Characterization of the Microenvironments of PRODAN Entrapped in

Kulwinder K. Flora, and John D. Brennan*. Department of .... Ana Rei , Graham Hungerford , Michael Belsley , M. Isabel C. Ferreira , Peter Schellenber...
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J. Phys. Chem. B 2001, 105, 12003-12010

12003

Characterization of the Microenvironments of PRODAN Entrapped in Tetraethyl Orthosilicate Derived Glasses Kulwinder K. Flora and John D. Brennan* Department of Chemistry, McMaster UniVersity, Hamilton, Ontario, L8S 4M1, Canada ReceiVed: July 9, 2001; In Final Form: September 28, 2001

6-Propionyl-2-(dimethylamino)naphthalene (PRODAN) has been widely used to probe the internal environment of sol-gel derived glasses. It is generally assumed that the entrapped probe reports primarily on the internal solvent environment, through changes in emission wavelength, lifetime, or anisotropy. However, we show that other effects, such as aggregation of the probe and adsorption of the probe onto the silica surface, can also alter the emission properties of PRODAN, providing further information on the evolution of sol-gel derived glasses. Both the steady-state and time-resolved fluorescence properties of PRODAN were examined when the probe was entrapped in tetraethyl orthosilicate (TEOS) derived glasses. The glasses were prepared using a two-step method that is commonly used for protein entrapment, and aged either in air without washing (dry-aged), in air after a washing step (washed), or in buffer (wet-aged). For all aging methods, the changes in the emission properties of the probe were consistent with at least three discrete microenvironments, reflecting free monomers, free aggregates, and adsorbed species (monomers and/or aggregates), the proportion of which changed as a function of drying time and conditions. The monomeric form of the probe underwent a polaritysensitive emission shift that reflected changes in the internal solvent composition. However, the aggregates/ adsorbates contributed unique features to both the steady-state and time-resolved emission properties of PRODAN that gave insights into changes in the solubility of the probe, consistent with loss of internal solvent as aging of the glass proceeded. This study clearly shows that significant new information can be obtained from studies of PRODAN emission, and demonstrates that time-resolved fluorescence measurements are critical to properly elucidate the environment(s) present within the sol-gel derived materials.

Introduction In the past few years several reports have appeared describing the use of fluorescence spectroscopy to characterize the internal environment(s) of sol-gel derived glasses and biomaterials prepared from tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS).1,2 Steady-state and time-resolved fluorescence spectroscopy have been able to provide detailed information regarding the polarity3, dynamics,4 and accessibility5 of the local microenvironment within the glass, and how these change as a function of the preparation and aging protocols used. One area where fluorescence methods have been particularly useful is for the determination of the pore-liquid composition of sol-gel derived materials.1 Information regarding the polarity and viscosity of the entrapped solvent allows for guidance in the development of improved materials that can be used to entrap functional biomolecules such as proteins. Previous studies from our group5d-h and others3,4 have shown that the microenvironment within TEOS and TMOS derived glasses tends to become more polar, more viscous, and more heterogeneous as aging continues, and that increased levels of water during aging tend to accelerate the evolution of the material toward its final state.6 It is well-known that only some entrapped probes accurately report on the pore-liquid composition and microviscosity within sol-gel derived materials, while others report on other, probe* Author to whom correspondence should be addressed. Phone: 905525-9140 (Ext. 27033). Fax: 905-522-2509. E-mail: [email protected].

specific phenomena. For example, some probes, such as 7-azaindole, adsorb to the surface of the glass, providing information on composition of the silica surface rather than the pore-liquid composition.3a,3b,6,7 Others, such as pyrene, report on the solubility of the probe, owing to shifts in the monomerexcimer equilibria when the probe is present at sufficiently high concentration.3c,8,9 Other probes can be sensitive to internal pH,10 oxygen content,11 or even surface charge.12 Given the vast number of parameters that may affect the emission of a fluorophore,13 detailed knowledge of the factors that determine the overall decay properties of a probe is of paramount importance for understanding the relationship between the emission properties of an entrapped probe and the properties of the host material. A fluorophore that has been used widely for examination of sol-gel derived materials is 6-propionyl-2-(dimethylamino)naphthalene (PRODAN).3b,4e,7,14 This probe is typically used to assess the polarity and microviscosity within sol-gel derived materials owing to its large solvatochromic shift (emission wavelength ranges from 401 nm in cyclohexane to 531 nm in water),15 and the sensitivity of the excited-state decay kinetics to the local environment of the probe.16 An interesting feature that has recently been reported for this probe is a concentrationdependent aggregation phenomena that is related to solvent composition, producing a diagnostic peak at ca. 430 nm.16a,16e,17 This blue-edge emission is typically weak in aqueous solution, but is prominent in sol-gel derived glasses,3b,7 in some cases being the dominant peak in the steady-state emission spectrum.

10.1021/jp012598i CCC: $20.00 © 2001 American Chemical Society Published on Web 11/03/2001

12004 J. Phys. Chem. B, Vol. 105, No. 48, 2001 While the origin of the peak for PRODAN in sol-gel derived materials has been assumed to be due to aggregates of the probe,3b,7 this has never been confirmed. Thus, a detailed photophysical characterization of the entrapped probe is required to determine the various forms of PRODAN in glass (monomer, aggregate, adsorbate) to better understand the nature of the information that is available from this probe. In this study, we have examined the steady-state and timeresolved fluorescence emission properties of PRODAN at relatively high concentrations in solution and when entrapped into TEOS-derived glasses. The glasses were prepared using a two-step method commonly used for protein entrapment, and aged either in air without washing (dry-aged), in air after a washing step (washed), or in buffer (wet-aged) to provide differences in the initial solvent composition, and hence the nature of the aging of the glasses.6 The results clearly show that PRODAN reports not only on internal solvent polarity and microviscosity, but that aggregation and adsorption of the probe to the silica occur, producing the emission peak at 430 nm. The formation of aggregated and adsorbed forms of PRODAN is shown to be dependent on the aging method employed for the sol-gel glasses, suggesting that PRODAN aggregation can provide new insights into the nature and location of dopants within sol-gel derived glasses. Experimental Section Chemicals. Tetraethyl orthosilicate (TEOS, 99.999+%), was supplied by Aldrich (Milwaukee, WI). 6-Propionyl-2-(dimethylamino)naphthalene (PRODAN) was purchased from Molecular Probes (Eugene, OR). Polymethacrylate fluorimeter cuvettes (transmittance curve C) were obtained from Sigma (St. Louis, MO). All water was twice distilled and deionized to a specific resistance of at least 18 MΩ cm. All other chemicals were of analytical grade and were used without further purification. Procedures. Entrapment of PRODAN. Sol-gel derived matrixes containing PRODAN were prepared by first mixing 9.0 mL of TEOS, 2.8 mL of H2O, and 0.2 mL of 0.1 N HCl and sonicating for an hour in a sealed scintillation vial until the mixture became monophasic. The solution was then stored at -20 °C for 5-7 days before addition of the probe so that complete hydrolysis of the TEOS could occur.5d A volume of 750 µL of hydrolyzed TEOS was mixed with an equal volume of a freshly prepared solution of phosphate buffered saline (PBS, 10 mM disodium phosphate, 100 mM KCl, pH 7.2) containing 10 µM PRODAN. The mixture was stirred and then immediately placed in a disposable methacrylate cuvette (1 cm path length) to produce blocks with initial dimensions of 1 cm × 1 cm × 1.5 cm. Blocks were used to minimize light scattering and to ensure that bulk properties of the glass were observed during fluorescence studies. The cuvettes were then sealed with Parafilm and the sol-gel derived blocks were aged at 4 °C using three different methods. The wet-aged and washed samples were filled with 2 mL of PBS containing 10 µM PRODAN (to avoid leaching of the entrapped probe) 20 min after gelation had occurred and allowed to stand overnight at 4 °C, whereas the dry-aged samples had no buffer added at any stage. Wet-aged and washed blocks were rinsed for 10 min two more times to exchange residual ethanol with buffer. Previous studies using the ethanol-sensitive fluorescent dye pyranine indicated that such a washing procedure was sufficient to remove all the entrapped ethanol.6 In all cases the washing solution contained 10 µM PRODAN. Washing samples without PRODAN in the rinse buffer led to a substantial loss of the entrapped probe, as

Flora and Brennan revealed by a decrease in the intensity of the emission peak at 520 nm from the glasses and a corresponding increase in the emission intensity of the rinse solution. In this case, the amount of entrapped probe decreased to the point where only moderate emission was obtained from the entrapped probe at 430 nm. In cases where PRODAN was present in the rinse solution at a concentration that was identical to that of the entrapped probe, there were no detectable changes in the emission spectrum of the PRODAN solution (i.e., no loss of the 430 nm peak in solution), consistent with no partitioning of the soluble dye into the matrix, and no loss of entrapped dye from the monolith. After rinsing, the washed blocks were aged in air while the wet-aged blocks were aged in the presence of the wash buffer for the duration of aging, but were tested in the absence of the wash buffer. Steady-State Fluorescence Measurements. Fluorescence measurements were performed by using a SLM 8100 spectrofluorimeter (Spectronic Instruments, Rochester, NY), as described elsewhere.5d PRODAN was excited at 363 nm with emission collected from 385 to 650 nm. All spectra were collected in 1 nm increments using 4 nm band-passes on the excitation and emission monochromators and an integration time of 0.3 s per point. Appropriate blanks were subtracted from each sample and the spectra were corrected for the wavelength dependence of the emission monochromator and photomultiplier tube. Steady-state fluorescence anisotropy measurements were performed as described previously.5d Single point fluorescence anisotropy measurements were generally made at an emission wavelength of 520 nm, with excitation at 363 nm, unless otherwise stated. All fluorescence anisotropy values were corrected for the instrumental G factor to account for any polarization bias in the monochromators. All fluorescence anisotropy values reported are the average of 5 measurements each on 2 different samples. Steady-state anisotropy measurements were converted to average rotational reorientation times (φSS) using the following equation:13

r)

r0 1 + (〈τ〉/φSS)

(1)

where r is the measured fluorescence anisotropy, r0 is the limiting anisotropy, and 〈τ〉 is the intensity-weighted mean fluorescence lifetime of the sample as determined using eq 5 below. A limiting anisotropy of 0.336 ( 0.002 (λex ) 363 nm) was used for PRODAN.4e The average rotational reorientation times were used to determine the volume of the rotating unit (V) (mL mol-1) for monomer and aggregate forms of the probe in solution and in the sol-gel derived glasses using the Perrin equation:

φSS )

ηV RT

(2)

where η is the average microviscosity (0.0010 kg m-1 s-1 for water at 293 K), R is the gas constant (8.314 kg m-2 s-2 K-1 mol-1), and T is the temperature in Kelvin, which was set to 293 K. Alternatively, this equation was used to determine microviscosity values using the volume of the rotating unit obtained for the monomeric form of the probe. Time-ResolVed Fluorescence. Time-resolved fluorescence intensity and anisotropy decay data was acquired in the timedomain using an IBH 5000U time-correlated single photon counting fluorimeter. A pulsed violet laser diode operating at a 1 MHz repetition rate with a 150 ps pulse duration and a

Microenvironments of PRODAN in TEOS Derived Glasses wavelength of 399 nm was used for excitation of the dye. The intensity decay data was collected under magic angle polarization conditions, passed through a monochromator (8 nm bandpass), and detected on a TBX-04 PMT detector. Single point intensity or anisotropy decay data was collected at 520 nm, while decay-associated spectra (DAS) were collected at emission wavelengths from 410 to 560 nm in 15 nm increments. Intensity decay data was collected into 4096 channels (12.5 ps per channel) until 10000 counts were obtained in the peak channel. Decays from blanks were collected at the same emission wavelength and were subtracted from the sample decay curves with appropriate weighting of the associated errors using software provided by IBH. The instrument response function was collected by measuring the Rayleigh scattering of the laser pulse from water (typical fwhm of 180 ps), and was used to deconvolute the instrument response profile from the experimentally determined decay trace. Appropriate time-shift parameters were obtained by allowing this to be a floating parameter in the fit. The fluorescence intensity decay was fit using both discrete and distributed fitting models, and in all cases a sum of three exponentials was found to provide the best fit to the data. The decay data was subjected to global analysis with linking of lifetimes across different aging times (for single point decay curves obtained at 520 nm) or emission wavelengths, according to the equation:18

I(λ,t) )

∑i Ri(λ) exp(- t/τi)

∑i Ri(λ)τi)

∑i fiτi

Figure 2. Changes in the ratio of the aggregate peak intensity (at ca. 430 nm) to the monomer peaks intensity (at ca. 520 nm) for entrapped PRODAN in dry-aged (b), washed (9), and wet-aged (2) glasses over a period of 55 days.

represent the fractional contributions to the total anisotropy decay from the slow and fast motions, respectively (Σβi ) 1).20 Results and Discussion

(4)

The intensity-weighted mean lifetime values, 〈τ〉, were obtained from single point decay data at 520 nm emission using the following equation:

〈τ〉 )

Figure 1. Emission spectra obtained from dry-aged, washed, and wetaged sol-gel glasses 1 day after gelation of the glass.

(3)

where τi is the decay time of the ith component and Ri(λ) is the preexponential factor at emission wavelength λ (or at a given aging time when linkage was done across aging times). Goodness-of-fit was determined using the reduced χ-squared parameter and by visual inspection of residual and autocorrelation plots.19 The fractional fluorescence of component i at wavelength λ (fi(λ)) (or at a given aging time) was calculated from

fi(λ) ) (Ri(λ)τi/

J. Phys. Chem. B, Vol. 105, No. 48, 2001 12005

(5)

Time-resolved decays of fluorescence anisotropy were constructed from intensity decays that were obtained using vertically polarized excitation and vertically polarized emission (IVV) or horizontally polarized emission (IVH), and were corrected for the instrument response profile and the instrumental G-factor, as described in detail elsewhere.20 The anisotropy decay was fit to a two-component hindered rotor model according to the following equation:

r(t) ) (r0 - r∞)[β1 exp(- t/φ1) + β2 exp(- t/φ2)] + r∞ (6) where φ1 reflects slow rotational motions associated with rotation of aggregates in solution, φ2 reflects rapid rotational reorientation of the monomeric form of the probe in solution, and r∞ is the residual anisotropy of the probe that is associated with an adsorbed fraction of the probe. The terms β1 and β2

Steady-State Fluorescence. Figure 1 shows the emission spectra obtained from dry-aged, washed, and wet-aged solgel glasses 1 day after gelation of the glass. The most obvious differences in the spectra are the slight red-shift in the emission maximum for washed and wet-aged samples as compared to dry-aged samples, the presence of a second peak centered at 430 nm in both the washed and wet-aged samples, and a lower intensity for the red-shifted peak (ca. 520 nm) in the washed and wet-aged samples. These spectral features are in agreement with those observed previously in TEOS derived glasses,3b,7 and have been interpreted as being due to aggregates of the probe, as demonstrated by Bright et al.17 The decrease in the intensity of the “monomer” peak at ca. 520 nm as the “aggregate” peak at 430 nm increases suggests an equilibrium between two forms of the probe, however, the exact origin of these peaks and the nature of the species that contribute to the peaks in sol-gel derived materials has not previously been examined in detail. Figure 2 shows the changes in the ratio of the emission intensity values for the peaks at 520 nm (assigned to solvated monomers of the probe) and at 430 nm (assigned to aggregated/ adsorbed probe based on time-resolved data, described below) as a function of aging time for the three different aging conditions over a period of 55 days. Measurements were terminated at this point since all samples had attained constant mass by this time (25 days for wet-aged samples, 50 days for

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Figure 3. Excitation spectra obtained as a function of emission wavelength for PRODAN in a 1-day-old wet-aged glass. 430 nm emission, 520 nm emission, 595 nm emission.

Figure 4. Changes in anisotropy with wavelength for 20 µM PRODAN in aqueous solution (b) and for a washed sample after 1 day of aging (9).

washed and dry-aged samples). The data show that the evolution of dry-aged samples was much different than that of washed or wet-aged samples, with the latter two samples evolving in a relatively similar manner. Wet-aged and washed samples initially showed a substantially higher proportion of the 430 nm peak as compared to dry-aged samples. This is consistent with the higher level of internal water, since formation of the aggregates occurs only when PRODAN reaches its solubility limit, which is ca. 3 µM in water but significantly higher in ethanol/water mixtures. The peak intensity ratio initially increased in all samples, reaching a plateau approximately 5 days after gelation for washed and wet-aged samples, and at a substantially longer time (day 25) for dry-aged samples, likely owing to slow loss of ethanol from this sample. The ratio then decreased for all samples over the next several weeks, consistent with loss of water and subsequent aggregation and/or adsorption of the probe onto the surface of the glass. Over this time, the emission wavelength of the monomer peak showed small shifts in all samples (data not shown). The emission wavelength of wet-aged and washed samples (517 nm) was initially red-shifted from that of the dry-aged samples (510 nm), consistent with the higher polarity expected upon removal of ethanol from the former samples. The emission maximum shifted very rapidly for wet-aged and washed samples, reaching a constant value of 514 nm by day 8, while dry-aged samples required 20 days to reach a constant emission wavelength (also 514 nm), consistent with slow loss of ethanol and then water. It is important to note that by day 25, all samples had similar emission maxima and emission peak ratios, regardless of the aging method, which is consistent with the probe experiencing a similar environment in all glasses. Hence, the specific aging method does not appear to dramatically alter the final environment reported by PRODAN emission maxima, even though previous reports suggest that significant differences should exist between such samples.6 Figure 3 shows excitation scans obtained as a function of emission wavelength for PRODAN in a wet-aged sol-gel derived glass. It is clear that the excitation spectrum associated with the 430 nm emission peak (solid line) is very different from that associated with the 520 nm (dashed line) or 595 nm (dotted line) emission peaks. Excitation spectra obtained from 20 µM PRODAN in solution showed similar behavior, while excitation spectra obtained from 1 µM PRODAN in solution were identical at all emission wavelengths tested (data not shown). These results clearly show that the blue-edge peak originates from a different source than the main emission peak seen at ∼520 nm, and confirm that there is a concentration dependence associated with the existence of the 430 nm peak,

as would be expected if this peak were associated with PRODAN aggregates. To further examine the origin of the two emission peaks, steady-state anisotropy measurements were done as a function of both aging time and wavelength. Figure 4 shows the changes in anisotropy with wavelength for 20 µM PRODAN in aqueous solution (circles) and for a washed sample after 1 day of aging (squares). The solution-based experiment was done to provide a situation where aggregates were present but adsorbed species were not, allowing us to selectively examine the aggregates. Two points merit special attention. First, the anisotropy is always much higher in the glass than in solution at all wavelength values, providing evidence for both adsorption of the probe and for a higher internal microviscosity of the solvent within the glass (this is discussed in more detail in the section on timeresolved anisotropy). Second, the anisotropy from both samples is significantly higher at shorter wavelengths than at longer wavelengths, consistent with slower rotation and hence a larger species emitting at the blue end of the spectrum. Using eqs 1 and 2, one can calculate the volume of the rotating unit in solution using data at 430 nm (aggregates) and 595 nm (which should correspond solely to the monomer). Using experimentally determined mean lifetimes for PRODAN in solution of 2.07 ns at an emission wavelength of 430 nm and 1.45 ns at 595 nm, steady-state rotational correlation times of φ430 ) 320 ps and φ595 ) 117 ps were obtained. From these values, the volumes of the rotating unit, assuming a viscosity of 1.00 cP for aqueous solution at 20 °C, are V430 ) 730 mL mol-1 and V595 ) 284 mL mol-1. Previous reports suggest that the volume of a PRODAN monomer is 140 mL mol-1,4e thus it appears that dimers of the probe may exist at long wavelengths while aggregates containing ∼5 PRODAN monomers are present in the blue edge of the spectrum. The higher anisotropy of the probe in the sol-gel derived glass is consistent with a higher microviscosity (ca. 6 cP using a volume of 140 mL mol-1,4e and lifetime and anisotropy values obtained for the entrapped probe at 595 nm), and, as was observed in solution, also reflect the presence of aggregates at shorter wavelengths. On the basis of time-resolved anisotropy data, described below, it is likely that the aggregates are also partially adsorbed, leading to the much higher anisotropy obtained at the blue end of the spectrum for the entrapped probe. Figure 5 shows the evolution of the steady-state anisotropy for PRODAN in glass (at 520 nm) as a function of aging time for the three aging methods. In general, the steady-state anisotropy data suggested that the probe had higher anisotropy (i.e., lower overall mobility) in washed and wet-aged samples than in dry-aged samples over the first 30 days of aging, after

Microenvironments of PRODAN in TEOS Derived Glasses

Figure 5. Steady-state rotational anisotropy for entrapped PRODAN in dry-aged (b), washed (9), and wet-aged (2) glasses over a period of 55 days.

which all samples had similar average anisotropy values. The higher rotational mobility for dry-aged samples is likely due to the presence of ethanol, which would solvate the probe and reduce the amount of aggregates. Both washed and wet-aged samples showed a rapid increase in rotational anisotropy over the first 25 days of aging, suggesting that the viscosity of the internal environment increased over time, that the proportion of aggregated or adsorbed PRODAN increased, or both. Dryaged samples remained relatively mobile for approximately 15 days, which corresponds to the time required to expel ethanol from the pores of the glass. Beyond this point, the mobility decreased rapidly, likely owing to loss of water, which would promote aggregation and adsorption of the probe and thus lower the overall rotational mobility. All samples showed identical average values for the rotational anisotropy by day 45, suggesting that the final environment within the glass did not depend on the aging method, in agreement with spectral data presented above. Time-Resolved Fluorescence. To better understand the origin of the two emission maxima, intensity and anisotropy decays were measured for entrapped PRODAN and for dilute and concentrated solutions of PRODAN. Figure 6 shows the mean emission lifetime data and the changes in the fractional contribution of each lifetime component (fi) at an emission wavelength of 520 nm for PRODAN entrapped within the different glasses. Discrete multicomponent fits were obtained in all cases, consistent with previous reports.16,17 Global analysis across all aging times clearly revealed the presence of three decay components with values of 3.87, 1.85, and 0.61 ns for wet-aged samples, 3.81, 1.70, and 0.45 ns for washed samples and 3.75, 2.11 and 0.73 ns for the dry-aged samples. (Note: the error in each lifetime value is approximately (0.05 ns.) Values close to the latter two lifetime components have previously been reported for the monomeric form of the probe in water, and hence both are tentatively assigned to the free monomer. The ∼3.8 ns component is not observed from dilute solutions of PRODAN, and thus is assigned to an aggregated/ adsorbed form or the probe. These assignments are discussed in more detail below. The lifetime data shown in Figure 6 clearly show that there are significant differences in the microenvironment of PRODAN during aging in dry-aged samples as compared to samples that are washed or wet-aged. The mean lifetimes are relatively similar on day 1 for all samples, even though the dry-aged sample has a much different internal environment than the other samples based on the steady-state spectral results. However, the emission lifetime of the dry-aged samples decreases over the first 15 days of aging, consistent with the loss of ethanol (τH2O

J. Phys. Chem. B, Vol. 105, No. 48, 2001 12007 ) 1.60 ns, τEtOH ) 3.48 ns), but then reaches a minimum and increases rapidly until day 30, and more slowly after that point. Both washed and wet-aged samples show increases in emission lifetime immediately, and both evolve similarly, reaching a plateau by about day 20. The increased lifetime corresponds to both the increase in anisotropy and the decrease in the ratio of the aggregate peak at 430 nm for the entrapped probe. Taken together, these results again suggest that the origin of the long lifetime is an aggregated/adsorbed form of the probe. To more accurately assess the origin of the 3.8 ns component, PRODAN aggregates were adsorbed onto a quartz slide from a solution containing 20 µM of the probe, and both a steadystate emission spectrum and intensity decay data were obtained from the sample (see Figure 7). Attempts to prepare samples of the adsorbed probe using solutions of 3 µM PRODAN (monomer form) did not provide sufficient adsorbed probe to allow either steady-state or time-resolved data to be collected. The emission spectrum of the adsorbed probe was centered at 430 nm, and showed no second peak at 520 nm, confirming that the blue-edge peak was indeed due to aggregated/adsorbed probe. Unfortunately, it was not possible to distinguish between adsorbed monomers and adsorbed aggregates, suggesting that these species may in fact emit in a similar wavelength range. The intensity decay from this sample (obtained at an emission wavelength of 430 nm) could be fit well to a 3 component decay (χ2 ) 1.09), providing decay components of 3.68 ns (34%), 1.02 ns (8%), and 0.39 ns (58%). The high weighting on the short component likely reflects some scattering, which was evident in the decay trace. On the basis of these data, it is clear that aggregates/adsorbates contribute significantly to the 3.8 ns decay component, but may also affect the values of the ∼1.8 ns and ∼0.7 ns components obtained from the entrapped probe. These lifetime assignments are also consistent with two other observations: (1) the proportion of the long lifetime component increases with aging time in a fashion that is consistent with the expectations for aggregate/adsorbate formation, and (2) decay-associated spectra (DAS), shown in Figure 8, reveal that the emission maximum of the long lifetime component is significantly blue-shifted relative to the other two lifetime components, and coincides with the peak of the “aggregate” form of the probe, at 430 nm. The DAS show an unexpectedly broad emission profile for both the long component (3.60 ns in the DAS) and the intermediate component (∼1.6 ns) suggesting that these lifetime components may in fact be associated with more than one form of the probe. DAS obtained from a 20 µM PRODAN solution in water, which contains aggregates but no adsorbates, also showed three decay components (5.13 ns, 1.48 ns, 0.44 ns), with the long component emitting toward the blue end of the spectrum (λmax ) 450 nm) and the medium and short components emitting with a maximum at 520 nm (data not shown). The 1.48 ns and 0.44 ns lifetime components agree well with those obtained by Bright et al.17 for dilute PRODAN solutions, and reflect monomers in solution. The longer lifetime component is therefore indicative of PRODAN aggregates. The increased value of the long component in solution relative to glass suggests that entrapment may alter the environment of the aggregates, perhaps through adsorption to the silica, such that emission is somewhat quenched in the glass relative to solution. On the basis of the above observations, the unusual “double hump” appearance of the DAS for the 3.60 ns and 1.58 ns components make it clear that at least two species must contribute these decay components. In fact, at least four species can simultaneously exist in the glass; free monomers, free

12008 J. Phys. Chem. B, Vol. 105, No. 48, 2001

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Figure 6. (a) Mean emission lifetime data for entrapped PRODAN in dry-aged (b), washed (9), and wet-aged (2) glasses over a period of 55 days and the changes in the fractional contribution of each lifetime component for PRODAN within the different glasses for (b) dry-aged, (c) washed, and (d) wet-aged glasses over a period of 55 days. (b) long τ, (9) medium τ, and (2) short τ.

Figure 7. Emission spectrum and intensity decay components obtained from a quartz slide containing PRODAN that was adsorbed from an aqueous solution containing 20 µM of the probe.

Figure 8. Decay-associated spectra for PRODAN in a 14-day-old wetaged glass. (b) 3.63 ns, (9) 1.58 ns, and (2) 0.59 ns. Errors in the lifetime values are ( 0.01 ns.

aggregates, adsorbed monomers, and adsorbed aggregates. It is clear that the long lifetime component is observed in both free aggregates (∼5 ns) and for adsorbed species (3.8 ns). Further-

more, the intermediate component (ca. 1.8 ns) is observed for free monomers, but a relatively similar component (1.0 ns) is observed from adsorbed aggregates, even though these have very different spectral maxima (430 nm for adsorbed/aggregated species, 520 nm for free monomer). These data indicate that fitting to three decay components likely results in a superposition of indistinguishable decay times from two or more different species, leading to the “double-hump” appearance. It should be noted that attempts to fit DAS to four decay components did not provide any better data in terms of statistics, and typically provided two components with identical lifetimes, reflecting the inability of the fitting program to distinguish between similar lifetimes from the different species. To further explore the origins of the decreased mobility of PRODAN in the different glasses, and to attempt to discriminate between soluble aggregates and adsorbed species, time-resolved decays of anisotropy were measured for PRODAN in solution and in glasses immediately after formation and after 30 days of aging, corresponding to the point where average rotational mobility became constant for all samples. Typical anisotropy decays and corresponding residual plots for PRODAN in solution (panel A) and in a 1-day-old wet-aged glass (panel B) are shown in Figure 9. The most striking difference between the decays is the residual anisotropy values, which are significant in the glass (ca. 0.25) but completely absent in solution. Timeresolved anisotropy decays of a 20 µM solution of PRODAN at 520 nm emission indicated that there were two rotational reorientation times, one on the order of 60 ps (97%), and the other on the order of 2.6 ns (3%) (χ2 ) 0.97). The viscosity obtained from the shorter component using eq 2 is 1.04 cP, in excellent agreement with the expected viscosity of water at 20 °C. The presence of the longer rotational reorientation time indicates that PRODAN aggregates exist, which would have a larger volume of rotation and hence a slower rotational reorientation time. This is expected since the PRODAN is present at 20 µM in water, and should show aggregates since it is above its solubility limit. This also suggests that even at 520

Microenvironments of PRODAN in TEOS Derived Glasses

J. Phys. Chem. B, Vol. 105, No. 48, 2001 12009

Figure 9. Typical anisotropy decays and corresponding residual plots for PRODAN in solution (panel A) and in a 1-day-old wet-aged glass (panel B).

TABLE 1: Time-Resolved Fluorescence Anisotropy Decay Parameters for PRODAN Entrapped in Dry-Aged, Washed, and Wet-Aged Sol-Gel Derived Glasses as a Function of Aging Time sample

φ1 (ns)a

β2

r∞c

χ2

1 day 2 days 14 days 28 days 30 days 40 days

5.74 5.98 4.33 2.61 0.84 0.89

Dry-Aged 0.35 0.03 0.33 0.04 0.34 0.06 0.38 0.07 0.30 0.16 0.29 0.31

0.97 0.96 0.94 0.93 0.84 0.69

0.004 0.005 0.013 0.034 0.149 0.134

1.02 1.04 0.94 0.99 1.16 1.12

1 day 2 days 14 days 28 days 30 days 36 days

2.12 5.06 7.04 6.13 4.65 5.22

Wet-Aged 0.27 0.04 0.16 0.09 0.15 0.17 0.16 0.13 0.17 0.16 0.15 0.08

0.96 0.91 0.83 0.87 0.84 0.92

0.25 0.25 0.25 0.27 0.27 0.28

1.05 1.23 1.22 1.23 1.11 1.16

1 day 2 days 14 days 28 days 30 days 36 days

5.08 6.12 4.50 5.23 5.34 2.66

0.30 0.16 0.15 0.18 0.14 0.14

Washed 0.12 0.10 0.14 0.13 0.03 0.03

0.88 0.90 0.86 0.87 0.97 0.97

0.14 0.27 0.27 0.27 0.29 0.26

1.12 1.14 1.17 1.15 1.16 1.17

φ2 (ns)

β1b

a Typical errors in rotational correlation times are (0.03 ns. b Typical errors in fractional contributions are (0.01. c Errors in r∞ values are (0.02.

nm, the steady-state anisotropy of a 20 µM solution does not reflect rotation of a monomer, but rather an average of monomer and aggregate forms. Time-resolved anisotropy data obtained at 520 nm for the entrapped probe is shown in Table 1. The data reveal that the probe exists in no fewer than three distinct environments. The fastest rotational component, which is generally on the order of a few hundred picoseconds, is associated with monomers that are present in the pore solvent within the glass. The slower rotational component, which is typically in the 2-6 ns range, is indicative of PRODAN aggregates that are still present in the pore solvent. This interpretation is consistent with the steadystate anisotropy measurements, which suggested the presence

of aggregates that emitted at 520 nm. Finally, the large residual anisotropy values that are present at all times in washed and wet-aged samples, and which grow in for dry-aged samples, are consistent with a significant fraction of adsorbed monomers and/or aggregated species. A few other points merit special attention. First, the value of the short rotational correlation time is consistent with an internal solvent microviscosity of ca. 2-4.5 cP, which remains constant as aging proceeds. Higher internal microviscosity values for pore solvents within sol-gel derived glasses have been reported previously,1 but the observation of a constant microviscosity has not been reported before. Second, the proportion of the longer rotational component generally increases with time (particularly for dry-aged samples), but then appears to decrease as aging continues, perhaps owing to adsorption of aggregates onto the pore walls. Third, the value of the long rotational component remains relatively constant, and in some cases even decreases as aging continues, suggesting that the aggregates reach a critical size and do not grow larger as aging continues. Finally, both β1 (reflecting long rotational reorientation times) and r∞, the residual anisotropy, increase with time in a manner that corresponds to the increase in f1, the fraction of the long lifetime component, particularly for dryaged samples. Washed and wet-aged samples also show increases in r∞ that correspond to the increase in f1, confirming that the 3.8 ns lifetime component predominantly reflects adsorbed species. Taken together, these results suggest that aggregates form slowly as aging continues, reach a critical size, and then adsorb onto the pore wall, producing hindered rotation. The presence of ethanol at early times in dry-aged samples results in solubilization of the probe, and thus prevents aggregate formation and adsorption over the first 14 days, at which point the entrapped ethanol has evaporated from the pores. Washing or wet-aging of glasses accelerates aggregate formation and adsorption, consistent with the lower solubility of the probe in aqueous solution. Conclusions 6-Propionyl-2-(dimethylamino)naphthalene (PRODAN) has been widely used to probe the internal environment of sol-gel

12010 J. Phys. Chem. B, Vol. 105, No. 48, 2001 derived glasses. Previous studies have generally assumed that the entrapped probe reports on the internal solvent environment alone, and have not generally considered the potential for aggregation or adsorption of the probe onto the surface of the glass. Both the steady-state and time-resolved fluorescence of the probe dispersed at moderate concentrations in tetraethyl orthosilicate (TEOS) derived glasses are consistent with the presence of at least three distinct forms of the probe within the glass: free monomers in solution, aggregates in solution, and adsorbed probe. The relative proportions of these different forms change as a function of both drying times and aging conditions, indicating that detailed information on the evolution of solgel derived materials can be obtained by monitoring the ratios of these three forms. Previous work from our lab has shown that the incorporation of either polymers or alkylsilane precursors into sol-gel derived materials results in complete removal of the peak at 430 nm, suggesting that the organic moieties are able to solubilize the probe and prevent aggregation and adsorption from occurring. Detailed studies on the photophysical behavior of PRODAN in organically modified silicates are underway, and will be reported in due course. An unexpected finding from the current work was the absence of distributions of local microenvironments within the glass, contrary to previous reports by Bright et al.4e This contradictory result is likely due to the significant differences in the methods used to prepare and age the glasses in our study as compared to that of Bright et al., with the latter materials being formed without a sonication step and with a higher ethanol level and a lower water:silicon ratio than in our studies. The sonication step and the higher level of water used in our study likely resulted in a much more homogeneous environment, producing the discrete emission properties observed. Overall, our results suggest that the emission properties of PRODAN can be used to report not only on internal solvent composition, but also on factors such as solubility and adsorption of the probe. Thus, PRODAN may provide useful information on the properties of sol-gel derived glasses, including their surface polarity, the ability of the internal solvent to solvate the probe, and the potential for hydrophobically modified glasses to partition the probe between the glass surface and the solvent phase. Such information may prove valuable in optimizing sol-gel based chromatographic media, solid-phase microextraction films, or biosensor coatings. Acknowledgment. Funding from the Natural Sciences and Engineering Research Council of Canada and MDS-Sciex is gratefully acknowledged. References and Notes (1) Dunn, B.; Zink, J. I. Chem. Mater. 1997, 9, 2280. (2) Keeling-Tucker, T.; Brennan, J. D. Chem. Mater., in press. (3) (a) Matsui, K.; Matsuzuka, T.; Fujita, H. J. Phys. Chem. 1989, 93, 4991. (b) Brennan, J. D.; Hartman, J. S.; Ilnicki, E. I.; Rakic, M. Chem.

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