Article pubs.acs.org/JPCC
Single-Molecule Fluorescence Detection of Effective Adsorption Sites at the Metal Oxide−Solution Interface Takashi Tachikawa,* Tatsuya Ohsaka, Zhenfeng Bian, and Tetsuro Majima* The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan S Supporting Information *
ABSTRACT: Nanoscale mapping of adsorption sites for molecules or ions at solid−liquid interfaces has not been explored in detail because of the difficulty in probing both stochastic adsorption/desorption events and heterogeneous surface structures. We report here the application of single-molecule-based super-resolution fluorescence microscopy using a catechol-modified boron−dipyrromethene dye (CA-BODIPY), which serves as a fluorescent reporter, to identify the locations of effective adsorption sites on metal oxide surfaces. Upon adsorption on a TiO2 nanoparticle, individual CA-BODIPY molecules exhibited detectable fluorescence because of the formation of chelating complexes between the catechol moiety and the surface Ti sites. Interestingly, a significant effect of the crystal face on the adsorption preference for CA-BODIPY was found in the case of anatase TiO2 microcrystals in neutral water: {101} > {001} ≈ {100}. In an aprotic solvent such as acetonitrile, however, the opposite crystal face effect was observed; this implies a significant contribution of solvent molecules to the adsorption of organic compounds on specific surfaces. From the quantitative analysis of the formation rate of fluorescent complexes per unit area, it was found that nanometer-sized TiO2 crystals have superior adsorptivity over micrometer-sized TiO2 crystals and an atomically flat TiO2 surface. This observation is consistent with the higher density of surface defects on the nanoparticles. Furthermore, it was revealed that CA-BODIPY molecules are preferentially adsorbed on the top branches of α-Fe2O3 micropines, where a high density of exposed Fe cations is expected. Our methodology and findings yield new insights into the mechanisms underlying the synthesis and (photo)catalytic activity of metal oxide particles with different sizes and shapes.
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INTRODUCTION Metal oxide semiconductors have been extensively studied because of their potential applications in photocatalysis and photovoltaics as well as in batteries and sensors.1,2 For instance, anatase TiO2, an n-type semiconductor with a band gap of 3.2 eV, finds applications in a wide variety of technical fields, for example, as a photocatalyst for environmental remediation and in dye-sensitized solar cells.3−6 Molecular adsorption on solid surfaces plays a key role in the synthesis and performance of these materials and hence has been studied experimentally by means of atomic force microscopy (AFM),7,8 scanning tunneling microscopy (STM),9−11 transmission electron microscopy (TEM),12,13 nuclear magnetic resonance (NMR) spectroscopy,14−16 sum frequency generation (SFG) spectroscopy, 17,18 and surface-enhanced Raman spectroscopy (SERS).19−22 Catechol has been employed as a model adsorbate in many studies because it is commonly used as the linker molecule in dye-sensitized solar cells.23,24 The use of catechol or its analogues has also been proposed as a promising method for anchoring biomolecules such as DNA and proteins onto metal oxide surfaces for medical and biological applications.25−27 Upon adsorption on a TiO2 surface, catechol donates H atoms from (one or both) its hydroxyl groups to 2-fold-coordinated bridging O (O2c) atoms, affording a catecholate bonded to © XXXX American Chemical Society
neighboring 5-fold-coordinated Ti (Ti5c) sites via Ti−O bonding. Solid-state magic angle spinning NMR spectroscopy showed that a large amount (ca. 65%) of 4-nitrocatechol is monodentately chemisorbed on nanocrystalline TiO2 powder in ambient air via one Ti−O bond, whereas a small amount (ca. 30%) is bidentately chemisorbed via two Ti−O bonds.16 The residual species (ca. 5%) is predominantly physisorbed on TiO2. Recent STM and density functional theory (DFT) calculations demonstrate that isolated catechol in vacuo adsorbs preferentially at the step edges and point defects on the TiO2 surface via monodentate and bidentate conformations.28 Monodentate catechol is mobile at room temperature and occupies the most stable adsorption sites, whereas bidentate catechol is essentially immobile. Nevertheless, there is limited information about molecular adsorption on solid surfaces that are fully immersed in solution because of the lack of in situ observation techniques with sufficient temporal and spatial resolution. In this study, we performed single-molecule fluorescence imaging analysis of a catechol-modified boron−dipyrromethene (CA-BODIPY) dye to detect the adsorption sites on individual Received: March 1, 2013 Revised: May 7, 2013
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Figure 1. Formation of fluorescent complexes from nonfluorescent CA-BODIPY and metal oxide (MOx). The possible binuclear bidentate, mononuclear bidentate, and mononuclear monodentate configurations for the adsorption of the catechol moiety are shown.
metal oxide crystals. Single-molecule fluorescence micro(spectro)scopy is emerging as a promising tool for exploring the inherent features of molecular interactions and reaction processes on heterogeneous solid catalysts such as metal and metal oxide nanoparticles.29−34 The catechol moiety of CABODIPY readily binds to metal oxide surfaces in neutral water to afford detectable fluorescent complexes. Single-moleculescale localization procedures revealed that the number and location of effective adsorption sites on TiO2 are largely dependent on the crystal face, crystal size, and type of environmental solvent. Furthermore, we examined the location of effective adsorption sites on α-Fe2O3 dendritic microstructures, which are closely related to their crystal growth mechanism.
Sample Preparation for Single-Molecule Fluorescence Experiments. Borosilicate and quartz cover glasses were purchased from DAICO MFG CO., Ltd. (Japan) and Matsunami Glass, respectively. Quartz cover glasses with numbered grids were prepared by conventional photolithography and reactive ion etching techniques (Figure S4). The details of the procedures are described in the Supporting Information. For single-molecule fluorescence experiments, the patterned cover glasses were cleaned by sonication in a 20% detergent solution (As One, Cleanace) for 6 h, followed by repeated washings with warm flowing water and Milli-Q water (Millipore). Well-dispersed aqueous suspensions of metal oxide particles were spin-coated on the cleaned cover glasses. The cover glasses were annealed at 100 or 150 °C for 1 h to immobilize the particles on the glass surface. A chamber was made with a cleaned silicon gasket (Invitrogen), filled with a sample solution, and then covered with a cleaned cover glass. The samples in the inverted configuration were prepared as follows.40 A clean cover glass or a TiO2 wafer was mounted on the bottom of a stainless steel holder designed for viewing specimens on the microscope. A chamber was made with a 12.5 μm thickness polyimide film (Nilaco), filled with a sample solution, and then covered with a cleaned cover glass. Single-Particle/Single-Molecule Fluorescence Measurements by Wide-Field Microscopy. The experimental setup was based on an Olympus IX81 inverted fluorescence microscope. The details of the experimental and analytical procedures are described elsewhere.40,41 A 488 nm CW laser (Coherent; 0.5 kW cm−2 at the glass surface) was used to excite the dye. The optical transmission and emission images were recorded on an electron-multiplying charge-coupled device (EMCCD) camera (Roper Scientific, Evolve 512) at a frame rate of 20 frames s−1 using MetaMorph (Molecular Devices). Suitable dichroic mirrors and bandpass filters were used to improve the signal-to-noise ratio. All experimental data were obtained at room temperature. With the aim to determine the locations of the reactive site distributed on the surface, the precise positions of the fluorescent spots were analyzed for each image using the ImageJ software and OriginPro 8.6 (OriginLab). A general approach is applied to define the intensity threshold in order to distinguish between the on and off states. The threshold was chosen to be 3σ greater than the background noise levels. Counts above the threshold were then considered to be the fluorescence signal. Single-Particle/Single-Molecule Fluorescence Measurements by Confocal Microscopy. Fluorescence lifetimes and spectra were recorded using an objective-scanning confocal microscope system (PicoQuant, MicroTime 200) coupled with
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EXPERIMENTAL METHODS Instruments. The samples were characterized using powder X-ray diffraction (XRD) (Rigaku, Rint-2500; Cu Kα source), SEM (JEOL, JSM-6330FT; operated at 5 or 15 kV), and TEM (JEOL, JEM 3000F; operated at 300 kV). Steady-state UV−vis absorption and fluorescence spectra were measured using a UV−vis−NIR spectrophotometer (Shimadzu, UV-3100) and a fluorescence spectrophotometer (HORIBA, FluoroMax-4). Materials. 8-(3,4-Dihydroxyphenyl)-1,3,5,7-tetramethyl-4,4difluoro-4-bora-3a,4a-diaza-s-indacene (CA-BODIPY) was synthesized according to established procedures.35 1H NMR (400 MHz, CD3OD): δ = 6.93 (d, 1H, J = 4.0 Hz), 6.68 (s, 1H), 6.58 (dd, 1H, J = 2.0, 4.0 Hz), 6.04 (s, 2H), 2.47 (s, 6H), 1.55 (s, 6H); MS (ESI): calcd for C19H19BF2N2O2. [M + Na]+ 379.1407, found 379.1397. Anatase TiO2 nanoparticles (A100; 100−200 nm size) were gifted from Ishihara Sangyo and used as received. Microsized anatase TiO2 crystals with dominant {001} or {100} facets were prepared by the hydrothermal method according to literature procedures.36,37 To remove surface fluorine species, the powders were heated at 600 °C for 90 min before use. Anatase TiO2 nanorods were prepared by the hydrothermal method as described elsewhere (Figure S1, Supporting Information).38 Atomically flat (110) wafers of rutile TiO2 were obtained from Shinko-sha and used as received. α-Fe2O3 micropines were synthesized according to the procedures reported in the literature.39 Powder XRD patterns of TiO2 nanorods and α-Fe2O3 micropines are given in Figures S2 and S3, respectively. Fresh Milli-Q ultrapure water (Millipore; 18.2 MΩ·cm at 25 °C resistivity) was used as the solvent throughout this study. Dimethyl sulfoxide (Wako) (30 mM) was used as the cosolvent to dissolve CA-BODIPY in water. B
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Figure 2. (A) Illustration of experimental setup for single-molecule fluorescence spectroscopy based on inverted fluorescence microscopy. (B) Typical wide-field fluorescence images showing on and off states observed during 488 nm laser irradiation of a single TiO2 particle on quartz cover glass in an aqueous solution containing CA-BODIPY (100 nM). The acquisition time of an image was 50 ms. Scale bars are 500 nm. (C) A typical trajectory of fluorescence intensity observed during 485 nm laser irradiation of a single TiO2 particle on quartz cover glass in an aqueous solution containing CA-BODIPY (100 nM). (D) Fluorescence decay profiles of burst emission (red) and background emission (black) in the time regions indicated by red and gray in panel C, respectively. Blue lines indicate single-exponential curves fitted to the data. (E) Fluorescence spectrum observed for a single TiO2 particle in an aqueous solution containing CA-BODIPY (0.1 μM) under 485 nm photoirradiation (red). The fluorescence spectrum of the bulk solution of BODIPY is also shown (blue). Note that the fluorescence below ∼520 nm is cut off by a long-pass filter. (F) CABODIPY concentration dependence of the formation rates of fluorescent species. The solid line was obtained from eq 1.
an Olympus IX71 inverted fluorescence microscope. The samples were excited through an oil-immersion objective lens (Olympus, UAPON 150XOTIRF; 1.45 NA, 150×) using a 485 nm pulsed laser (PicoQuant, LDH-D-C-485) controlled by a PDL-800B driver (PicoQuant). The emission from the sample was collected using the same objective and detected by a single photon avalanche photodiode (Micro Photon Devices, PDM 50CT) through a dichroic mirror, a long-pass filter (Chroma, HQ510LP), a bandpass filter (Semrock, FF01−531/40−25), and a 50 μm pinhole for spatial filtering to reject out-of-focus signals. An instrument response function (IRF) of ∼100 ps was obtained by measuring the scattered laser light in order to analyze the temporal profile. For the fluorescence spectral measurements, only the emission that passed through a longpass filter and a slit entered the imaging spectrograph (ActonResearch, SP-2356) that was equipped with an EMCCD camera (Princeton Instruments, ProEM). The spectrum detected by the EMCCD camera was stored and
analyzed by using a personal computer. The spectra were typically integrated for 10 s. All experimental data were obtained at room temperature.
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RESULTS AND DISCUSSION
CA-BODIPY is composed of a BODIPY fluorophore and a catechol group (Figure 1 and Figure S5).35 BODIPYs have promising applications in bioimaging because of various attractive properties such as high extinction coefficients (ε = 40 000−110 000 cm−1 M−1), high fluorescence quantum yields (Φfl = 0.6−0.9), good chemical stability and photostability, and facile chemical modification.42,43 Prior to adsorption onto metal oxide surfaces, BODIPY fluorescence is strongly quenched by intramolecular electron transfer from the catechol moiety to the excited BODIPY (Φfl ≈ 10−3).35 The free energy change for the electron transfer (ΔGET) can be calculated by the Rehm− Weller equation:44 C
dx.doi.org/10.1021/jp402144h | J. Phys. Chem. C XXXX, XXX, XXX−XXX
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containing the reference BODIPY dye (1,3,5,7-tetramethyl-4,4difluoro-4-bora-3a,4a-diaza-s-indacene) (Invitrogen; D3921), again confirming the fluorescence from CA-BODIPY adsorbed on TiO2. The formation rate of fluorescent species, i.e., the adsorption rate of CA-BODIPY, increased with the concentration of CABODIPY ([CA-BODIPY]). An example is demonstrated in Figure 2F. At high concentrations of CA-BODIPY, adsorption equilibrium is attained rapidly. According to the Langmuir adsorption isotherm, the [CA-BODIPY] dependence of the number of detected fluorescent bursts per unit area (nad) can be described as
(1)
where E(D+/D) and E(A/A−) are the oxidation potentials of the electron donor (catechol, +0.98 and +1.24 V vs NHE at pH 6 for free and adsorbed species, respectively45) and the reduction potential of the electron acceptor (BODIPY, −0.98 V vs NHE46), respectively, and ΔE0,0 is the zero−zero transition energy of the fluorophore (BODIPY, 2.41 eV46). The work term, wp, which corresponds to the Coulomb stabilization energy of the radical ion pair, is neglected because it is much smaller (∼0.02 eV in the present system) than the other parameters in polar environments. The ΔGet values calculated for free and adsorbed CA-BODIPY are −0.45 and −0.19 eV, respectively, thus suggesting that intramolecular electron transfer accompanying fluorescence quenching is energetically suppressed by adsorption on metal oxides such as TiO2. Preliminary, confocal fluorescence images showed bright fluorescent spots representing CA-BODIPY molecules on glass in air (Figure S6). The increased fluorescence intensity and lifetime (1.1 ns main component) implied that the catechol moiety of CA-BODIPY binds to metal or metal oxide impurities such as TiO2, ZnO, or Al2O3 in borosilicate glass. No such significant enhancement in fluorescence was observed for CA-BODIPY molecules dispersed on cleaned quartz because there were no suitable binding sites available. These findings are analogous with those in the case of N-(1nonyldecyl)-N′-(p-aminophenyl)perylene-3,4,9,10-tetracarboxylbisimide, as reported by Adams and co-workers.47 A schematic for single-molecule fluorescence imaging of CABODIPY molecules adsorbed on individual TiO2 particles is shown in Figure 2A. The position and morphology of the TiO2 particles immobilized on a custom-made quartz cover glass, which has numbered grids, were determined from the correlated optical transmission and field-emission scanning electron microscopy (SEM) images.41 Figure 2B shows typical fluorescence images captured for a single TiO2 nanoparticle (anatase, 100−200 nm size) in an aqueous solution containing CA-BODIPY (100 nM) under 488 nm laser irradiation. Individual particles show a number of fluorescence bursts that show signals over the background (Figure 2C). Control experiments confirmed that both TiO2 and CA-BODIPY are necessary to generate the fluorescence bursts. The fluorescence lifetimes of the in situ generated bursts over single TiO2 particles were measured by combining confocal microscopy with time-correlated single-photon counting (TCSPC) observations. The fluorescence bursts exhibited a much longer lifetime (τfl ∼ 1.3 ± 0.4 ns) than did the background signal from free CA-BODIPY in solution (τfl < 0.2 ns), suggesting that such a sudden intensity increase is due to the suppression of intramolecular electron transfer from the catechol moiety to the excited BODIPY chromophore, as discussed above (Figure 1). The characteristic time for which persistent emission is exhibited is possibly related to the dissociation of CA-BODIPY from the surface rather than photobleaching or blinking.36,41 Oxidation of catechol to quinine or semiquinone under intense laser irradiation may also induce the dissociation of CA-BODIPY (the chargetransfer complexes show a characteristic absorption band in the visible-wavelength region48) because these products show poor affinity for the TiO2 surface.49 Figure 2E shows the normalized emission spectrum measured for a single TiO2 particle in the CA-BODIPY solution under 485 nm laser excitation. The observed spectra were identical to that of the bulk solution
nad =
nsK ad[CA‐BODIPY] 1 + K ad[CA‐BODIPY]
(2)
where Kad is the equilibrium adsorption constant for CABODIPY and ns is the total number of effective binding sites on one particle.40,50 From eq 2, the average value of Kad is determined to be (4.5 ± 1.5) × 106 M−1, which is 1−3 orders of magnitude larger than those for catechol and related enediol compounds on TiO2.48,51−53 On a heterogeneous surface, a variety of adsorption sites with different adsorption energies may coexist. The discrepancy between the Kad values may arise from the difference in the surface structures of the particles or the analysis methods used. For example, Moser et al. obtained a Kad value of 8 × 104 M−1 for catechol on nanocrystalline TiO2 powder (Degussa P-25, particle diameter: ca. 25 nm) suspended in a water−methanol mixture, from the Langmuir adsorption isotherm.51 Rajh et al. determined two distinct Kad values of 54 M−1 (high concentrations) and 7.9 × 103 M−1 ( {001} ≈ {100} (Table 1).
Very recently, experimental evidence for the crystal-face dependency of TiO2 photocatalytic reactions has been provided by means of single-molecule fluorescence spectroscopy.40 In that experimental system, the reaction sites for the photocatalytic reduction of fluorogenic dye molecules were preferentially located on the {101} facets of the crystal rather than on the {001} facets, which have higher surface energy. Moreover, it has been demonstrated that the {001} facets exhibit similar or slightly higher oxidation reactivity than do the {101} facets.37,40 However, the role of the crystal faces in (photo)catalytic processes is still not completely verified61 because of the lack of quantitative analysis methods for facedependent molecular adsorption under various conditions. In order to assess this issue, we observed the fluorescence images for a single TiO2 microcrystal with dominant {001} facets in aqueous solution containing CA-BODIPY under 488 nm laser irradiation. The locations of the fluorescence bursts were determined with an accuracy of ∼50 nm using a twodimensional Gaussian function to fit the distribution of the fluorescence intensity.41 As demonstrated in Figure 3A, a
Figure 4. (A) Crystal structure of TiO2 crystal with dominant {100} facets. (B) Locations of the reactive sites on the {101} (red dots) and {100} (green dots) surfaces of TiO2 crystal. The optical transmission image of the crystal analyzed is shown. The scale bar is 2 μm.
Table 1. Surface Properties and Adsorptivity of TiO2 Crystals TiO2 microcrystal microcrystal microcrystal wafer A-100d nanorode a
phase (facet) anatase (101) anatase (001) anatase (100) (or 010) rutile (110) anatase anatase
surface energy,a J m−2
formation rate, molecules μm−2 s−1
0.44 0.90 0.53
0.10 ± 0.04b 0.02 ± 0.01b 0.03 ± 0.01b
0.31
