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Microscopic Structure and Mobility of Guest Molecules in Mesoporous Hybrid Organosilica: Evaluation with Single-Molecule Tracking† Syoji Ito,*,‡,§ Shohei Fukuya,‡ Takatsugu Kusumi,‡ Yukihide Ishibashi,‡,| Hiroshi Miyasaka,*,‡,| Yasutomo Goto,⊥,| Masamichi Ikai,⊥,| Takao Tani,⊥,| and Shinji Inagaki*,⊥,| DiVision of Frontier Materials Science, Graduate School of Engineering Science and Center for Quantum Materials Science under Extreme Conditions, Osaka UniVersity, Toyonaka, Osaka 560-8531, Japan, PRESTO, Japan Science and Technology Agency, CREST, Japan Science and Technology Agency, and Toyota Central R&D Laboratories, Inc., Nagakute, Aichi, 480-1192, Japan ReceiVed: March 12, 2009; ReVised Manuscript ReceiVed: April 27, 2009
Single-molecule tracking (SMT) was applied to the evaluation of a microscopic heterogeneous structure by observing the diffusivity of guest molecules in phenyl-bridged periodic mesoporous organosilica films with one-dimensional (1D) nanoscale channels filled with template surfactant micelles. SMT clearly visualized the trajectories of individual fluorescence dyes, N,N′-dipropyl-1,6,7,12-tetrakis(4-tert-butylphenoxy)-3,4,:9,10perylenetetracarboxdiimide (BP-PDI) and N-(2,6-diisopropylphenyl)-N′-(n-octyl)-terrylene-3,4:11,12-tetracarboxidiimide (TDI), encapsulated in the channels of the mesoporous material. The 1D trajectory indicated successful formation of bundled channel structures and encapsulation of the guest molecules into the channels. The encapsulation ratio of the guest dyes was strongly dependent on template surfactants. The difference was discussed in terms of the affinity between the guest molecules and surfactants. Statistical analysis for the diffusion of the guest molecules also revealed that diffusion coefficient in the channels was strongly dependent on the guest molecules and surfactants. The validity and applicability of the present approach for elucidation of a microscopic structure was discussed on the basis of the comparison between the trajectories of BP-PDI and TDI in the same sample. 1. Introduction Since the invention of ordered mesoporous silicas in early 1990s,1-4 their interesting properties such as periodic uniformsized pores with 1-10 nm diameters, huge specific surface areas, and promising applications to catalysts, molecular sensor, drug delivery, and so forth have been stimulating a large variety of intensive research on mesoporous materials. Recently, organic groups such as ethane,5 benzene,6 and biphenyl7,8 were successfully incorporated into the framework of mesoporous materials and some of them formed regular arrangements by themselves like crystal.9-12 These organic mesoporous materials have expanded functionalities and possible applications that could not be attained using conventional mesoporous material solely with inorganic silica. Mesoporous materials are generally synthesized by selforganization of precursors and surfactant micelles, followed by polycondensation of precursors. The configuration (spherical, channel-like, and lamellar) and size of the mesostructures strongly depend on the type and concentration of the template surfactants. For evaluation of these mesostructures and their sizes, X-ray diffraction (XRD) analysis and transmission electron microscopy (TEM) have been used as common methods. XRD provides averaged information on periodicity of the mesostruc* To whom correspondence should be addressed. E-mail: sito@ chem.es.osaka-u.ac.jp. Telephone: +81-6-6850-6243. Fax: +81-6-68506244. (S. Ito) and E-mail:
[email protected]. Telephone: +81-6-6850-6241. Fax: +81-6-6850-6244. (H. Miyasaka). † Part of the “Hiroshi Masuhara Festschrift”. ‡ Osaka University. § PRESTO. | CREST. ⊥ Toyota Central R&D Laboratories, Inc.
tures, while TEM can visualize the fine structure of the mesoporous materials at nanometer-scale resolution. Although these two methods are powerful tools, it is difficult to obtain information of the detailed microscopic structures by XRD, and the imaging by TEM is limited to about several hundred nanometers. Hence, it is difficult to evaluate the homogeneity and heterogeneity of materials on a scale of hundreds of nanometers and micrometers. In addition, both methods do not possess temporal resolution sufficient to the tracking of the motion of guest molecules diffusing in the mesostructures. The information on the diffusion process of the guest molecules is indispensable to the elucidation of the functionality of these mesoporous materials leading to the development of advanced systems. Hence, a method with high spatiotemporal resolution is required. Single-molecule fluorescence imaging is a powerful tool for evaluating the microscopic structure of mesoporous materials and investigating the dynamic motion of molecules in the nanoscale structure. This method stores sequential fluorescence images of individual fluorescence molecules as digital movies. Positions of the individual molecules can be precisely determined at the nanometer level by single-molecule tracking (SMT).13-15 Time course of the positions provides trajectories of single molecules that are closely related to microscopic configuration and spatial heterogeneity of mesoporous materials. Actually, Bra¨uchle et al. applied this method to the first observation of the confined diffusion of single molecules in mesoporous silica with characteristic internal structures such as bundled channels and multilamellar.16-18 They successfully overlaid the nanostructure of mesoporous silica obtained by electron microscopy and individual trajectories of guest fluorescence molecules by SMT.19 In addition, this method has
10.1021/jp902219d CCC: $40.75 2009 American Chemical Society Published on Web 05/21/2009
Guest Molecules in Mesoporous Hybrid Organosilica recently proved its powerful ability in the space- and timeresolved measurement of other nanostructured systems such as sol-gel-derived silica materials by Ye et al.,20 spatial selectivity of catalysts on the surface of a micrometer-scale crystal by Roeffaers et al.,21,22 and quantitative evaluation of inhomogeneity accompanied with the polymerization of styrene reported by Wo¨ll et al.23 Although SMT is a powerful tool to provide information inaccessible by conventional methods, the photodegradation of probe fluorescence dyes limits the temporal and spatial window of the exploration. Because the area size covered by SMT is dependent also on the diffusion velocity of the probe dyes, the result by SMT is significantly influenced by the size of guest molecules and the interaction between these probe dyes and environments. Hence, in the present study, we have employed two guest molecules and have applied SMT to the evaluation of the mesoscopic structure of periodic mesoporous organosilica films filled with template surfactant micelles and to the elucidation of diffusional motions for individual guest molecules in the nanoscale channels. By comparing results depending on guest molecules and by referencing the studies on inorganic mesoporous silica,16-19 we will discuss the dynamics of the guest molecules and the structure of the mesoporous organosilica prepared solely from an organosilane monomer.
J. Phys. Chem. C, Vol. 113, No. 27, 2009 11885 SCHEME 1: Chemical Structures of BTEB, Brij76, P123, BP-PDI, and TDI
2. Sample Preparation Chemical structures of the reagents used in the present study are illustrated in Scheme 1. A template surfactant, Brij76 (1.42 g, Aldrich) was dissolved into a mixed solution of water (0.72 mL), 2N HCl (0.02 mL), and ethanol (3.0 g) and stirred for 30 min. An organosilane precursor, 1,4-bis(triethoxysilyl)benzene (BTEB, 2.01 g, Aldrich) was added into the above Brij76 solution and was stirred well for 90 min. Then ethanol (2.0 g) and water (2.0 g) were added into the solution and stirred for a further 30 min to obtain a sol solution by hydrolysis and partial condensation of the precursor. Mixing 20 µL of an ethanol solution of fluorescence dye (5 × 10-8 M) with 980 µL of the sol solution provided the final sol solution for spin casting.24 N,N′-Dipropyl-1,6,7,12-tetrakis(4-tert-butylphenoxy)-3,4:9,10perylenetetracarboxydiimide (BP-PDI, Yamada Chemical) and N-(2,6-diisopropylphenyl)-N′-(n-octyl)-terrylene-3,4:11,12-tetracarboxydiimide (TDI, Yamada Chemical) were doped as guest molecules in the present study.25,26 A thin film of periodic mesoporous organosilica was prepared by spin casting of the final sol solution at 4000 rpm for 30 s via evaporation induced self-assembly. For the sample preparation using a template surfactant P123 (Aldrich), the same procedure was applied except that the initial amount of the surfactant was 1.45 g of P123. All of the sample preparation processes were conducted at room temperature (276 K). Optical measurements were carried out after drying the samples at least for 48 h at room temperature without any calcination process. 3. Apparatus Figure 1 schematically shows the single-molecule optical detection system consisting of an inverted optical microscope (IX70, Olympus), three CW lasers emitting at 488 nm, 532 nm, and 633 nm, and highly sensitive photodetectors. The system can be used in two ways: (1) wide-field fluorescence microscopy and (2) confocal laser microscopy. An electron-multiplying charge-coupled devise (EM-CCD) camera (Cascade II 512B, Roper) can provide wide-field fluorescent images of individual dye molecules, while two avalanche photodiodes (SPCM-AQR-
14, PerkinElmer) are used as single-photon counters for fluorescence correlation spectroscopy (FCS). 3.1. Wide-Field Microscope for Single-Molecule Imaging. In the wide-field fluorescence microscope, an argon ion laser at 488 nm (LGK7872M, LASOS Lasertechnik GmbH) and a He-Ne laser at 633 nm (25 LHP 925, Melles Griot) were used for excitation of BP-PDI and TDI, respectively. Laser beams were guided into an objective (UPlanApo Oil Iris, X100, NA1.35) through a pair of lenses (f: 200), realizing wide-field epi-illumination with the spot size of several tens of micrometers in diameter for almost uniform illumination for the imaging area of the EM-CCD camera. The laser power introduced to the objective was 6.0 mW for both light sources. Circular polarization at the sample plane of the objective was attained for each of laser beams using a Soleil-Babinet compensator and quarter wave plates for 488 and 633 nm. Scattered light from the sample plane of the objective was blocked by long-pass filters (Semrock, LP01-488RU for the 488 nm light and LP01-633RU for the 633 nm light). Sequential fluorescence images for the single fluorescence molecules were acquired with the EM-CCD camera as digital movies. The exposure times for the imagings were
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[] [
x1 cos θ -sin θ ) y1 sin θ cos θ
][
x - x0 y - y0
]
(2)
Here, x0 and y0 are coordinates of the centroid of the 2D Gaussian. θ is the rotating angle of the 2D Gaussian distribution from the x-axis. Values x1 and y1, by image analysis with eq 1, provided the positions of individual dye molecules. The diffusion coefficient for the individual molecules was calculated from time courses of their positions. 3.2. Confocal Laser Microscope for FCS. We employed a CW DPSS laser (Excelsior 532, SpectraPhysics) at 532 nm as an excitation light source for FCS measurement. The SoleilBabinet compensator and quarter wave plates for 532 nm placed in front of the incident aperture of the objective ensured the circular polarization of the green light. The laser beam was focused into the sample solution by the microscope objective after passing through a pair of lenses (f: 200) and polarization compensators. The detection volume (confocal volume) of the FCS measurement was regulated by a pinhole (diameter: 40 µm) attached to the side port of the optical microscope. Photons emitted from dye molecules inside the confocal volume were detected with the two avalanche photodiodes connected to a counting board (M9003, Hamamatsu photonics) in order to avoid the after-pulsing effect. Scattered light from the sampling volume was blocked by an edge (long-pass) filter (Semrock, LP03-532RU). The correlation function between the detected fluorescent intensities of APD1 and APD2 was calculated using FCS software (U9451, Hamamatsu photonics). The fluorescence autocorrelation function thus obtained, G(τ), was analyzed by an analytical model of FCS as expressed by eq 330-32
G(τ) ) 1 +
Figure 1. Diagram of the single-molecule imaging system used in the present study.
250 ms for BP-PDI and 500 ms for TDI. Because of the image transfer time from the CCD to a personal computer, typically 34.8 ms per frame, the image sequences were recorded every 284.8 ms for BP-PDI and 534.8 ms for TDI. Fluorescence images of individual dye molecules are regarded as point spread functions (PSF). The intensity distribution of the PSF on the CCD chip, I(x,y), is approximately reproduced by a two-dimensional (2D) Gaussian function expressed by eq 113,14,27-29
(
I(x, y) ) I0exp -
x12 2σx2
-
y12 2σy2
)
+ IBG
(
( )) ( )(
1 p τ 1+ × exp N 1-p τT τ -1 τ 1+ 1+ 2 τD w τD
)
-1/2
(3)
Here, N is the average number of molecules in the confocal volume, Vconf, with a cylindrical shape. p is the fraction of the contribution of the triplet state. τT is the triplet lifetime. w is structure parameter defined by w ) wz/wxy. Here, wz and wxy are the axial and radial radii of the cylindrical confocal volume (Vconf ) 2πwzwxy2), respectively. τD is average residence time for a molecule to pass through the confocal area. The average residence time, τD, is related to the translational diffusion coefficient, D, as represented by eq 4
wxy2 τD ) 4D
(4)
3.3. XRD and TEM Measurements. A X-ray diffractometer (X’Pert MPD, Philips) and tunneling electron microscope (JEM200CX, JEOL) were used for the measurements of the XRD patterns and TEM images, respectively.
(1)
Here, I0 is the peak of 2D Gaussian distribution. σx and σy are widths of the 2D Gaussian distribution in the x- and y-axes, respectively. IBG is the background signal intensity of the CCD image. Parameters x1 and y1 are defined by the eq 2
4. Results and Discussion 4.1. Evaluation of Mesoporous Organosilica with XRD and TEM. Figure 2 shows the XRD diffractogram of the specimen prepared in the present study, clearly indicating the successful formation of periodic mesostructures in the thin films. From Figure 2, we estimated the periodicity (d100) of the channels in the mesoporous organosilica thin films before
Guest Molecules in Mesoporous Hybrid Organosilica
Figure 2. XRD signals for mesoporous organosilica prepared with Brij76 (a) and with P123 (b). The samples were calcinated in the following manner using an electric oven. First, we put the samples in the oven at room temperature (296 K), and then we gradually increased the temperature in the oven up to 473 K in 6 h. After that, we kept the temperature at 473 K for 2 h.
calcination as 6.4 and 9.6 nm for those prepared with Brij76 and P123, respectively. These values are in agreement with the results by Morell et al.,33 where the periodicity of 5.7 and 9.6 nm were reported for benzene-bridged periodic mesoporous organosilica powders prepared with Brij76 and P123, respectively. Figure 2 also shows that peaks in the XRD signals still remained even after the calcination at 473 K, although these peaks shifted to larger theta values. This result supports the stable formation of nonlamellar periodic mesostructures in the specimen. Formation of the bundled channel structure (hexagonal P6mm) was also confirmed by TEM, after the calcination at 673 K, leading to complete elimination of the surfactants as shown in Figure 3. Although the lattice shrinkage33 during the calcination decreased lattice parameters to about 5 and 7 nm for the specimens prepared with Brij76 and P123, respectively, we can still find bundled channel structures. For the optical measurement, we used samples without elimination of the surfactants. 4.2. SMT in the Mesoporous Organosilica. Figure 4a shows typical trajectories of BP-PDI in the mesoporous organosilica prepared with the surfactant, Brij76. The fluorescence molecules exhibited one-dimensional (1D) diffusion, which is clear evidence for encapsulation of the dyes into the mesochannels. In the present study, 44% of BP-PDI molecules exhibited clear 1D diffusion in the mesoporous organosilica prepared with Brij76 (Table 1). More than half of the dyes, however, showed two-dimensional (2D) diffusion; typical examples are shown in Figure 4b. The 2D diffusion may be interpreted as due to the structural disorder in the mesoporous materials and/or the position of the guest dyes in the materials. The disorder of the mesostructure in the organosilica as observed in the TEM images in Figure 3 could lead to the 2D diffusion of the dyes in the disordered amorphous area. However, the dyes adsorbed onto the surface of the mesoporous thin film may move more freely than those inside the material because the surfactants are not likely to form the micelles on the hydrophilic organosilica surface. Hence, diffusion coefficient for the molecules on the
J. Phys. Chem. C, Vol. 113, No. 27, 2009 11887 surface could be larger than that for molecules inside the material. To compare the two types of diffusion coefficients, we plotted histograms of the diffusion coefficients in histograms c and d of Figure 4, where relatively fast diffusing molecules, with a diffusion coefficient of 30 × 10-3 µm2s-1, are recognized only in the histogram in Figure 4d for 2D diffusion. This result strongly implies that the molecules showing 2D diffusion are adsorbed onto the surface and not encapsulated in the pores inside. In the case of the mesoporous organosilica prepared with P123, the number of BP-PDI molecules that underwent 1D diffusion was about 10% (13/129 molecules), whose averaged diffusion coefficient was 7.3 × 10-3 µm2s-1 (Table 1). This result indicates that the number of the guests successfully encapsulated in the mesochannels was smaller, although the XRD results showed a more prominent and stronger diffraction peak corresponding to the well-aligned mesochannel structures in the specimen prepared with P123 (Figure 2b). This result also supports 2D diffusion on the surface of the specimen and suggests a low affinity of BP-PDI for the surfactant, P123, due to its weaker hydrophobicity than that of Brij76. When the surfactants form micelles with evaporation of the solvent, most of BP-PDIs might be diffused to the surface of the thin film without being incorporated in the micelles because of the low affinity, resulting in the localization of BP-PDIs on the surface. The proposed mechanism is also supported by the result that BP-PDIs exhibiting 2D diffusion have a one-order larger average diffusion coefficient than that of the dyes diffusing onedimensionally as shown in Table 1. Figure 5 shows the relation between mean square displacement (MSD) and time for individual BP-PDI and TDI in the fine pores of the mesoporous organosilica prepared with Brij76, indicating that the translational diffusion coefficient strongly depends on the guest molecule. Because the time window for SMT is limited due to photodegradation of the dyes, the number of data points decreases with increasing lag time. This small number of data points at a longer time range, >10 s for BP-PDI and >30 s for TDI, leads to scattering MSD-time plots as in Figure 5. Histograms of the diffusion coefficients in panels b and c of Figure 5, which were obtained from Figure 5a and Table 1, clearly show there is one-order of difference in the translational diffusion coefficient between BP-PDI and TDI in the mesoporous organosilica prepared with Brij76. Additionally, it is worth noting that no fast 2D-diffusing molecule was observed in the case of TDI in the mesoporous organosilica prepared with Brij76. The averaged diffusion coefficient for 2Ddiffusing TDIs in the system is much smaller than that of TDIs diffusing one-dimensionally as shown in Table 1, and most TDIs showed 1D diffusion. This is why we did not observe 2Ddiffusing TDIs in a defocused image as described later. To explore the difference in the diffusion coefficient between PB-PDI and TDI, we compared diffusion coefficients for the two dyes in several solvents using fluorescence correlation spectroscopy (FCS). Because the focused area of a laser beam by an objective depends on several properties such as the thickness of a coverslip, refractive index of the sample solution, and parameters of laser light such as wavelength, incident beam waist to an objective, and transverse mode, the same excitation condition was required for accurate comparison of diffusion velocity between BP-PBI and TDI. Hence, we used a common light source (532 nm) for photoexcitation of the dyes and a common glass vessel in this series of experiment. Figure 6 shows fluorescence autocorrelation functions for ethanol solutions of BP-PDI and TDI. The dotted lines in the traces are curves
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Figure 3. Transmission electron micrographs of mesoporous organosilica thin films prepared with Brij76 (a) and with P123 (b) and a schematic image of the mesostructure.
TABLE 1: Diffusion Modes (1D or 2D) of Guest Mmolecules, Number of Molecules Exhibiting Corresponding Diffusion Modes, and Average Diffusion Coefficients diffusion number of molecules analyzed (percentage average diffusion mode dye/surfactant (1D or 2D) in all molecules) coefficient (µm2s-1) BP-PDI/Brij76 BP-PDI/P123 TDI/Brij76
Figure 4. Typical trajectories (a, b) of individual BP-PDIs in the mesoporous organosilica prepared with Brij76 (without calcination). Trajectories for 149 molecules were analyzed in the present study. Individual diffusive motions of the dyes were categorized as 1D or 2D motions. The diffusion coefficient for the 1D and 2D motions are summarized in histograms c and d, respectively.
analyzed by the nonlinear least-squares method with eq 3. Residuals plotted in the top of the traces indicate that the experimental results were well-reproduced by the calculated curve. For other solvents, toluene and chloroform, it was confirmed that the analysis based on eq 3 well-reproduced the experimental results. Average residence times of BP-PDI and TDI in the three solvents are summarized in Table 2, indicating that the differences in the averaged residence times between BP-PDI and TDI stayed less than 20% in all solutions examined.
1D 2D 1D 2D 1D 2D
65 (44%) 84 (56%) 13 (10%) 113 (90%) 63 (82%) 14 (18%)
4.2 × 10-3 1.3 × 10-2 7.3 × 10-3 2.2 × 10-2 2.0 × 10-4 1.1 × 10-4
Hence, the one-order difference in the diffusion coefficient between BP-PDI and TDI in the mesochannels cannot be accounted for only by the size of the molecule, namely, the hydrodynamic diameter, and it is strongly suggested that diffusion velocity of guest molecules in the mesochannels is severely affected by the interaction between the guests and the surfactants under the assumption that the mesochannels have almost the same pore diameters in the presence of the two dyes. 4.3. Microstructure of Mesoporous Organosilica Evaluated by SMT. The experimental result in Figure 4a shows that the 1D trajectories of individual molecules are not aligned with each other in the same direction, indicating that mesoporous organosilica consisted of many small domains. This kind of information is not easily obtained by ensemble measurements such as XRD and indicates the usefulness of the present approach using a single-molecule detection method for elucidation of the microscopic heterogeneity of mesoporous materials. It is worth noting that the “apparent” trajectories for individual dyes in the mesoporous organosilica are not complete copies of the shape of the mesochannels because of the limitations of the probing procedures. As mentioned in the previous sections, we can obtain trajectories for individual molecules only during the duration in which the dyes can emit photons. Hence, the observation time of the SMT is intrinsically limited by photobleaching of the dyes. Of course, when the time before photobleaching is longer than the time needed to map all of the mesochannels, the apparent trajectories really reflect the shape of the mesochannels. In the present experimental conditions, BP-PDI survived typically for 10 to several tens of seconds, and TDI could survive longer than BP-PDI, typically several tens to 100 s. Panels a and b of Figure 7 show typical trajectories of BP-PDI and TDI, respectively, in the mesoporous organosilica prepared with Brij76. A comparisons of the figures demonstrates
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Figure 6. Typical fluorescence autocorrelation curves of ethanol solutions of BP-PDI (a, black solid lines) and TDI (b, black solid lines) with their fitting curves (gray dotted lines) and residuals.
TABLE 2: Translational Diffusion Coefficients of Guest Molecules in Ethanol, Toluene, and Chloroform
Figure 5. MSD versus time plots (a) for individual BP-PDI (blue lines) and TDI (red lines) molecules that underwent 1D diffusion. Corresponding histograms of diffusion coefficients for the individual BPPDI (b) and TDI (c) molecules.
that trajectories of slower diffusing molecules provide shorter 1D trajectories. This is statistically confirmed by histograms of the apparent length of the 1D channels shown in Figure 7c, although the lengths of the mesochannels have distribution depending on the positions and lot of the samples. Thus, an optimized condition, namely, long survival time of probe dyes and a proper diffusion coefficient, is necessary for exploration of the shapes of mesostructures. 4.4. Rotational Motion of the Guest Molecules in the Mesochannels. It was recently demonstrated that the defocused pattern of a single fluorescence molecule on a CCD chip shows not coaxial but anisotropic intensity distribution reflecting the three-dimensional orientation of the fluorescent transient dipole of a single molecule.34-36 To obtain the information on rotational motion and to elucidate the relation between the translational and rotational motion in the present systems, we applied the defocused imaging method to the guests in the mesoporous organosilica prepared with Brij76. Unfortunately, because of opaque defocused images for BP-PDIs in the mesoporous organosilica due to low S/N ratio and fast diffusion velocity, we could not obtain clear images of the rotational motion for individual BP-PDIs. However, the defocused images for individual TDIs was obtained with 200 ms exposure time, indicating distinguishable anisotropic intensity distributions of their transient dipoles as shown in Figure 8. The different z-positions of the TDIs in the relatively thick mesoporous organosilica film, typically several hundreds of nanometers, hindered the simul-
mean residence time in ethanol (µs)
mean residence time in toluene (µs)
mean residence time in chloroform (µs)
71 ( 1.0 71 ( 5.8
56 ( 1.6 46 ( 2.7
48 ( 1.0 55 ( 2.4
BP-PDI TDI
taneous observation of defocused images for all of the dyes at the same defocusing depth as was observed in polymer thin film with a thickness
