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J. Phys. Chem. B 2009, 113, 5720–5727
ARTICLES Balance between Coulombic Interactions and Physical Confinement in Silica Hydrogel Encapsulation Yongyao Zhou and Wai Tak Yip* Department of Chemistry and Biochemistry, UniVersity of Oklahoma, Norman, Oklahoma 73019 ReceiVed: April 25, 2008; ReVised Manuscript ReceiVed: January 20, 2009
We examined the behavior of various entrapped guest molecules within silica hydrogel and evaluated the effect of Coulombic interactions and physical confinement on molecular mobility. Although rhodamine 6G (R6G) and fluorescein (FL) share similar size and molecular structure, their behavior in silica hydrogel was found to be dramatically different. A good majority of R6G was immobilized with little to no exchangeable molecules, whereas FL displayed a considerable amount of mobility in silica hydrogel. Moreover, silica hydrogel encapsulated R6G failed to gain mobility even under low pH or high ionic strength conditions to minimize Coulombic interactions, implying that encapsulated R6G molecules were inaccessible and likely trapped deep inside the silica matrix of a hydrogel. On the contrary, FL was relatively free to rotate and translate inside a silica hydrogel, implying that FL remained solvated in the solvent phase and was able to maintain its mobility throughout the hydrogel formation process. Fluorescence recovery after photobleaching measurements put the diffusion coefficient of FL in silica hydrogel at ca. 2.1 × 10-6 cm2 s-1, about a factor of 3 slower than that in solution. The substantial difference in mobility between cationic R6G and anionic FL led us to conclude that the effect of Coulombic interactions on mobility is more dominating in hydrogel than in alcogel. Our results also suggest that Coulombic interactions are strong enough to influence the eventual placement of a guest molecule in a silica hydrogel, causing R6G and FL to reside in different microenvironments. This has a profound implication on the use of molecular probes to study silica hydrogel since a slight difference in physical attribute may result in very diverse observations even from identically prepared silica hydrogel samples. As demonstrated, the repulsion between FL and silica renders FL liquid-bound, making FL more suitable for monitoring the change in viscosity and physical confinement during hydrogel formation, whereas other researchers have shown that silica-bound R6G is more suitably used as a reliable probe for monitoring the growth of silica colloids because of its strong attraction toward silica. Introduction Understanding the behavior of an encapsulated guest in sol-gel silica has been an active research area ever since the interest in silica sol-gel biocomposite materials took off in 1990 when alkaline phosphatase was shown to be 30% active in a silica hydrogel.1 While silica hydrogel encapsulation helps suppress the degradation of a guest enzyme by proteases, the very confinement that protects the enzyme may impose too much mobility constraint and completely abolish bioactivity. As a result, a wide variety of strategies have been developed to encapsulate different enzymes in hydrogel while maximizing bioactivity.2,3 Evidently, different enzymes require different degrees of mobility to carry out their biological functions. At issue are factors that control the mobility of an encapsulated guest molecule. In this regard, both Coulombic interactions and physical confinement have emerged as the leading candidates that control mobility. At neutral pH, the surface of a silica hydrogel is negatively charged. As a result, positively charged guest molecules tend * To whom correspondence should be addressed. E-mail: ivan-yip@ ou.edu.
to adhere strongly to a silica surface through Coulombic attraction and become immobilized. Thus, positively charged rhodamine 6G (R6G) has been shown to exhibit high fluorescence anisotropy value when bound to silica particles.4 Furthermore, it has been suggested recently that R6G can be firmly locked inside a silica pore by a maximum of four hydrogen bonds that effectively eliminate local wobbling, which leads to high residual anisotropy in time-resolved fluorescence anisotropy measurements.5 Interestingly, using fluorescence recovery after photobleaching (FRAP) measurements, it was shown that R6G is able to diffuse on a silica surface under a humid atmosphere.6 Similar observations have also been confirmed by singlemolecule studies of R6G diffusion on silica thin films where R6G adsorption/desorption time of 67 ms was reported.7 Collectively, these studies imply that even Coulombic attraction and hydrogen bonding may not be sufficient to account for the complete immobilization of R6G and that physical confinement may also be responsible for R6G’s immobility found in silica hydrogel. The situation is rather different in anionic guest molecules. Due to Coulombic repulsion, anionic dyes were shown to display much higher mobility than cationic dyes. Thus, negatively
10.1021/jp8036473 CCC: $40.75 2009 American Chemical Society Published on Web 04/03/2009
Coulombic Interactions vs Physical Confinement
Figure 1. Molecular structures of rhodamine 6G and fluorescein.
charged pyranine exhibits rapid anisotropy decays with negligible residual anisotropy in silica hydrogels,8 and diffusion coefficient as high as 3.8 × 10-7 cm2 s-1 for FL in an aged hydrogel has been reported.9 On the other hand, translational motion is completely arrested in silica alcogels regardless of the charge of a guest molecule, suggesting that Coulombic interaction is completely dominated by physical confinement.10 These observations lead us to believe that the balance between Coulombic interaction and physical confinement strongly depends on the size of a guest molecule relative to the size of an encapsulating silica pore. Accordingly, for small molecules such as R6G and FL in silica hydrogel, it may be possible for Coulombic interaction to dominate their mobility, whereas physical confinement is the major factor to their immobilization in alcogel, which is typically filled with much smaller silica pores than hydrogel. In this study, we compare the behavior between cationic and ionic dyes in silica hydrogel. Cationic R6G and anionic FL are chosen in view of their similar molecular structures (Figure 1), so that any difference in their behavior could be confidently attributed to their opposite molecular charges. Both R6G and Oregon Green 514 (a more photostable FL derivative) are reportedly immobilized in silica alcogel because of physical confinement.10,11 Despite their similarity, surprisingly, a direct comparison between R6G and FL encapsulated inside identically prepared silica hydrogels has never been reported. The change in fluorescence anisotropy in R6G and FL during the hydrogel formation process will be compared in order to assess the influence of Coulombic interaction on mobility. In addition, to evaluate the relative contribution of physical confinement to R6G’s immobility in a silica sol-gel host of higher porosity, fluorescence anisotropy of silica hydrogel encapsulated R6G will be measured under low pH or high salt conditions where the effect of Coulombic interaction is considerably weakened. Results from the latter measurements revealed that R6G became inaccessible after encapsulation, providing further insights to the eventual placement of R6G in a solidified hydrogel. Experimental Section Materials. Tetramethyl orthosilicate (99+%, TMOS), R6G, and FL were purchased from Aldrich. All R6G and FL solutions were prepared using phosphate buffer pH 7.0. The phosphate buffer and other aqueous reagents were prepared using deionized water (18.1 MΩ · cm). All reagents were used as received. Microscope cover glasses (Fisher Premium) were purchased from Fisher Scientific and were thoroughly cleaned by consecutive sonication in a 10% sodium hydroxide solution, distilled water, acetone, and deionized water for 1 h each, respectively, before use. Hydrogel Preparation. A sol solution was prepared by mixing TMOS, H2O, and HCl (0.01 N) with volumes of 562.5, 120, and 11.25 µL, respectively. To facilitate acid hydrolysis, the sol solution was sonicated in an ice bath for a half-hour. Trapping of the dye molecules was made before polycondensation of the sol solution. For hydrogel formation, 40 µL of sol
J. Phys. Chem. B, Vol. 113, No. 17, 2009 5721 solution was added to 400 µL of 10 mM phosphate buffer, pH 7.0, that contained either R6G or FL. A hydrogel monolith can be prepared by transferring all 440 µL sample mixtures to a plastic cuvette and have the cuvette sealed for gelation. Alternatively, a sandwiched sample can be made by first stacking two cover glasses together using double-sided tape as a spacer to form a thin solution chamber. A 40 µL aliquot of the sample solution was then transferred and spread inside the chamber. The opening of the solution chamber was then sealed to prevent the rapid drying of hydrogel after gelation. The sandwich-structured sample was then aged for 12 h before use. Single Molecule Spectroscopy. Fluorescence images and kinetics traces of encapsulated molecules were obtained by a home-built confocal microscope under continuous argon-ion laser excitation as previously described.12 The laser excitation was delivered to the epi-illumination port of an inverted microscope through an optical fiber. The excitation was then collimated by a 10× objective, cleared an interference filter, reflected by a dichroic beam splitter, and finally focused onto a diffraction-limited spot by a 100×, 1.25 N.A. oil immersion objective. Upon excitation, single-molecule fluorescence collected by the same objective was directed to a side exit port and cleared a 100 µm aperture before it was collimated. Longpass filters were used to eliminate scattered laser excitation from the collimated fluorescence. The fluorescence was detected by an avalanche photodiode (APD) detector. The size of every fluorescence image was maintained at 10 µm × 10 µm throughout the entire investigation. Single FL and R6G molecules were excited at 488 and 514 nm, respectively. Fluorescence Recovery after Photobleaching. FRAP9 of FL in hydrogel was performed using the same confocal microscope as described above. An area on the sample was chosen and moved into the laser focus. A probe laser light was used to obtain a stable initial fluorescence intensity (Fi) first. The laser power was then increased by 10000-fold to photobleach the molecules for 10 s. Immediately after photobleaching, the laser power was decreased back to the probe level to begin monitoring the recovery of fluorescence due to diffusion of FL from adjacent areas into the bleached region. The fluorescence immediately after photobleaching would drop to its lowest intensity (F0). The experiment was stopped when the fluorescence recovery reached a plateau (F∞). The procedure would be repeated 5 times to obtain FRAP data at other areas. For comparison, FRAP of FL in solution was also studied. The FRAP data (fluorescence intensity vs time) were fit using the following equation,
I(t) ) A(1 - exp(-krecovert)) + C
(1)
where I(t) is the fluorescence intensity, krecover is the recovery rate constant, A is the difference between the final plateau intensity and the initial intensity after photobleaching (F∞ F0), and C is F0. Steady-State Fluorescence Measurements. Fluorescence anisotropy measurements were performed using a Shimadzu RF3101PC fluorimeter. Spectra were recorded at λex ) 488 nm for FL and λex ) 514 nm for R6G. To determine steady-state fluorescence anisotropy, λem ) 513 and 551 nm were used for FL and R6G, respectively. All fluorescence anisotropy values were calculated on the basis of the following equations,
r ) (IVV - GIVH)/(IVV + 2GIVH)
(2a)
5722 J. Phys. Chem. B, Vol. 113, No. 17, 2009
G ) IHV /IHH
Zhou and Yip
(2b)
where r is anisotropy, IVV, IVH, IHH, and IHV are the fluorescence intensity measured with different polarized excitation and emission schemes. G is a correction factor used to account for any polarization bias in the fluorimeter. The R6G anisotropy as a function of the pH of a silica hydrogel was investigated over a 3-month period. To change the acidity of a hydrogel matrix, a 100-300 µL aliquot of HCl solution at pH 2.0 was added to the top of a R6G-encapsulated silica hydrogel monolith and allowed to equilibrate for at least 1 week before the anisotropy was measured again. During the 3 month period, hydrogel monolith was gradually brought down from pH 7.0 to 3.5. In a separated experiment, NaCl was introduced to the hydrogel in an attempt to weaken the Coulombic attraction between R6G and the hydrogel. A 300 µL aliquot of 1 M NaCl was added to the top of a R6Gencapsulated hydrogel monolith and allowed to equilibrate for 2 weeks before the anisotropy of R6G was measured again. Time Evolution of Fluorescence Anisotropy of FL and R6G during Hydrogel Formation. R6G and FL anisotropy evolution during the gelation process of the hydrogel were examined by adding 150 µL of liquid TMOS sol and 1.5 mL of buffer solution containing 10-6 M dye to a 2.5 mL cuvette. Before the experiments, a G factor was first measured by recording the IHV and IHH from a 10-6 M R6G solution. Monitoring of fluorescence intensity time course was started 15 s after the dye-containing buffer was mixed with the TMOS sol and lasted for approximately 30 min. The fluorescence anisotropy evolution as a function of time was plotted. The data were fit according to the following equation,
r(t) ) A exp(-kanisot) + C
(3)
where A is the change of anisotropy during the gelation process from 15 s to 30 min, kaniso is the rate of change of anisotropy, and C is the initial anisotropy. Results and Discussions Fluorescence Images. The fluorescence images shown in Figure 2 exemplify the different mobility experienced by FL and R6G in silica hydrogels that were prepared under identical conditions. The R6G image in Figure 2A displays easily discernible, distinct fluorescent spots. The well-defined circular fluorescent spots indicate that many R6G molecules were immobilized in the hydrogel. Photobleaching or blinking is probably responsible for the noncircular fluorescent spots in the image. Also apparent is the presence of fluorescence streaks, which suggest that despite being immobilized to a great extent, yet few R6G molecules are still quite mobile in the hydrogel.13 R6G molecules have been shown to be mostly immobilized when encapsulated inside,10 but diffuse quite freely with a diffusion coefficient of 4.89 × 10-7 cm2 s-1 on,7 thin films of mesoporous silica alcogel. As demonstrated recently, a thin layer of absorbed water is known to facilitate R6G diffusion on glass surfaces.6 With a more porous silica framework practically filled with water, physical confinement in the hydrogel employed in this study is expected to be much relaxed and permit substantial R6G diffusion. Instead, the vast number of fluorescent spots relative to the very few number of fluorescence streaks in the image suggests otherwise, indicating that despite the small molecular size and the moderate solubility of R6G in water, the majority of R6G was immobilized in the extremely porous
Figure 2. Fluorescence images of (A) R6G and (B) FL encapsulated in silica hydrogels.
hydrogel. Strong Coulombic attraction between cationic dyes and silica surface has been demonstrated and utilized to monitor the growth of nanosize silica colloids during the hydrogel formation process.8,14-19 Expectedly, R6G molecules in our case are likely to attract to the silica surface through Coulombic attraction, which renders them mostly immobilized despite being trapped inside an extremely porous hydrogel framework. Most recently, time-resolved fluorescence anisotropy measurements on R6G ionically adsorbed on various silica substrates imply that R6G can be rigidly bound to a silica surface via a maximum of four hydrogen bonds.5 This will further enhance the propensity for R6G to become immobilized in our hydrogel, which favors the observation of stationary fluorescence spots in Figure 2A. Quite the contrary, the FL image in Figure 2B is characterized by a surprisingly featureless fluorescent background. Any attempt to obtain an image of immobilized FL in the hydrogel by decreasing FL concentration only resulted in a corresponding decrease in the featureless fluorescence background, implying that the FL molecules in silica hydrogel were too mobile for raster scanning imaging. Moreover, the absence of distinct fluorescence streak implies that FL diffusion in the hydrogel is considerably faster than that of R6G. A recent report put the diffusion coefficient of FL encapsulated inside a 2 day aged hydrogel with a higher silica content at 3.93 × 10-7 cm2 s-1,9 comparable to that obtained from R6G diffusing in a thin water film on top of an alcogel.7 For FL in our freshly prepared hydrogel that contains more than 90% water (≈3.0 wt % SiO2), diffusion is expected to be even faster. As Figure 1 illustrates, both FL and R6G belong to the broader family of xanthene dyes; they share similar molecular properties including size, shape, and mass. The completely different diffusion behavior of FL and R6G depicted in Figure 2 suggests that the Stokes-Einstein equation alone is no longer an adequate model to describe their diffusion in silica hydrogels. It is influenced by other factors that clearly go beyond the physical dimension of FL and R6G. At neutral pH, we expect that strong Coulombic attraction of cationic R6G toward negatively charged silica surfaces would severely impair R6G diffusion inside the hydrogel. Whereas anionic FL would be sufficiently repelled from the silica surfaces and remains relatively mobile in the solvent phase. Being constantly solvated by liquid, FL therefore exhibited mildly hindered diffusion caused only by the presence of mesopores and channels that make up the hydrogel. The completely opposite mobility behavior observed is therefore likely caused by Coulombic interactions alone that force R6G and FL to reside in totally different microenvironments within a hydrogel. Similar observations have been reported from a 3.1 wt % SiO2 sodium silicate gel where R6G was found to be effectively immobilized while the anionic pyranine dye only experienced a modest drop in mobility.8 In a previous study, we observed a similar but less
Coulombic Interactions vs Physical Confinement
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TABLE 1: Single-Molecule Mobility Distributions of R6G in Silica Hydrogel and Alcogela fix (%) hydrogel dry alcogel wet alcogel a
77 ( 2 23 ( 3 69 ( 3
tumbling (%)
intermediate (%)
4(1 2(1 4(1
19 ( 2 75 ( 3 27 ( 3
Alcogel data are obtained from ref 10.
prominent Coulombic effect on the mobility of R6G and Oregon Green 514 (ORG, a FL derivative) in alcogel thin films, where the silica framework is thought to be much more constricted than that of a hydrogel. In that study, both molecules were found to be incapable of translational diffusion, with ORG displaying a moderate increase in rotational diffusion only after the alcogel was equilibrated with a neutral pH buffer.10,11 The observation of a significantly higher FL mobility in the present work indicates that the influence of Coulombic interactions on mobility is strongly related to the porosity of a silica sol-gel host, with higher porosity tends to favor Coulombic interactions over physical confinement when determining mobility. While it is tempting to apply a similar argument to attribute the lack of R6G mobility to Coulombic attraction alone, however, there is evidence from subsequent experiments that physical confinement may also play a significant role in the immobilization of R6G in hydrogel. Although translational diffusion is prohibited in most silica hydrogel encapsulated R6G molecules, it is still possible to examine their rotational mobility by performing emission polarization measurements on single R6G molecules.12 The mobility of 296 R6G molecules were classified, and the results are compared to those previously obtained from alcogel encapsulated R6G also shown in Table 1. While the mobility distributions of R6G in hydrogel and dry alcogel do not resemble one another, there is a striking similarity in R6G mobility between those measured from hydrogel and wet alcogel. Both silica gels are dominated by fixed R6G molecules with significantly less contributions from intermediate and then tumbling molecules. This similarity implies that despite the very different structural architecture between silica alcogel and hydrogel, once the more mobile R6G molecules (e.g., those loosely adsorbed on thin film surface) in a dry alcogel are washed off by water, the remaining molecules that left in the wet alcogel are probably residing in microenvironments that are similar to those surrounding the hydrogel encapsulated R6G molecules. It is known that physical confinement is responsible for the low rotational mobility of R6G in alcogel. A similar microenvironment to the alcogel-encapsulated R6G would suggest that physical confinement may also have a considerable influence on R6G in a solidified hydrogel. On top of physical confinement, it has also been pointed out recently that R6G molecules entrapped inside silica pores of comparable physical dimension are less capable of free rotations because of the formation of multiple hydrogen bonds with the pore surface.5 The combined effect of physical confinement and hydrogen bonding may help explain why a dramatic increase in the percentage of fixed R6G molecules is observed in both hydrogel and wet alcogel relative to that encapsulated in dry alcogel, regardless of gel architecture. Fluorescence Recovery after Photobleaching. Figure 3A illustrates two fluorescence transients of FL in hydrogel after photobleaching, revealing two distinct recovery behaviors. The rapid fluorescence recovery in both transients solidly points to a high FL translational mobility in the hydrogel. The failure of a complete recovery back to its original intensity within the
Figure 3. Fluorescence recovery traces of FL in hydrogel after photobleaching: (A) example of full (gray) and partial (black) recovery of fluorescence intensity; (B) FL fluorescence recovery traces obtained from six separate locations inside a silica hydrogel. Solid curves are obtained from a global fitting to the recovery traces. For illustration purposes, all recovery traces are vertically displayed to remove congestion.
TABLE 2: Fluorescence Recovery Rate of FL Encapsulated in Hydrogel and Free FL in Water A1 (%)
τ1 (s-1)
46 ( 10 46 ( 10 40 ( 10 46 ( 10 43 ( 10 40 ( 10
0.23 ( 0.01 0.38 ( 0.01 0.50 ( 0.01 0.42 ( 0.01 0.48 ( 0.01 0.69 ( 0.01
In Hydrogel 54 ( 10 0.01 ( 0.01 54 ( 10 0.07 ( 0.01 60 ( 10 0.09 ( 0.01 54 ( 10 0.07 ( 0.01 57 ( 10 0.07 ( 0.01 60 ( 10 0.08 ( 0.01
0.11 ( 0.01 0.21 ( 0.01 0.25 ( 0.01 0.23 ( 0.01 0.24 ( 0.01 0.33 ( 0.01
52 ( 10 29 ( 10 34 ( 10 37 ( 10 26 ( 10 34 ( 10
0.50 ( 0.01 1.12 ( 0.01 0.74 ( 0.01 0.85 ( 0.01 1.35 ( 0.01 1.23 ( 0.01
48 ( 10 71 ( 10 66 ( 10 63 ( 10 74 ( 10 66 ( 10
In Water 0.09 ( 0.01 0.15 ( 0.01 0.14 ( 0.01 0.15 ( 0.01 0.17 ( 0.01 0.15 ( 0.01
0.30 ( 0.01 0.43 ( 0.01 0.34 ( 0.01 0.41 ( 0.01 0.48 ( 0.01 0.52 ( 0.01
A2 (%)
τ2 (s-1)
τav (s-1)
τbleach (s-1)
3.08 ( 0.01 1.17 ( 0.01 2.22 ( 0.01 1.18 ( 0.01 0.85 ( 0.01 0.48 ( 0.01
measurement time in one case, despite the high FL mobility, is a vivid reminder of the heterogeneous pore structure inside the hydrogel. The incomplete recovery could be due to the photobleaching of irreplaceable FL that was embedded deep inside the silica matrix of the hydrogel. Alternatively, incomplete recovery may be due to the photodepletion of FL inside a big, well-isolated hydrogel domain, within which are mesopores and channels that are large enough to allow FL diffusion to fuel a rapid recovery. However, if the bleached domain is well-isolated from the remaining hydrogel such that infusion of external FL is either completely excluded or significantly impaired, the dwindling supply of FL inside the isolated domain would result in a partial fluorescence recovery. On the other hand, if photobleaching is performed at pore domains where mesopores are well-connected to the vast hydrogel structure, the continuous supply of FL would make it possible to achieve 100% recovery within the short experimental time frame. Regardless of the final percentage recovery, all transients are characterized by a fast and a slow recovery rate, possibly due to FL traveling through
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Zhou and Yip TABLE 3: Steady-State Fluorescence Anisotropy Values of FL and R6G FL R6G
Figure 4. Fluorescence recovery traces of FL in water after photobleaching. FL fluorescence recovery traces were collected from six separate locations inside a sample cell. Solid curves are obtained from a global fitting to the six recovery traces. For illustration purposes, all recovery traces are vertically displayed to remove congestion.
the center and near the surface of silica channels, respectively. Table 2 summarizes the recovery rate extracted from six FRAP curves fit individually. A global fitting of the same six curves (Figure 3B) yields 0.069 and 0.411 s-1, respectively, as the slow and fast recovery rate, which suggests a 6-fold decrease in FL diffusion near a silica surface. With 53% contributed by the slow recovery, this gives an averaged recovery rate of 0.229 s-1 in the hydrogel. When the same FRAP experiment was performed on an aqueous solution of FL near a glass substrate, recovery rates of 0.129 and 0.714 s-1 were obtained from a global fit of six recovery curves (Figure 4). The faster recovery rates for FL in water as opposed to those in hydrogel indicate that despite constituting more than 90% water, the hydrogel still imposes a considerable drag to FL diffusion. The close to 6-fold difference between the fast and the slow rates here again suggests that diffusion near the surface of a glass substrate is responsible for the slower recovery rate. A major difference between the FL recovery curves in water and in hydrogel is that under the same probe laser intensity, all recovery curves in water contain a distinct, gradually declining component, which amounts to a continuous depletion of FL even under the weak probe laser excitation. On the other hand, the recovery curves in hydrogel continue to remain steadily up at the end of every measurement illustrated in Figure 3. The gradual decrease in FL fluorescence intensity in water is attributed to a less photostable FL. Once hydrogel bound and rendered less mobile, FL becomes more photostable because of impaired oxygen diffusion as well as the freezing of dynamic motions that facilitate photodegradation. Using 6.4 × 10-6 cm2 s-1 as the diffusion coefficient of FL in water to relate to the fast 0.714 s-1 recovery rate we recorded,20 the diffusion coefficient of FL near the glass substrate would be ca. 1.2 × 10-6 cm2 s-1, about a factor of 5 faster than that of R6G.6 The slower R6G diffusion is attributed to a strong electrostatic attraction between R6G and the surface of a glass substrate. On the basis of the same calculation, the averaged diffusion coefficient of FL in our hydrogel was found to be ca. 2.1 × 10-6 cm2 s-1. This is about 5 times higher than that obtained from a 2-day-old hydrogel with a significantly higher silica content and therefore a much denser framework to slow FL diffusion.9 The diffusion coefficients of FL in hydrogel and in water also indicate, according to the Stokes-Einstein equation, that the viscosity in the hydrogel is approximately 3 times higher than that in water. Our estimate is slightly higher than that reported from a 3.2 wt % sodium silicate hydrogel using time-resolved fluorescence anisotropy (TRFA) measurement on pyranine. In that report, viscosity was estimated from its effect on rotational diffusion using the Debye-Stokes-Einstein
solution
hydrogel
fluorophore infusion
0.0103 ( 0.0008 0.0077 ( 0.0012
0.0222 ( 0.0009 0.3248 ( 0.0017
0.02 0.18
equation and was found to be marginally higher in the hydrogel. Collectively, this suggests that while an anionic probe may experience slightly hindered rotation in a hydrogel, its translational diffusion can still be considerably impaired by the hydrogel framework. The discrepancy is attributed to the very different time scales associated with TRFA and FRAP measurements. Fast dynamics like rotational diffusion appears to be less sensitive to the presence of a surrounding silica matrix if a guest molecule is residing in a large enough silica pore, where the molecule is unlikely to encounter a silica surface within subnanosecond rotational time scales. On the other hand, when the relatively slower translational dynamics is considered, numerous collisions between a guest molecule and its surrounding silica matrix could occur in microsecond time scales that are typical of molecular diffusion. Consequently, translational diffusion experiences a more pronounced surface effect than molecular rotation, thereby registering a higher microviscosity in the hydrogel. A similar effect has also been observed in a more constricted silica hydrogel.9 Instead of focusing on viscosity, the slower translational diffusion of FL can be viewed as the result of hindered diffusion through the mesoporous matrix of a hydrogel. It is known that translational diffusion decreases monotonically with decreasing pore size. A pore 10 times the size of a molecule can dramatically reduce molecular diffusion by ∼40%.21 According to Figure 3 in ref 21, a 3-fold decrease in diffusion (i.e., ∼67% drop) would imply a molecule-to-pore ratio of ∼0.22. As a semiquantitative estimate, for a flat molecule approximately 10 Å × 7.5 Å such as FL, that would put the average pore size in our hydrogel at no less than 50 Å. Steady-State Fluorescence Anisotropy. The reduction of translational and rotational diffusion is expected to result in a corresponding increase in fluorescence anisotropy of FL and R6G. Table 3 compares the steady-state fluorescence anisotropy (r) values of free and hydrogel encapsulated FL and R6G. As expected, R6G registers a bigger increase in r value after encapsulation because of a significant loss in mobility. On the other hand, the marginal increase in r seen in FL after encapsulation suggests that FL still enjoys a high degree of freedom in the hydrogel. Both observations are consistent with results obtained from the fluorescence images and FRAP measurements. Also included in Table 3 are the anisotropy values of FL and R6G infused into the hydrogel long after gelation set in. While FL remains free to rotate regardless of when and how it is introduced into the hydrogel, the data from R6G suggest otherwise. The anisotropy of infused R6G almost drops by half to a value of 0.18 relative to 0.32 obtained from those introduced into the hydrogel before gelation, implying a higher mobility for the infused R6G. Evidently, silica pores that strongly impair R6G mobility are inaccessible to dyes infusion after a hydrogel has solidified. This indicates that the local environment of R6G depends strongly on when R6G is introduced into a hydrogel. Unlike FL, R6G is probably directed toward a more restrictive environment during the solidification of a hydrogel. In view of the possible difference between FL and R6G, this alternative placement is most likely related to the opposite charges carried by the two fluorophores. Controlling the placement of a guest molecule based on its chemical attributes has been demonstrated before. When lanthanide and
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Figure 6. Fluorescence anisotropy values of hydrogel encapsulated R6G as the pH of the hydrogel decreases. Figure 5. Time evolution of fluorescence anisotropy of FL (top) and R6G (bottom) during the hydrogel formation process. Solid curves are fitting to anisotropy increase according to A(1 - exp(-kanisot)) + C.
ruthenium complexes were functionalized with triethoxysilane, they were mainly found in the silica matrix of alcogel, whereas other organic probes were mostly localized in hydrophobic domains formed by surfactant templates.22-25 If opposite Coulombic interactions can direct FL and R6G to reside in different microenvironments in silica hydrogel, then it may be possible to use FL and R6G to examine the gelation process of hydrogel from two different perspectives. Specifically, the lack of attraction between FL and silica should allow FL to exclusively monitor how viscosity and physical confinement increase as the silica framework builds up during gelation, whereas the strong attraction between R6G and silica should be more suitably used to reveal the growth rate of silica colloids during gelation.4 Figure 5 shows the r values of FL and R6G as a function of time after adding 10 volumes of dye containing buffer to 1 volume of liquid TMOS sol. Both dyes exhibit increasing r as gelation proceeded, with R6G displaying a bigger increase relative to that of FL. Despite the very different final r values, FL and R6G produce comparable gelation rates of 0.025 ( 0.004 and 0.012 ( 0.001 s-1, respectively. Although a 2-fold difference between the two rates may imply that the gelation process could be influenced by the nature of the encapsulated probe, this explanation is deemed unlikely as the concentrations of FL and R6G used in these measurements were far below that of the silane precursor used. A more probable explanation is that oppositely charged FL and R6G were encapsulated in different types of local environment and sampling the gelation process therein. Presumably, the faster rate observed in FL indicates that, immediately after mixing, the viscosity of the sol-buffer mixture increases rapidly. The formation of oligosilane at this early stage of reaction was probably responsible for the increase in viscosity. Unfortunately, FL soon lost the ability to interrogate its local environment after 3 min as the increasing physical confinement imposed by the hydrogel was eventually out-weighted by the strong repulsion between FL and silica. Because FL was forced to remain solvated, the minor drop in mobility due to the occasional encounters of oligosilane and larger silica colloids could only result in a marginal increase in anisotropy. This continued to be the case throughout the gelation process, as the collapsing pores inside the hydrogel were nowhere near the dimension that is small enough to significantly impair FL mobility through physical confinement. In the case of R6G, the much bigger increase in anisotropy (0.306 ( 0.011) suggests that, on top of the anticipated initial rise in viscosity, R6G was drawn to a constricting environment
that eluded FL’s detection. The influence of this restrictive environment appears to completely overwhelm the viscosity effect and raise the anisotropy of R6G far beyond what viscosity alone can account for. In view of the widely adapted nanoparticle metrology approach, the gradual rise to large anisotropy value can be readily explained by the Coulombic attraction of R6G toward rapidly formed oligosilane, followed by the coalescence of oligosilane to secondary/higher order particles at a slower rate. The slower gelation rate and higher final anisotropy value registered by R6G are therefore more reflective of the slower secondary/higher order particle formation rate during the gelation process. The coalescence of oligosilane to larger particles raises the prospect that surface-adsorbed R6G at the beginning of a gelation process may eventually end up burying deep inside the particle and become well-insulated from any external stimulus. To determine whether R6G in a hydrogel remains accessible, we tried to exchange encapsulated R6G with H+ by equilibrating a solidified hydrogel with a pH 2.0 HCl solution and monitored the change in R6G anisotropy value as the pH of the hydrogel gradually decreased with time. As shown in Figure 6, the anisotropy values of R6G were practically unchanged as the pH of the hydrogel decreased from 7 to 3. This indicates that even as the charges on a silica surface were neutralized at low pH, R6G remained immobilized in a hydrogel and was unavailable for exchange with H+, hence no change in the anisotropy values. The slowly upward trend in anisotropy value in Figure 6 is probably caused by continuous hydrogel aging or accelerated shrinking of silica matrix as surface charges are neutralized by H+ to eliminate repulsive interactions between silica surfaces, which further reduce R6G mobility. We also equilibrated R6G doped hydrogel with a 1.0 M NaCl solution in an attempt to weaken Coulombic attraction and hydrogen bonding to free R6G from the hydrogel surface to no avail.26 The anisotropy value of R6G remained unchanged despite extended hours of equilibration. Collectively, these results indicate that once a hydrogel is formed, encapsulated R6G becomes inaccessible, an outcome that is made possible only if all R6G molecules are permanently embedded deep inside the silica matrix of a hydrogel. This leads us to hypothesize a model (Figure 7) for the placement of cationic molecules such as R6G in silica hydrogel, in which strong Coulombic attraction not only causes R6G adsorption on silica surface initially but also helps direct the eventual encapsulation of R6G inside well-insulated pores templated by the dye itself. In this model, R6G is attached exclusively onto the surface of oligosilane primary particles at the early stage of a gelation process because of Coulombic attraction. Subsequent coalescence between primary particles as well as continuous polymerization of silanol on the surface
5726 J. Phys. Chem. B, Vol. 113, No. 17, 2009
Figure 7. Encapsulation model of R6G (red star) during hydrogel formation.
of primary/secondary particles may easily trap those once surface-bound R6G molecules inside the secondary/higher order particles. As the particles continue to grow in size, R6G eventually becomes deeply embedded inside the silica matrix of a hydrogel and physically confined, displaying highly restrained rotational and translational motions and also incapable of undergoing exchange reaction with any external reagent. As a final note, this interpretation is consistent with the observation that R6G infused into a solidified hydrogel exhibits higher mobility and thus a lower anisotropy value compared to that from R6G added before gelation set in, as indicated in Table 3. Presumably, R6G that infuses into a silica matrix after gelation can adhere to the silica surface through Coulombic attraction, resulting in lower mobility hence increasing fluorescence anisotropy relative to that in free solutions. However, fluorescence anisotropy from these infused molecules is still noticeably lower than that from R6G added before a hydrogel solidified, indicating that the latter R6G molecules are subjected to more stringent conditions than Coulombic interactions alone in the solidified hydrogel. We believe that the additional loss in mobility is due to physical confinement when R6G molecules are trapped inside their self-templated pores during the hydrogel formation process. Conclusions Moving from silica alcogel to the more porous hydrogel, we found that Coulombic interactions became more dominating than physical confinement as the dimension of silica pore was increased. While both cationic and anionic dyes found mostly immobilized in alcogel, anionic FL appears to exhibit a high degree of mobility in hydrogel. Surprisingly, cationic R6G remains mostly immobilized in hydrogel despite larger pore dimensions. In view of the similar physical properties between FL and R6G except for molecular charge, we attribute the dramatic difference in their mobility to Coulombic interactions, with R6G firmly attracted to but FL repelled from silica surfaces at neutral pH during and after the gelation process. The opposite Coulombic interactions thus cause FL and R6G to reside in different microenvironments within a hydrogel. Consequently, anionic fluorophores such as FL are more suitable for monitoring physical confinement effect and viscosity change, whereas cationic fluorophores such as R6G are ideal for following the growth of silica colloid during hydrogel formation. We also demonstrated that FL is capable of unhindered rotation (negligible anisotropy) as well as relatively free diffusion inside a hydrogel. The latter is confirmed by FRAP measurements through which the diffusion coefficient of FL in hydrogel was estimated at 2.1 × 10-6 cm2 s-1, about a factor of 3 slower than in solution. This translates to a physical confinement by silica pores of 50 Å or bigger, or alternatively, the slower diffusion points to a 3-fold increase
Zhou and Yip in viscosity in hydrogel according to the Stokes-Einstein equation. Although the viscosity in hydrogel should be slightly above that in water according to TRFA measurements by others, the discrepancy can be readily understood in terms of the different intrinsic length scales FRAP and TRFA are sensitive to. While TRFA (molecular rotation) reports the microviscosity within the first few solvent shells surrounding a fluorophore, FRAP (molecular translation) is inherently used for revealing viscosity pertaining to bigger dimensions. In this case, the presence of silica matrix in a hydrogel slows the averaged translational diffusion of FL despite the free rotational diffusion revealed by the TRFA measurements. The difference in steady-state anisotropy values for R6G added before and after the solidification of hydrogel helps reveal the influence of Coulombic attraction in directing the placement of a guest molecule in a hydrogel. The larger anisotropy value for R6G added before gelation agrees with the notion that R6G binds to the oligosilane surface through Coulombic attraction initially. Subsequent coalescence between oligosilane to form larger secondary/higher order particles then physically trapped the once surface-bound R6G underneath a growing layer of silica coat. Access to the encapsulated R6G molecules then becomes almost impossible because they are located deep inside the silica matrix and trapped in pores templated by the molecules themselves, leading to an additional drop in mobility due to physical confinement. On the other hand, R6G that diffuses into a silica matrix after gelation is only attached to a silica surface through Coulombic attraction. The absence of any additional physical confinement in these R6G molecules results in a smaller anisotropy value relative to that from R6G added before gelation. Collectively, this implies that the extent of physical confinement experienced by a molecule can be influenced by Coulombic interactions during gelation, with cationic and anionic molecules producing tighter and looser encapsulating silica pores correspondingly and therefore leading to stronger and weaker physical confinement effect, respectively. Acknowledgment. This study was supported by the Oklahoma Center for the Advancement of Science and Technology (Grant HR03-144) and NSF-IC (Grant CHE-0442151). References and Notes (1) Braun, S.; Rappoport, S.; Zusman, R.; Avnir, D.; Ottolenghi, M. Mater. Lett. 1990, 10, 1. (2) Pierre, A. C. Biocatal. Biotransform. 2004, 22, 145. (3) Avnir, D.; Coradin, T.; Lev, O.; Livage, J. J. Mater. Chem. 2006, 16, 1013. (4) Tleugabulova, D.; Duft, A. M.; Zhang, Z.; Chen, Y.; Brook, M. A.; Brennan, J. D. Langmuir 2004, 20, 5924. (5) Tleugabulova, D.; Sui, J.; Ayers, P. W.; Brennan, J. D. J. Phys. Chem. B 2005, 109, 7850. (6) Mitani, Y.; Shimada, A.; Koshihara, S.; Fukuhara, K.; Kobayashi, H.; Kotani, M. Chem. Phys. Lett. 2006, 431, 164. (7) Mahurin, S. M.; Dai, S.; Barnes, M. D. J. Phys. Chem. B 2003, 107, 13336. (8) Tleugabulova, D.; Zhang, Z.; Brennan, J. A. J. Phys. Chem. B 2003, 107, 10127. (9) Hungerford, G.; Rei, A.; Ferreira, M. I. C.; Suhling, K.; Tregidgo, C. J. Phys. Chem. B 2007, 111, 3558. (10) Gilliland, J. W.; Yokoyama, K.; Yip, W. T. Chem. Mater. 2004, 16, 3949. (11) Gilliland, J. W.; Yokoyama, K.; Yip, W. T. J. Phys. Chem. B 2005, 109, 4816. (12) Viteri, C. R.; Gilliland, J. W.; Yip, W. T. J. Am. Chem. Soc. 2003, 125, 1980. (13) Bardo, A. M.; Collinson, M. M.; Higgins, D. A. Chem. Mater. 2001, 13, 2713. (14) Geddes, C. D. J. Fluoresc. 2002, 12, 343.
Coulombic Interactions vs Physical Confinement (15) Geddes, C. D.; Karolin, J.; Birch, D. J. S. J. Fluoresc. 2002, 12, 113. (16) Geddes, C. D.; Karolin, J.; Birch, D. J. S. J. Fluoresc. 2002, 12, 135. (17) Geddes, C. D.; Karolin, J.; Birch, D. J. S. J. Phys. Chem. B 2002, 106, 3835. (18) Birch, D. J. S.; Geddes, C. D. Phys. ReV. E 2000, 62, 2977. (19) Geddes, C. D.; Birch, D. J. S. J. Non-Cryst. Solids 2000, 270, 191. (20) Furukawa, R.; Arauzlara, J. L.; Ware, B. R. Macromolecules 1991, 24, 599. (21) Beck, R. E.; Schultz, J. S. Science 1970, 170, 1302.
J. Phys. Chem. B, Vol. 113, No. 17, 2009 5727 (22) Hernandez, R.; Franville, A. C.; Minoofar, P.; Dunn, B.; Zink, J. I. J. Am. Chem. Soc. 2001, 123, 1248. (23) Minoofar, P. N.; Hernandez, R.; Franville, A. C.; Chia, S. Y.; Dunn, B.; Zink, J. I. J. Sol-Gel Sci. Technol. 2003, 26, 571. (24) Minoofar, P. N.; Dunn, B.; Zink, J. I. J. Am. Chem. Soc. 2005, 127, 2656. (25) Minoofar, P. N.; Hernandez, R.; Chia, S.; Dunn, B.; Zink, J. I.; Franville, A. C. J. Am. Chem. Soc. 2002, 124, 14388. (26) McCain, K. S.; Harris, J. M. Anal. Chem. 2003, 75, 3616.
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