffused from the TiO2 surface. A rapid decline in the detected fluorescein molecules after a burst is due to the photobleaching. Interestingly, significant •OH molecules were detected even in a brief UV irradiation time ( 14 s), of which the decay rate corresponds to the pseudo-first-order rate constant (kOH) between the HPF and •OH molecules.

by the self-sensitization reaction.17,53 As shown in the right panel of Figure 4B, unfortunately, the emissive products due to the oxidized TDI were observed after the flow, although the reaction mechanism between the excited TDI and H2O2 is inexplicit at the present stage. On the other hand, when the 532 nm excitation and H2O2 flow are not simultaneous, no significant fluorescent spots were observed after the flow. This indicates the ground state TDI hardly reacts with H2O2. The time trajectories of the fluorescence intensity of the oxidized TDI during 1 s before and after the flow are also shown in the bottom of Figure 4B. These clearly indicate the increase in the fluorescent products after the flow and its blinking behavior (defined as the intermittent fluorescent state).69,70 This blinking behavior during the simultaneous excitation hides the signal-on arising from the 1O2 molecules. These results reveal the serious problems of the application of TDI to the real-time fluorescence imaging of 1O2 diffused from TiO2. Thus, for the single-molecule detection of 1O2 the TDI molecule is available only in the alternative excitation in which the excited state generated by the continuous 532 nm irradiation is not involved. Consequently, we have performed the real-time single-molecule fluorescence imaging on the detection of airborne •OH with HPF. Real-Time Single-Molecule Imaging of the Spatial Distribution of Airborne •OH Molecules. A typical example of the real-time single-molecule detection of the airborne •OH diffused from the pure TiO2 film under UV irradiation is shown in Figure 5 (see Supporting Information, Movie S1). The vertical axis in Figure 5 is the number of the detected fluorescein molecules emerged per bin time (33 ms). Thus, this corresponds

Naito et al. to the count rate of the •OH molecules. The few fluorescence spots observed before the UV irradiation would be ascribed to the self-oxidation caused by the 488 nm excitation of HPF. The UV excitation started at t ) 14 s, as shown in the top of Figure 5. Interestingly, the significant airborne •OH molecules were immediately detected after the UV excitation. This infers that the time scales of the generation and diffusion processes of the airborne •OH molecules are much faster than that of the manual switching during the UV- and visible-light irradiations in this experiment (∼100 ms). This result is consistent with the fact that during the alternative excitation, significant •OH molecules were detected even in a brief UV irradiation time (Figure 3). Considering the typical diffusion coefficient of •OH in air (very similar to that of H O in air, 0.22 × 10-4 m2 2 s-1 at 1 atm)71,72 and the intervening gap (12.5 µm) between the HPF-coated cover glass and TiO2 film, the time-of-flight until the •OH diffused from the TiO2 surface reached the cover glass can be estimated to be about 4 µs, which is quite short compared with our time resolution (∼100 ms). Therefore, we assumed that the time scale of the generation process of the airborne •OH molecules is within 100 ms, and the concentration of the airborne •OH molecules under UV irradiation (t > 14 s) has almost reached the steady state. In other words, the concentration of •OH at the HPF-coated cover glass at the intervening gap will be constant as long as the intensity of the UV light remains constant, although a small amount of •OH molecules are consumed by the reaction with HPF (a pseudofirst-order approximation). In this condition, as shown in the inset of Figure 5, the integrated •OH molecules that is the summation of the number of the detected •OH molecules emerged per bin time (33 ms) should increase in the pseudo-first-order rate constant under UV irradiation. The inset also clearly indicates that the integrated •OH molecules increased with the increasing UV irradiation time. The saturated value was determined to be about 2700 molecules. This maximum value is somewhat meaningless for the estimation of the number of the airborne •OH molecules, because this value depends on the heterogeneous coating of HPF molecules on the cover glass. The rate constant derived from the evolution of the integrated •OH molecules is important for discussing the estimation of the concentration of airborne •OH molecules. For example, the inset of Figure 5 shows the fitting curve by a single-exponential function (red line), which means the pseudo-first-order reaction, and this pseudo-first-order rate constant, kOH (in this case, the value is (6.2 s)-1), corresponds to the steady-state concentration of the airborne •OH molecules at the intervening gap. Therefore, the photocatalytic activities of the pure and N-doped TiO2 films can be evaluated by using kOH. Figure 6 shows the single-molecule detection of the airborne •OH molecules that diffused from the surface of the pure and N-doped TiO2 films under simultaneous irradiation (365 or 435 nm). For both TiO2 films, the detected number of •OH per bin time (33 ms) immediately increased upon UV irradiation and decreased to the background level within 30 s. In contrast, the detected •OH molecules were almost the same as the background level under visible-light irradiation. As mentioned above, the saturated value of the integrated number of •OH molecules should be different due to the heterogeneous coating of HPF on the cover glass (the inset of Figure 5). To compare the photocatalytic activities of the pure and N-doped TiO2 films, the normalized integrated •OH molecules were examined as shown in Figure 6C. It should be noted that the kinetics of the



Figure 6. Real-time single-molecule detection of the airborne •OH diffused from the surface of the pure (A) and N-doped TiO2 films (B) with the simultaneous 365 nm or 435 nm irradiation. The intervening gap is 12.5 µm. The 365 nm or 435 nm excitation of the TiO2 film was started at t ) 12 s in all the experiments (Top). The airborne •OH molecules were clearly detected only in the 365 nm excitation. (C) The normalized integrated number of airborne •OH diffused from the surface of the pure and N-doped TiO2 films irradiated at 365 nm. The kinetics of the reaction between the •OH and HPF, that is, the photocatalytic activity under the 365 nm irradiation, are almost the same.

formation of •OH for the pure and N-doped TiO2 films are almost the same (the kOH value of pure is 0.15 s-1, and N-doped is 0.15 s-1), in line with the results for the detection of 1O2 molecules (Figure 2). Next, we investigated the spatial distribution of the airborne •OH molecules diffused from the TiO films using the k 2 OH values determined from the simultaneous single-molecule detection method. For this purpose, the kOH values were obtained by changing the intervening gap between the HPF-coated cover glass and pure TiO2 film from 12.5 to 125 µm. The dependence of the normalized integrated •OH molecules on the intervening gap under UV irradiation is shown in Figure 7. As expected, the kOH decreased with the intervening gap (Figure 7A). From the data regarding the influences of the intervening gap, the spatial distribution was examined by postulating the Gaussian distribution (eq 6)

kOH ) A exp(- (kq/2DOH)d2)

(6)

which was applied by Midden et al. to describe the concentration of the airborne 1O2 molecules generated from the photoirradiated sensitizers, such as rose bengal and methylene blue, coated on silica gels.73 It was assumed in this study that the •OH molecules

generated by the photolysis of H2O2 on the TiO2 surface, which have a finite lifetime, diffuse to the cover glass coated with HPF through air in one dimension. In this equation, kOH is the pseudo-first-order rate constant of the reaction between 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 (thus, kq-1 is the lifetime of the •OH molecules in ambient air), and DOH is the diffusion coefficient of •OH molecule in ambient air (0.22 × 10-4 m2 s-1). A semilog plot of the dependence of the intervening gap according to eq 6 is shown in Figure 7B. kq can be estimated from the slope of the straight line. Using kq, 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 reported elsewhere (ca. 20 ms).74 This would be explained by the escape of the airborne •OH molecules from the detection region by the spatial diffusion and the scavenging of •OH by H2O2 (bimolecular reaction rate constant, kb ) 1.7 × 10-12 cm3 molecules-1 s-1 at 298 K)75,76 or other airborne species in the gas phase, which are accompanied by the formation of •OH during the TiO2 photocatalytic reactions.


Naito et al.

Figure 7. (A) The influences of the intervening gap between the cover glass and TiO2 film-coated slide glass on the normalized integrated number of airborne •OH diffused from the surface of the pure TiO2 film under the 365 nm irradiation. The reaction rate decreased with the increasing of the intervening gap. (B) The dependence of the reaction rate on the intervening gap. Inset indicates semilog plot of the dependence of the intervening gap. The lifetime of the airborne •OH in ambient air is estimated to be approximately 170 µs from the slope of the straight line.

In this section, we have examined the spatial and temporal distribution of the airborne •OH molecules generated from the UV-irradiated TiO2. Because the observed region of the •OH molecules (the diameter is ca. 50 µm) is broader than the UVirradiated region (the diameter is ca. 10 µm, Figure 1C), the •OH molecules should be generated from the photolysis of H O 2 2 and not in air or on the glass surface but rather on the UVirradiated TiO2. Next, we have more clearly revealed the diffusion processes of •OH molecules from the TiO2 surface using the real-time single-molecule imaging, which can directly monitor the diffusing species with both the time and spatial resolutions. Real-Time Single-Molecule Imaging of the Lateral Diffusion of •OH Molecules at the Interface. We have successfully demonstrated the single-molecule fluorescence imaging of the diffusing •OH molecules at the glass-air interface. This result is shown in Figure 8. Figure 8A shows the successive fluorescence images of the HPF-coated cover glass during the UV irradiation. Figure 8B also shows the schematic representation of the lateral diffusion at the glass-air interface. The pure TiO2 film was placed on the edge of a cover glass and irradiated with UV light to form •OH molecules. As shown in Figure 8A, immediately after the UV irradiation, the single-molecule fluorescence emerged from the edge of the cover glass irradiated with UV light (see Supporting Information, Movie S2). These images and movie clearly indicate the real-time observation of the generation of the •OH molecules from the UV-irradiated region. 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 diffuse twodimensionally (see Supporting Information for details). To roughly estimate Dsurf OH , the mean-square displacement versus the arrival time of •OH molecules was plotted, as shown in Figure 8C. By fitting the data using the mean-square displacement in one dimension, ) 2Dsurf OH t (solid line), -10 m2 s-1. The estimated we calculated Dsurf to be 7.5 × 10 OH Dsurf OH is approximately consistent with the diffusion coefficient of water adsorbed in near-monolayer films on a silica surface (3-6 × 10-10 m2 s-1 at 298 K).77,78 This fact presumably

suggests that the •OH molecules 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.79-81 However, by considering the typical DOH in the adsorbed water (3-6 × 10-10 m2 s-1), the lifetime of •OH molecules in water (ca. 830 µs) calculated from the bimolecular reaction rate between •OH and OH- (kb ) 1.2 × 1010 M-1 s-1, measured at pH 11)82 and the concentration of OH- molecules in the buffer solution ([OH-] ) 10-7 M, at pH 7), the diffusion length of •OH molecules in the adsorbed water can be estimated to be about 1 µm, which is quite shorter compared to the observed diffusing region (∼50 µm, Figure 8A).83 This infers that all the •OH molecules cannot diffuse through the adsorbed water layer. Otherwise, it is probably considered that •OH molecules diffuse through the air and react with HPF on the -10 m2 s-1) is cover glass, although the apparent Dsurf OH (7.5 × 10 -4 2 -1 much lower than that in air (0.22 × 10 m s ). The 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 reported elsewhere.79,84 In addition, the accommodation and the surface activity of airborne •OH molecules at the air-water interface were previously predicted by Tobias et al.85 They concluded that airborne •OH molecules play an important role in the heterogeneous reactions at the thin water film on solid surfaces. Consequently, we hypothesized that the airborne •OH molecules generated from the TiO2 film might diffuse laterally on the cover glass across a number of the air-water interfaces of the domains or networks, accompanied by the 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 that linearly increases with time, ) 2Dsurf OH t , where R ) 86-88 1. On the other hand, such an air-mediated effective surface diffusion through the adsorbed water domains is considered to obey the superdiffusive behavior (so-called Le´vy walk) in which R ) 1.5.89-93 The fitting line using the superdiffusive model is shown in Figure 8C (dotted line). The observed data can be well fitted by the superdiffusive model, compared to the Brownian motion (solid line). The Dsurf OH calculated from the fitting line of this model is 7.0 × 10-10 m2 s-1. These results



Figure 8. (A) Real-time single-molecule imaging of the lateral diffusion of •OH molecules from the UV-irradiated pure TiO2 film placed on the edge of a cover glass. Immediately after the UV irradiation, •OH molecules spread on the cover glass within a couple of seconds (see Supporting Information, Movie 2). (B) Schematic representation of the observation of the lateral diffusion of •OH molecules from the UV-irradiated pure TiO2 film. The fluorescein molecules generated by the reaction between HPF and the •OH molecules are shown in red. (C) Plot of the mean-square displacement versus the arrival time of the •OH molecules. Fitting lines using the mean-square displacement in one dimension, ) 2 R Dsurf OH t , are shown by the solid line (Brownian motion model, R ) 1) and dotted line (superdiffusive model, R ) 1.5) (see text for details). The -10 diffusion coefficients of the •OH molecules on the cover glass (Dsurf and 7.0 × 10-10 m2 s-1, OH ) calculated from the fitting lines are 7.5 × 10 respectively.

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 the fluorescence microscope. However, the reliable evidence to fully explain the mechanism has not yet been obtained at the present study. A further investigation will be required for a deeper understanding of the diffusion and reaction processes of the airborne ROS at the interface. Conclusion We have successfully demonstrated the single-molecule detection of airborne ROS, such as 1O2 and •OH, generated on and diffused from the surface of a TiO2 film under UV- and visible-light irradiation. In the present study, we discussed two methods to excite the TiO2 film for the single-molecule fluorescence imaging as follows: (i) the alternative excitation and (ii) the simultaneous excitation of the fluorescent probes and the TiO2 film. We clarified the contribution of the excitedstate of single fluorescent probes in the real-time detection of ROS. For the alternative excitation, we clarified that the formation of ROS was confirmed to be sensitive to the wavelength (UV or visible) irradiated on the TiO2 film, as expected. In particular, 1O was detected from the N-doped TiO film even under 2 2 visible-light irradiation, consistent with the photocatalytic activity of the visible-light responsive TiO2. On the other hand, the

•OH

molecules were exclusively detected under UV irradiation. These results, which can be expected from their formation mechanisms but not obtainable from the conventional measurements, strongly indicate the validity of the methodology in the single-molecule detection of ROS performed in this study. During the simulteneous excitation, we succeeded in detecting single airborne •OH molecules in real-time. The bimolecular reaction between •OH and HPF was found to obey pseudo-firstorder kinetics, and thus the kOH value can be regarded as a steady-state concentration of •OH. The spatial and temporal distribution of airborne •OH molecules from the UV-irradiated TiO2 surface in ambient air was clarified using the kOH value. The lifetime of the •OH molecules also can be directly determined from their spatial distribution. Additionally, using the real-time single-molecule imaging technique, we revealed that the heterogeneous, complicated diffusion processes of the •OH molecules across the air-water interfaces of the adsorbed water domains or networks were directly demonstrated. Our finding in this single-molecule study provides new insights into the generation, diffusion, and reaction processes of the airborne ROS at the heterogeneous interfaces including TiO2 photocatalysts, air, and oxidizing substrates. Acknowledgment. The authors wish to thank Professor Yoshio Nosaka and Dr. Yoshinori Murakami, Nagaoka University of Technology, for valuable discussion. This work has been partly supported by a Grant-in-Aid for Scientific Research (Project 17105005 and others) from the Ministry of Education,

1058 J. Phys. Chem. C, Vol. 112, No. 4, 2008 Culture, Sports, Science, and Technology (MEXT) of the Japanese Government. One of the authors (K.N.) expresses his thanks for JSPS Research Fellowship for Young Scientists and the Global COE Program “Global Education and Research Center for Bio-Environmental Chemistry” of Osaka University. Supporting Information Available: Movies showing the real-time single-molecule fluorescence imaging of the diffusion of •OH molecules in air (Movie S1) and at the interface (Movie S2). Experimental details (PDF) including the description of the movies, the synthesis and characterization of the TiO2 films (S1), the measurements of the photocatalytic activities of the pure and N-doped TiO2 films by time-resolved diffuse reflectance spectroscopy (S2), the control of the relative humidity and oxygen concentration (S3), the influence of O2 and H2O2 on the formation of airborne •OH molecules (S4), and the estimation of the diffusion coefficient of the •OH molecules on a cover glass (S5). This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Zang, L.; Liu, R.; Holman, M. W.; Nguyen, K. T.; Adams, D. M. J. Am. Chem. Soc. 2002, 124, 10640-10641. (2) Kiel, A.; Kovacs, J.; Mokhir, A.; Kra¨mer, R.; Herten, D.-P. Angew. Chem., Int. Ed. 2007, 46, 3363-3366. (3) Opitz, N.; Rothwell, P. J.; Oeke, B.; Schwille, P. Sens. Actuators, B 2003, 96, 460-467. (4) Mei, E.; Vinogradov, S.; Hochstrasser, R. M. J. Am. Chem. Soc. 2003, 125, 13198-13204. (5) Erker, W.; Sdorra, S.; Basche´, T. J. Am. Chem. Soc. 2005, 127, 14532-14533. (6) Tinnefeld, P. Chem. Phys. Chem. 2006, 7, 1189-1191. (7) Nie, S.; Zare, R. N. Annu. ReV. Biophys. Biomol. Struct. 1997, 26, 567-596. (8) Xie, X. S.; Trautman, J. K. Annu. ReV. Phys. Chem. 1998, 49, 441480. (9) Seinfeld, J. H.; Pandis, S. N. Atmospheric Chemistry and Physics: From Air Pollution to Climate Change; Wiley & Sons: New York, 1998. (10) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239-247. (11) Fujishima, A.; Rao, T. N.; Tryk, D. A. J. Photochem. Photobiol. C: 2000, 1, 1-21. (12) A Ä lvaro, M.; Atienzar, P.; Bourdelande, J. L.; Garcıá, H. Chem. Commun. 2002, 3004-3005. (13) Wang, S.; Gao, R.; Zhou, F.; Selke, M. J. Mater. Chem. 2004, 14, 487-493. (14) Kovalev, D.; Fujii, M. AdV. Mater. 2005, 17, 2531-2544. (15) 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. (16) Edman, L.; Fo¨ldes-Papp, Z.; Wennmalm, S.; Rigler, R. Chem. Phys. 1999, 247, 11-22. (17) Naito, K.; Tachikawa, T.; Cui, S.-C.; Sugimoto, A.; Fujitsuka, M.; Majima, T. J. Am. Chem. Soc. 2006, 128, 16430-16431. (18) Fujishima, A.; Honda, K. Nature 1972, 238, 37-38. (19) Hoffmann, M. R.; Martin, S. T.; Choi, W.; Bahnemann, D. W. Chem. ReV. 1995, 95, 69-96. (20) Mills, A.; Le Hunte, S. J. Photochem. Photobiol. A 1997, 108, 1-35. (21) Thompson, T. L.; Yates, J. T., Jr. Chem. ReV. 2006, 106, 44284453. (22) Tatsuma, T.; Tachibana, S.; Miwa, T.; Tryk, D. A.; Fujishima, A. J. Phys. Chem. B 1999, 103, 8033-8035. (23) Lee, M. C.; Choi, W. J. Phys. Chem. B 2002, 106, 11818-11822. (24) Lee, S.-K.; McIntyre, S.; Mills, A. J. Photochem. Photobiol. A 2004, 162, 203-206. (25) Lee, J. P.; Sung, M. M. J. Am. Chem. Soc. 2004, 126, 28-29. (26) Kubo, W.; Tatsuma, T.; Fujishima, A.; Kobayashi, H. J. Phys. Chem. B 2004, 108, 3005-3009. (27) Ishikawa, Y.; Matsumoto, Y.; Nishida, Y.; Taniguchi, S.; Watanabe, J. J. Am. Chem. Soc. 2003, 125, 6558-6562. (28) Kubo, W.; Tatsuma, T. J. Am. Chem. Soc. 2006, 128, 1603416035. (29) Anpo, M.; Shima, T.; Kubokawa, Y. Chem. Lett. 1985, 17991802. (30) Howe, R. F.; Gra¨tzel, M. J. Phys. Chem. 1987, 91, 3906-3909. (31) Micic, O. I.; Zhang, Y.; Cromack, K. R.; Trifunac, A. D.; Thurnauer, M. C. J. Phys. Chem. 1993, 97, 13284-13288.

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Single-Molecule Detection of Reactive Oxygen Species arising from the charge carriers generated by the visible-light irradiation was exclusively observed only for the N-doped TiO2 film. This result strongly indicates the photocatalytic activity of N-doped TiO2 under visiblelight irradiation (see Supporting Information for details). (64) Hirakawa, K.; Hirano, T. Chem. Lett. 2006, 35, 832-833. (65) (a) On the other hand, it has been reported that •OH molecules would be generated from the H2O2-coated pure TiO2 under visible-light irradiation through the reduction reaction of H2O2.65b The H2O2-adsorbed species on TiO2 leading to the visible responsibility are probably Ti-OOH65b or TiOO (Ti-η-peroxide), which is characteristic of rutile surface.65c,65d The excitations of Ti-OOH and TiOO under visible-light irradiation probably result in the radical oxygen species, such as •OH and •O. Therefore, we should confirm weather or not the reduction process of H2O2 molecules contributes in the formation of airborne •OH molecules. The fact that no significant •OH molecules were detected under visible-light irradiation even in the N-doped TiO2 film (Figure 3) might be due to the insufficient amount of H2O2 generated by the brief irradiation time. For these purposes, the detected number of airborne •OH molecules from H2O2-coated pure TiO2 irradiated with visible-light was investigated. As a result, no significant •OH molecules were detected from the H O -coated pure TiO film irradiated 2 2 2 with the visible light, although in the UV irradiation significant •OH molecules were detected even under Ar atmosphere. These results confirm that the photolysis of H2O2 molecules formed on the TiO2 surface is the plausible mechanism for the formation of •OH molecules in ambient air (see Supporting Information for details; the diffuse reflectance spectrum of the H2O2-coated pure TiO2 film is also shown). (b) Li, X.; Chen, C.; Zhao, J. Langmuir 2001, 17, 4118-4122. (c) Ohno, T.; Masaki, Y.; Hirayama, S.; Matsuura, M. J. Catal. 2001, 204, 163-168. (d) Takahara, Y. K.; Hanada, Y.; Ohno, T.; Ushiroda, S.; Ikeda, S.; Matsuura, M. J. Appl. Electrochem. 2005, 35, 793-797. (66) The detected number of •OH molecules decreased with the decreasing concentration of oxygen (under this condition of the lower concentration of oxygen, the number of •OH molecules would be lower than that of the HPF molecules coated on the cover glass. Thus, the discussion of the intrinsic detected number of •OH molecules is meaningful). In particular, at [O2] ) 0.2 vol% ([Ar] ) 99.8 vol%), no significant •OH molecules were observed. This infers that the water oxidation mechanism on the TiO2 surface is not involved in the formation of the airborne •OH molecules. With a slightly increasing concentration of oxygen from 0.2 to 0.8 vol %, the •OH molecules started to diffuse from the pure TiO2 film. This result indicates that the formation of •OH molecules is very sensitive to the oxygen concentration,22,27,68 and the reduction process of oxygen, which results in the formation of O2•- leading to H2O2, plays an important role in the formation of •OH. Thus, it was revalidated that the photolysis of H2O2 molecules on the TiO2 surface is the plausible mechanism for the formation of •OH molecules in ambient air (see Supporting Information for details). (67) Palacios, R. E.; Fan, F.-R. F.; Bard, A. J.; Barbara, P. F. J. Am. Chem. Soc. 2006, 128, 9028-9029. (68) Kubo, W.; Tatsuma, T. Anal. Sci. 2004, 20, 591-593. (69) Panzer, O.; Go¨hde, W.; Fischer, U. C.; Fuchs, H.; Mu¨llen, K. AdV. Mater. 1998, 10, 1469-1472.

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Real-Time Single-Molecule Imaging of the Spatial and Temporal

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