J. Phys. Chem. B 2009, 113, 12067–12074
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Monitoring Sol-to-Gel Transitions via Fluorescence Lifetime Determination Using Viscosity Sensitive Fluorescent Probes Graham Hungerford,*,†,§ Archie Allison,† David McLoskey,† Marina K. Kuimova,‡ Gokhan Yahioglu,‡,| and Klaus Suhling§ HORIBA Jobin YVon IBH Ltd., Skypark 5, 45 Finnieston Street, Glasgow G3 8JU, U.K., Chemistry Department, Imperial College London, Exhibition Road, London SW7 2AZ, U.K., Department of Physics, King’s College London, Strand, London WC2R 2LS, U.K., and PhotoBiotics Ltd., 21 Wilson Street, London, EC2M 2TD, U.K. ReceiVed: March 26, 2009; ReVised Manuscript ReceiVed: June 8, 2009
The sol-to-gel transition was monitored via the use of time-resolved recording of the fluorescence emission of viscosity-sensitive probes. Two dyes were chosen for the study, water-soluble DASPMI and a hydrophobic BODIPY, and steady-state, time-resolved and time-tagged fluorescence measurements were performed. These techniques, coupled with the probes different solubility, allowed complementary fluorescence lifetime and intensity data to be obtained from the dyes introduced into the matrix-forming mixture to produce sol-gel derived monoliths. Two different precursors were used as examples. A hydrogel was formed from a commercially available gellan gum (Gelrite), and a glass-like monolith was formed using tetraethyl orthosilicate. Changes in fluorescence lifetime could be related to those in the local viscosity sensed by the probe. The combination of this type of probe with time-resolved measurements is extremely useful in monitoring the microscopic changes that occur during the sol-to-gel transition within this important class of materials. Introduction The sol-to-gel transition is of great importance in areas such as biology and materials science.1-5 It involves the transformation of a solution or biopolymer into an integrated network, which can either be composed of discrete particles or interlinked polymer chains. Many substances are known to undergo this kind of transformation, ranging from proteins and polysaccharides to metal alkoxides, with the outcome a gel-like biological polymer or a porous glass-like material. Many hybrid materials incorporating biologically significant compounds within a robust protective host have also been produced for the purpose of biosensing,6 drug delivery,7,8 and study of protein confinement.9 During the sol-to-gel transition dramatic changes in viscosity occur as a microheterogeneous medium is formed. Because of the diversity and significance of substances that can undergo this transformation, and the wealth of associated applications, its study is of importance. Many lines of investigation have been pursued, involving the use of rheology to monitor changes in bulk viscosity,10,11 as well as calorimetric methods.12 Also fluorescence anisotropy has been used to monitor the chain mobility in a polysaccharide.13 This example demonstrates a way of monitoring more local microenvironmental changes. Fluorescence depolarization measurements, although mainly the measurement of rotation from which a viscosity can be determined, can be relatively complex to perform.14 They require the measurement of at least two planes of polarization (four, if the G-factor correction is included). Thus, because of the measurement time they can be affected by photobleaching and * To whom correspondence should be addressed. E-mail:
[email protected]. Tel.: 0044-(0)1412296796. Fax: 0044(0)1412296790. † HORIBA Jobin Yvon IBH Ltd. § King’s College London. ‡ Imperial College London. | PhotoBiotics Ltd.
are not simple to apply to time course experiments. Fluorescence lifetimes on the other hand are simpler to record and more suited for time course and imaging applications. Recently there have been reports on the usage of viscosity sensitive probes,15-20 whose photophysics are governed by molecular twisting, to estimate the local viscosity within solutions or microheterogeneous media. Hence the employment of this type of fluorescence probe is well suited to monitor the sol-to-gel transition. The use of fluorescence lifetime to measure microviscosity has several advantages, as it is practically independent of the fluorophore concentration, thus allowing a clear distinction between probe concentration and quenching effects. The most popular technique for fluorescence lifetime determination is that of time-correlated single-photon counting.21 This allows the precise determination of the probe’s fluorescence lifetime. However, it suffers from a drawback; it is by definition inefficient, historically resulting in relatively long acquisition times, which can hamper its application in the study of dynamic processes. Obviously, as the transition is a continuous process there is a high probability that the probe environment will be changing during the measurement. One way to obtain an uninterrupted view of the probes fluorescence lifetime is to employ the time-tag fluorescence method.22 In this technique the arrival time of individual photons from the probe’s fluorescence at the detector is labeled. These data are constantly streamed and this allows an uninterrupted record of the timeresolved fluorescence to be constructed. In this work we employ two viscosity sensitive probes, a hydrophobic BODIPY (4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene) and a more hydrophilic probe, DASPMI (4-(4-(dimethylamino)styryl)-N-methyl-pyridiniumiodine). These molecules have previously been used to report on viscosities in different complex systems.17,18,20 As DASPMI is water-soluble and the BODIPY hydrophobic, combining their use can provide complementary information in microheterogeneous systems.
10.1021/jp902727y CCC: $40.75 2009 American Chemical Society Published on Web 08/13/2009
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Here this pair was used to probe the local viscosities, both in the entrapped waterpools and at the bulk material-water interface in two sol-gel systems. The first system selected uses a tetraethyl-orthosilicate (TEOS) precursor to form a glass-like xerogel.1,2 Fluorescent probes have been employed to study sol-gel derived media23,24 and use has been made of highly solvatochromic dyes, such as PRODAN,25 and Nile red,26 which have been successfully applied to study both the number and polarity of local environments present within sol-gel derived media.27,28 Application of this type of material has been found, for example, for the incorporation of dyes29 and biologically active molecules; the latter for use in the field of biosensing.30 The second system involved the study of the viscosity behavior of a gellan gum solution. Upon heating the helical polysaccharide backbone undergoes a helix-to-coil transition4 accompanied by a large change in viscosity. Addition of metal ions has been found to affect this through interhelix aggregation.31-33 This gum is versatile and has found usage in the food industry,34 as a substance for controlled drug release7 and for tissue engineering applications.35 The systems incorporating the probes were studied by time-resolved fluorescence and, when monitoring the TEOS system, by time-tag measurements. Experimental Section Theoretical Framework. The fluorescence quantum yield of molecular rotors, Φf, is a function of the viscosity (η) of the medium they are dissolved in and is described well by the Fo¨rster-Hoffmann equation36
( Tη )
Φf ) z
x
(1)
where z and x are constants and T is the temperature. Using the definition of the quantum yield
kr kr + knr
Φf )
(2)
and the fluorescence lifetime τf
τf )
1 kr + knr
(3)
where kr is the radiative rate constant and knr is the nonradiative rate constant, eq 1 can then be expressed37 as
τf )
z η kr T
SCHEME 1: Overview of a Time-Tag Experiment to Monitor the Arrival of Photons from the Viscosity Sensitive Probes during the Sol-to-Gel Transitiona
a The stop pulses originating from the detection of fluorescence photons are correlated to the start pulses (source pulsed at 1 MHz) and binning used to construct histograms from which to obtain the decay parameters.
Measurements. Absorption spectra were recorded using a Perkin-Elmer Lambda 2, and steady-state fluorescence measurements were made on a HORIBA Jobin Yvon Fluorolog 3. Timeresolved measurements were performed on a HORIBA Jobin Yvon IBH TemPro Fluorescence Lifetime System using either a NanoLED N-488 L diode laser or a N-495 LED excitation source (for time-tagged measurements). Detection was made via a 550 nm cutoff filter with a TBX-04 detection module. Time-tagged fluorescence data were recorded using in-house software to control the equipment’s data acquisition electronics, with a microtime resolution of 55 ps per channel, at a clock rate of 12 MHz. The instrumental response was obtained by removing the emission filter and replacing the sample with a scattering solution before the end of the experiment. This created one file containing the data with the arrival time of the individual photons recorded. The time-tagged data were binned into different time segments (shorter duration close to the beginning of the experiment and longer toward the end) and the recovered decay reconvoluted with the instrumental response to yield the fluorescence lifetime data.38 This means that, nominally, fluorescence lifetimes from 100 ps could be determined. Intensity data was obtained by integrating the number of counts in the decay (normalized for bin time width). A schematic overview of this type of measurement procedure is given in scheme 1. Data analysis was performed with IBH DAS6 software and the goodness of fit judged in terms of a χ2 value and weighted residuals. The decay time data were analyzed using a sum of exponentials, employing a nonlinear least-squares reconvolution analysis of the form n
I(t) )
∑ Ri exp(-t/τi)
(6)
i)1
x
()
(4)
Taking logarithms in eq 4 leads to
( Tη ) + log( kz )
log τf ) x log
(5)
r
A plot of log τf versus log η thus allows conversion of fluorescence lifetimes into viscosity, that is, it serves as a calibration graph. In the case of the dyes used in this study a fuller photophysical characterization of their viscosity behavior related to this work has been published elsewhere.17,18,20
The pre-exponential components (R) are given normalized to unity and errors are given as three standard deviations. Average fluorescence lifetimes were calculated as τave ) ΣRiτi. Temperature was maintained using a Neslab RTE 7 Digital One recirculating bath. Data for 1 (scheme 2) in methanol-glycerol mixtures were measured as previously reported.18 Sample Preparation. Sol-gel derived monoliths were manufactured as previously reported39 and were based on the method given by Flora and Brennan27 making use of a tetraethyl-orthosilicate precursor (Aldrich). Equal volumes of sol and buffer solution (phosphate buffered saline, pH 7.4) were mixed by inverting (the start point for the experiment) and allowed to gel. The probes (see scheme 2) were introduced prior to mixing.
Monitoring Sol to Gel Transitions SCHEME 2: Probe Structures: 1 (4,4′-Difluoro-4bora-3a,4a-diaza-s-indacene) and 2 (4-(4(Dimethylamino)styryl)-N-methyl-pyridiniumiodine). Also Indicated Are the Principal Rotations Giving Rise to Their Fluorescence Decay Behavior40-42
4-(4-(Dimethylamino)styryl)-N-methyl-pyridiniumiodine (DASPMI), from Invitrogen, was dissolved into the buffer, while 4,4′difluoro-4-bora-3a,4a-diaza-s-indacene18 was dissolved in the sol. Polysaccharide, gellan gum solutions were made from a 1% solution of Gelrite in water (HPLC grade), both from Sigma. The Gelrite was dissolved at ca. 85 °C by stirring, and initial gelation occurred on cooling. Measurements were then performed upon heating of the “solution”. Magnesium ions, in the form of MgSO4, were added to the water prior to mixing. Results and Discussion Fluorescence Lifetime Behavior of 1 and 2 in Solution upon Changing Viscosity. To be able to make use of the two chosen probe molecules to sense changes in viscosity, the fluorescence lifetime behavior of the two molecules was measured in glycerol-solvent mixtures of differing viscosities. This was previously reported for 1, in glycerol-methanol mixtures18 and for 2 in glycerol-water mixtures.17 Figure 1 compares the previous data for 1, with that for 2. This was remeasured using an extended viscosity range, similar to that used for 1 in a previous study.17 From these data it is obvious that dramatic changes in fluorescence lifetime are evident for both probes upon changing viscosity. The mechanisms behind this behavior and the photophysics of these probes have previously been reported17,18,41,42 and will not be gone into here. However, it should be noted that for 1 the monomer fluorescence lifetime (single exponential decay) and for 2 the average fluorescence lifetime (obtained from analyzing as a sum of three exponentials) are used to monitor changes in viscosity. Monitoring the Sol-to-Gel Transition in TEOS-Based Systems. This transition involves a colloidal suspension (sol) of the precursor material undergoing a hydrolysis and condensation reaction to form a gel. This involves the polymerization and interlinking of these polymer chains to form a porous SiO2 based material. Upon further aging this can result in the formation of a glass-like monolith. This type of reaction is initially exothermic relating to the hydrolysis of TEOS, followed by a small endothermic reaction which diminishes as polymerization increases.43 However, overall observed temperature changes are small (>1 °C).43 These media have shown promise for protein entrapment and for the incorporation of a range of light addressable entities.9,44 Before discussing the time-resolved fluorescence results, note should be taken of the steady state fluorescence spectra, taken before and at the end of the time-tag study. These are shown in Figure 2. The sol-gel reaction commences on mixing of an equal quantity of sol with phosphate buffered saline solution. Probe 1 was introduced into the reaction mixture in the sol and 2 in the buffer (in separate experiments) because of their
J. Phys. Chem. B, Vol. 113, No. 35, 2009 12069 solubility. Figure 2a shows the spectrum of 1 in sol before the addition of buffer and at the end of the experiment in the gel, after approximately 3 h. It can be seen that the spectrum of 1 in gel exhibits a shoulder at 600 nm which is most likely attributed to the presence of aggregates.45 This is important to consider when analyzing the time-resolved data. Figure 2b shows 2 in buffer and in gel at the end of the experiment. Although the two spectra are not exactly comparable (because of a factor 2 dilution on combining sol and buffer, plus a slight wavelength change related to a reduction in solvent polarity) a drastic change in the fluorescence intensity can be discerned, which cannot be explained by the above factors. The evolution of the change in fluorescence intensity during the transition can be estimated by integration of the time-resolved decay data (inset Figure 2a). The form of this curve is, as we have previously reported, similar to that obtained from steady state measurements and indicative of a two-step process.17 A similar form of analysis of the time-resolved decays of 1 did not show this change in fluorescence intensity. We can hypothesize that the difference stems from the different phases in which probes 1 and 2 are embedded, for example, aqueous and organic phases. Another contributing factor could be aggregation of 1, as indicated by the shoulder in fluorescence spectra at 600 nm. The presence of the aggregates of 1 in sol-gel mixtures are further confirmed by its multiexponential behavior (see below). For probe 1, the analysis of the time-resolved fluorescence data obtained from binning the time-tag data showed that the decays were complex and required the sum of three exponentials to obtain a satisfactory fit. This is strikingly different to the monoexponential decays obtained in methanol/glycerol mixtures, see Figure 1. The outcome of the fluorescence lifetime analysis is shown in Figure 3. The two shorter-lived components appear invariant during the gelation period. This behavior is consistent with their emission originating from aggregated probe molecules. The longer-lived component exhibits an increase in fluorescence lifetime and it is this emission that we will consider in more detail. It is interesting to note that the average fluorescence lifetime remains constant, in accordance with the observation of no significant change in the emission intensity, thus indicative of a dynamic aggregation process during the gelation process. The analysis of the emission of 2 also required the sum of three exponential components to fit the decay data. However in this case, the origin of the fluorescence is still within keeping of the regime we and others have reported of charge transfer and potential bond twisting.17,41 A general increase in all fluorescence lifetimes is observed (see Figure 4) and a concomitant decrease in the amount of shorter-lived (emanating from the planar form) with the rise of the longer-lived fluorescence emission occurs. This is in keeping with the stabilization of charge transfer states upon increasing viscosity. The trend for the change in the time-resolved decay parameters is shown more clearly in Figure 5, which shows the change in average fluorescence lifetime during the gelation process. Inset is also the ratio between the pre-exponential components for the longest and shortest lived decays, which demonstrates an increased stabilization of the twisted charge transfer state emission. Combining the time-tag data with that obtained from the glycerol mixtures allows an estimate of the change in viscosity during the sol-to-gel transition to be made. Visually the solution has been seen to gel within the first few minutes after combining the sol with the buffer solution. The viscosity data as calculated from eq 5, shown in Figure 6, does not exhibit any abrupt
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Figure 1. Plot showing variation in fluorescence decay in different viscosity media (a) for compound 1 in methanol-glycerol mixtures and (b) for 2 in water-glycerol mixtures.
change, instead it reports on what appears to be a two step process; an initial fast increase in viscosity followed by a slower gradual increase. This is in accordance with what we have previously observed using steady state measurements for gelation and the commencement of matrix consolidation.17,46 From the figure it is clear that the two probes sense different local viscosities over the time scale of the experiment. Probe 1, which is expected to associate with the silica polymer chains, reports an increase of ∼50% in viscosity, with most of this increase observed within the first hour. For the latter part of the experiment the probe’s fluorescence lifetime does not show any significant change. This is in contrast to the data obtained with 2, where greater changes in local viscosity are reported. Again there is an initial “fast” increase followed by a slower increase, which toward the end of the experimental time scale is discernibly augmenting. These data show that initially both polymer chains and waterpools experience similar rates of increasing viscosity, after which the main influence is sensed by the more hydrophilic probe 2. This can be indicative of a fast polymerization process followed by interlinking of the polymer chains which in turn influences the entrapped buffer solution. The consolidation of the polymer network to form a more defined structure with associated changes in morphology
thus elevates the viscosity of the aqueous phase. Obviously we have limited the time scale of these experiments to the initial aging process, which has been made possible by the use of the time-tag technique, but we have observed changes, which occur over an extended aging period (several weeks) in terms of morphology and the behavior of entrapped probes and proteins by monitoring molecular diffusion Via fluorescence anisotropy and fluorescence recovery after photobleaching in addition to usage of molecular rotors.47,48 It is clear that the extension of time-resolved fluorescence techniques along with the use of complementary probes is advantageous in elucidating nanoscale viscosity changes in this system. Temperature Induced Changes in Gellan Gum Structure. Gellan gum consists of a linear tetrasaccharide repeating unit [f4)-L-rhamnopyranosyl-(R-1f3)-D-glucopyranosyl-(β-1f4)D-glucuronopyranosyl-(β-1f4)-D-glucopyranosyl-(β-1f]) and it is the commercial form, Gelrite, that is used in this work. This versatile polysaccharide has found a myriad of applications, ranging from the food industry to drug delivery and tissue engineering applications.7,8,34,35,49 On cooling it undergoes a solto-gel transition at 33-34 °C50 and there have been many reports evaluating its viscosity behavior over a range of concentrations and upon addition of metal ions.31,50,51 Cations help to cross-
Monitoring Sol to Gel Transitions
Figure 2. Emission spectra of (a) 1 in sol and gel at the end of experiment, (b) 2 in PBS, prior to gelation, and in the gel at the end of the experiment (∼3 h). (Inset) Change in intensity of 2 monitored via the integrated fluorescence from the decay data (per minute). Note for 1, after gelation the spectrum was slightly distorted by Raman scattering (treated).
Figure 3. Time-resolved decay data for 1 showing trend in the longestlived decay component (open circles) and average lifetime (τave, crossed squares). Note that before 40 min it is not possible to accurately distinguish between the two shorter-lived components.
link gellan’s negatively charged polysaccharide helices, forming helix-helix aggregates.4 Most work has made use of rheology to monitor changes in the bulk viscosity, although there is a report of fluorescence anisotropy employed to monitor chain conformation,13 as well as calorimetry.52 Manufacturer’s data shows that a dramatic (∼20 fold) decrease in bulk viscosity is expected over the temperature range of our study.53
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Figure 4. Time resolved data and trends for 2 in TEOS sol-gel, (a) lifetimes (τi) from analysis as a sum of three exponential components, (b) corresponding normalized pre-exponentials (Ri).
Figure 5. Average lifetime of 2 and inset the ratio of the preexponential components, R3 to R1, calculated from Figure 4.
The steady state spectra for the probes incorporated within a cooled 1% solution of Gelrite in water is shown in Figure 7. Again, because of the highly aqueous environment, the emission of 1 is indicative of the presence of a small quantity of aggregates (emission bands >600 nm). Figure 8 shows the decay curves for the two probes in the gellan system at different temperatures. It can be seen that the change in fluorescence lifetimes and intensities for each probe follow the same trend (Figure 8c) and dramatic decreases in the decay times (and
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Figure 6. Viscosities for the sol-to-gel transition for TEOS calculated from lifetime data for the two probes.
Figure 7. Steady-state absorption (dotted line) and emission (solid line) for 1 and 2 in 1% gelrite solution at room temperature.
intensity) occur. It should be noted that, as for the TEOS study, the average fluorescence lifetime is used as an indicator for 2 and the longest-lived component, assigned to monomer emission, is used for 1. The fact that an intensity change is noted for 1 is indicative that the aggregates are stable and that no further increase in their number or size occurs. The resolution of the effect of temperature alone as well as viscosity on the probe’s behavior is problematic. Increasing temperature is known to augment the nonradiative rates, thus decreasing the lifetime; however, the documented dramatic change in the viscosity of the host over this temperature range would be the overriding influence. To confirm if this is the case a study of the lifetime of 1 in a low viscosity solvent (ethanol, see Supporting Information) was performed. Using the parameter x obtained in the ethanol study and applying eq 5 to the data for 1 in gelrite, however, did not prove conclusively that the effect that we are observing has viscosity as the overriding influence. A fuller more detailed study is required to separate the exact influence of temperature and viscosity on the probes photophysical behavior, although relative differences in fluorescence lifetime at any temperature could reflect the local changes in viscosity. Comparing the two probes, it can be seen that the trend in fluorescence change with temperature for 1 is linear, while that for 2 clearly deviates from linearity, as evident in Figure 8c.
Figure 8. Time-resolved fluorescence decays for probes 1 (a) and 2 (b) in hydrogels at different temperatures. (c) Relative change in intensity and lifetime for the two probes with temperature.
This behavior must relate to different morphological changes sensed by the two probes, with 1 associated with the polysaccharide helices and 2 located in a more aqueous environment. These data, however, do not show any pronounced changes close to 30 °C, where other studies have reported significant structural changes associated with chain melting. These data relate to the presence of thermal hysteresis in the polysaccharide, as our measurements were taken on heating.10 The analysis of local microviscosity upon addition of Mg2+ ions, is given in Figure 9. Here the fluorescence lifetime data have been combined with the calibration plot in Figure 1 to allow an estimate of the initial viscosity to be made, using eq 5. As further deconvolution of the direct effect of temperature on the probe, as well as that on the viscosity, is required, the lifetimes will be used as a tool to reflect changes in viscosity. From Figure 9 several distinct trends can be discerned, with differences revealed by the two probes. Upon increasing Mg2+
Monitoring Sol to Gel Transitions
J. Phys. Chem. B, Vol. 113, No. 35, 2009 12073 Summary The use of complementary fluorescence probes, whose fluorescence lifetimes are viscosity sensitive has proven useful in elucidating changes in microviscosity that occur during solto-gel transitions. Since the fluorescence lifetime is independent of the probe concentration, viscosity and concentration effects can be readily separated. In the TEOS-based system differences are seen in the microviscosities sensed by the two probes and time-tag fluorescence allows for this dynamic process to be continuously monitored. In the case of the gellan gum system, again differences in viscosity between the aqueous phase and the polymer structure can be seen, and upon addition of cations the disaggregation of gellan helices noted. The use of these complementary probes combined with time-resolved techniques, such as time-tagging, are promising for the study of these complex systems.
Figure 9. Effect of [Mg2+] on the two probes lifetime values at different temperatures. The values in the brackets are the corresponding calculated viscosities at 20 °C, and the log of the lifetime plotted against log (1/temperature) is in accordance with eq 5.
concentration at 20 °C, probe 2 senses a dramatic increase in viscosity of its local environment, which is in direct contrast to probe 1. The addition of divalent cations causes the polysaccharide double helix to aggregate with other helices before, upon heating, a helix-coil transition occurs. Any nonaggregated helices will melt out before the aggregated ones which results in a second transition.10 Therefore probe 2 can either be reflecting the fact that the increase in polysaccharide aggregation is causing a contraction in the size of the entrapped water leading to increased viscosity (similar in magnitude experienced by probe 1 in absence of Mg2+) or the probe is migrating toward the aggregates. This increase in viscosity is only prominent for the higher concentration samples; the addition of 1 mM Mg2+ has no significant influence on 2’s fluorescence lifetime. Although different in magnitude, the trend of the change in the average fluorescence lifetime and hence viscosity sensed by 2 is similar for all Mg2+ concentrations with temperature. Probe 1, on the other hand exhibits a more interesting behavior. At 20 °C, surprisingly there is an apparent decrease in viscosity upon the addition of Mg2+, with nearly a 50% reduction sensed by the probe. This most likely rules out the probe being incorporated within an aggregate, as the viscosity would be expected to increase not decrease. The observed results could relate to microgel growth caused by the insoluble aggregates forming a weakened network,10 which is sensed by 1. Upon heating the differences between the cation containing samples reduce and a different trend is seen, as compared to the sample without Mg2+ ions added. Rather than the linear dependency seen with that sample, below 50 °C there is a dip followed by an increase. As this is absent without added Mg2+ these data are consistent with probe 1 sensing the melt out of the aggregated helices; the helix coil transition for this sample is not seen because of thermal hysteresis.10 At 50 °C and above there is no significant difference between samples, as sensed by the probe, in keeping with what we have observed with 2 at higher temperature. Thus, the two complementary probes have shown the ability to report on local differences within the samples and provide information relating to processes within the samples. This information is not accessible from bulk measurements. Our use of two complementary probes, which preferentially localize in two different environments allows the comprehensive set of information of the sol-gel structure and gelation process to be obtained.
Acknowledgment. The authors wish to thank David Campbell of HORIBA Jobin Yvon IBH Ltd for technical assistance. M.K.K. thanks the U.K.’s Engineering and Physical Sciences Research Council (EPSRC) Life Sciences Interface programme for a personal Fellowship. Supporting Information Available: Full fluorescence lifetime analysis for the data presented in Figures 3, 4, and 9. Change in fluorescence lifetime of probe 1 in ethanol with temperature. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Brinker, C. J.; Scherer, G. W. Sol-Gel Science; Academic Press: New York, 1989. (2) Hench, L. L.; West, J. K. Chem. ReV. 1992, 90, 33. (3) Lushnikov, A. A. Phys. D 2006, 222, 37. (4) Jeong, B.; Kim, S. W.; Bae, Y. H. AdV. Drug DeliVery ReV. 2002, 54, 37. (5) Gill, I.; Ballesteros, A. Trends Biotechnol. 2000, 18, 282. (6) Livage, J.; Coradin, T.; Roux, C. J. Phys.: Condens. Matter 2001, 13, R673. (7) Balasubramaniam, J.; Kant, S.; Pandit, J. K. Acta Pharm. 2003, 53, 251. (8) Deasy, P. B.; Quigley, K. J. Int. J. Pharm. 1991, 73, 117. (9) Jin, W.; Brennan, J. D. Anal. Chim. Acta 2002, 461, 1. (10) Paulsson, M.; Ha¨gerstro¨m, H.; Edsman, K. Eur. J. Pharm. Sci. 1999, 9, 99. (11) Miyoshi, E.; Nishinari, K. Colloid Polym. Sci. 1999, 277, 727. (12) Fukada, H.; Takahashi, K.; Kitamura, S.; Yuguchi, Y.; Urakawa, H.; Kajiwara, K. J. Therm. Anal. Calorim. 2002, 70, 797. (13) Horinaka, J.-I.; Kani, K.; Honda, H.; Uesaka, Y.; Kawamura, T. Macromol. Biosci. 2004, 4, 714. (14) Steiner, R. Topics in Fluorescence Spectroscopy; Lakowicz, J. R., Ed.; Plenum Press; New York, 1999; Vol. 2, pp 1-52. (15) Haidekker, M. A.; Ling, T.; Anglo, M.; Stevens, H. Y.; Frangos, J. A.; Theodorakis, E. A. Chem. Biol. 2001, 8, 123. (16) Alamiry, M. A. H.; Benniston, A. C.; Copley, G.; Elliott, K. J.; Harriman, A.; Stewart, B.; Zhi, Y. G. Chem. Mater. 2008, 20, 4024. (17) Rei, A.; Hungerford, G.; Ferreira, M. I. C. J. Phys. Chem. B 2008, 112, 8832. (18) Kuimova, M. K.; Yahioglu, G.; Levitt, J. A.; Suhling, K. J. Am. Chem. Soc. 2008, 130, 6672. (19) Kuimova, M. K.; Botchway, S. W.; Parker, A. W.; Balaz, M.; Collins, H. A.; Anderson, H. L.; Suhling, K.; Ogilby, P. R. Nat. Chem. 2009, 1, 69. (20) Levitt, J. A.; Kuimova, M. K.; Yahioglu, G.; Chung, P.-H.; Suhling, K.; Phillips, D. J. Phys. Chem. C 2009, 113, 11634-11642. (21) Eaton, D. F. Pure Appl. Chem. 1990, 62, 1631. (22) Felekyan, S.; Ku¨hnemuth, R.; Kudryavtsev, V.; Sandhagen, C.; Becker, W.; Seidel, C. A. M. ReV. Sci. Instrum. 2005, 76, 083104. (23) Zink, J. I.; Dunn, B. Chem. Mater. 1997, 9, 2280. (24) Unger, B.; Rurack, K.; Mu¨ller, R.; Jancke, H.; Resch-Genger, U. J. Mater. Chem. 2005, 15, 3069. (25) Weber, G.; Farris, F. J. Biochemistry 1979, 18, 3075. (26) Greenspan, P.; Fowler, S. D. J. Lipid Res. 1985, 26, 781. (27) Flora, K. K.; Brennan, J. D. J. Phys. Chem. B 2001, 105, 12003.
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