Effect of Gelatin Gelation Kinetics on Probe Diffusion Determined by

Biomacromolecules 2010, 11, 3359–3366

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Effect of Gelatin Gelation Kinetics on Probe Diffusion Determined by FRAP and Rheology Joel Hagman,†,‡ Niklas Loreń,*,†,‡ and Anne-Marie Hermansson†,‡ Department of Applied Surface Chemistry, Chalmers University of Technology, Go¨teborg, Sweden, and Department of Structure and Material Design, The Swedish Institute for Food and Biotechnology, SIK, Go¨teborg, Sweden Received July 23, 2010; Revised Manuscript Received October 8, 2010

The time-dependent diffusion and mechanical properties of gelatin in solution, in the gel state, and during the sol/gel transition were determined using fluorescence recovery after photobleaching (FRAP) and rheology. The parameters in the experimental design were 2% w/w and 5% w/w gelatin concentration; 15, 20, and 25 °C end quench temperatures; and Na2-fluorescein, 10 kDa FITC-dextran, and 500 kDa FITC-dextran as diffusion probes. The samples were monitored in solution at 60 °C, during quenching, for 75 min at end quench temperatures and after 1, 7, and 14 days of storage at the end quench temperature. The effect of temperature on the probe diffusion was normalized by determining the free diffusion of the probes in pure water for the different temperatures. The results gained by comparing FRAP and rheology showed that FRAP is able to capture structural changes in the gelatin before gelation occurs, which was interpreted as a formation of transient networks. This was clearly seen for 2% w/w gelatin and 20 and 25 °C end quench temperatures. The structural changes during sol/gel transition are detected only by the larger probes, giving information about the typical length scales in the gelatin structure. The normalized diffusion rate increased after 7 and 14 days of storage. This increase was most pronounced for fluorescein but was also seen for the larger probes.

Introduction The mechanical and mass transport properties of gels are of great importance for the food, pharmaceutical and cosmetics industries. Gels are commonly used as thickeners and stabilizers, as different concentrations and types can give products with a range of rheological behaviors. Gels are also used as a matrix for controlled release of functional molecules (such as flavor or drug molecules), where the gel structure can be tailored to some extent to obtain the desired release profiles.1 The gel structure acts like a sieve, where the gel strands obstruct the diffusing molecules.2 A denser gel structure gives greater obstruction and retards the diffusion rate and a less dense structure offers less obstruction and retardation. Studies have shown a strong connection between the gel structure and the diffusion rate.3,4 The diffusion rate is also influenced by the rheological properties of the material. The Stokes-Einstein’s relation, which relates the diffusion rate of molecules in solution to the viscosity, is a well-known example. However, little is known about the connection between diffusion properties, microstructures, and mechanical properties during gelation. Gelatin is one of the most common gels used industrially and is one of the most investigated gels. The gelation of gelatin involves a formation of triple helices, where the individual gelatin strands are stabilized by hydrogen bonds in a righthanded helix.5 The gelation of gelatin follows the percolation theory, where the gelatin triple helices form aggregates in the solution. The aggregates then grow, interconnect and form larger and larger domains until the whole volume is percolated and the gel point is reached.6,7 Gelatin gelation is sensitive to temperature and gelatin concentration such that lower temper* Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected]. † Chalmers University of Technology. ‡ SIK.

atures and higher concentrations lead to faster gel formation and stronger gels.5 Transient networks are believed to rapidly form locally in the system, rearrange, and disappear. The lifetime of these networks increases when the system moves toward gelation and, at the gelation point, the transient networks percolate the whole system. Previous work has shown that transient networks form in carrageenan,8 which causes the gel to rearrange after gelation. Exactly what happens in the gelatin before percolation is not fully known but well investigated.6,7,9-11 If transient structures exist during the gelation of gelatin, they should be able to be seen as a change in diffusion rate before the gel has percolated. Because it is possible to alter the gelation kinetics of gelatin by means of temperature and concentration, gelatin is an appropriate material for determining transient networks during gelation. Fluorescence recovery after photobleaching (FRAP)12 is an excellent method for following dynamic changes because it is possible to control parameters, such as the temperature, of the sample with high precision and a diffusion measurement can be made in a few minutes. In FRAP, the sample is stained with a fluorescent probe and placed under a confocal laser scanning microscope (CLSM).13 As the CLSM is a high precision microscope, FRAP gives local information and can be carried out in any stainable material that is sufficiently photo stable to be irradiated with a laser. At measurement, a region of interest (ROI) is chosen wherein an area to bleach is selected. The CLSM then scans the ROI with a low intensity setting on the laser for a couple of frames to determine that the sample is not affected by the laser during the recovery phase. After a set amount of prebleach frames have been analyzed, the laser intensity is increased to maximum intensity inside the region of bleaching so that the fluorescence of the probes is deactivated. The intensity outside the bleaching region is kept low during bleaching. Immediately after the bleaching step, the laser

10.1021/bm1008487  2010 American Chemical Society Published on Web 11/05/2010

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intensity is reduced again while the laser is swept across the ROI for a set number of postbleach frames in order to study the recovery without further bleaching. The deactivated molecules in the bleached area diffuse out from the bleached area while nonbleached molecules diffuse in. The diffusion rate is then determined by analyzing how rapid the recovery of fluorescence is. Depending on the size and shape of the bleached area and what assumptions have been made regarding the diffusion mechanisms and sample response, different models for calculating the diffusion rate from the recovery information exist. Earlier models were not as accurate as later ones, partly because some important parameters were unknown and partly because of a lack of sufficient computational power. Early FRAP models did not include the scanning of the laser beam during bleaching. Braeckmans et al.14 developed a quantitative FRAP model that fully utilizes the scanning of the laser beam to increase precision of the diffusion rate estimate. One of the latest addition to FRAP is the pixel-based most likelihood estimation models developed by Jonasson et al.,15,16 bringing it to a par with diffusion NMR in the precision of the diffusion rate estimate. The main objective of this study is to combine diffusion and rheological measurements in order to explore structure transient changes during gelation of gelatin. Another objective is to demonstrate that FRAP is a useful tool for measuring the changes in diffusion rate in a dynamic system. In this work, an experimental design with two different gelatin concentrations, three different end temperatures and three different probe sizes was used to determine the effect of gelatin gelation on probe diffusion. The effect of temperature on probe diffusion was eliminated using normalization by the temperature dependent probe diffusion rate in free water. A comparison was made between the gelation kinetics of gelatin, measured by rheology, and the time-dependent probe diffusion, measured by FRAP.

Materials and Methods Materials. The gelatin used was pig skin, type A, with a bloom number of 300 (Sigma, Sweden). The probes used were Na2-fluorescein (Fluka, U.S.A.), anionic FITC-dextran, 10 kDa MW (Invitrogen Molecular Probes, U.S.A.), and anionic FITC-dextran, 500 kDa MW (Invitrogen Molecular Probes, U.S.A.). The secure-seal spacers used were 120 µm thick and 9 mm in diameter (Invitrogen, U.S.A.). Sample Preparation. For the practical experimental setup, all samples were made fresh at the start of the day and analyzed in the afternoon. To keep the samples as fresh as possible, they were all locked between two cover slides with the aid of a secure-seal spacer. All samples were handled such that they were exposed to as little light as possible and were stored in light-blocking sample holders. The samples were prepared by making a 50-100 ppm solution of the probe in distilled water, where the Na2-fluorescein only needed 50 ppm to be strongly fluorescent, while the FITC-dextran probes were somewhat weaker in fluorescence and, thus, needed 100 ppm solutions. These concentrations are well in the linear regime of the fluorescence dependence on the concentration.15 The gelatin powder and probe solution were then mixed together in 10 mL vials and placed to rest for 1 h with the vial lid fastened. The vials were subsequently put in a heater in 70 °C water for 15 min to melt the gelatin completely. After the gelatin had melted, it was stirred vigorously for a few seconds and 6 µL were placed into secure-seal spacer grids between two cover glass slides. Two grids were made from each sample and each was then put on an individual temperature stage held at 60 °C. One of the double samples was put under the microscope for FRAP analysis while the other was given the same temperature treatment, but without exposing it to FRAP analysis. This was done to

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Figure 1. The experimental space with the dimensions of end temperature, probe size, and gel concentration.

Figure 2. Temperature profile for the FRAP and rheological analysis. Note that the rheological measurements were made only for the first 24 h of the gelation, whereas the FRAP measurements continued for up to 2 weeks (at selected intervals of 24 h, 7 d, and 14 d).

ensure that nothing had happened to the samples during the storage. The duplicates were analyzed with FRAP at the same time as the originals at 24 h, 7 d, and 14 d of storage. After moving the samples to the temperature stage, but before starting the FRAP experiments, the samples were kept at 60 °C for about 10 min while randomizing the run order and adjusting the microscope settings. FRAP measurements were made every fifth minute for 1.5 h at a new ROI each time. The depth in the sample was kept at 40 µm for all ROIs. Three recovery sequences were taken at 60 °C; the fourth started at the same time as the temperature stages were starting to quench the samples, and the following 16 recovery sequences were taken at the designated quench temperature (15, 20 or 25 °C). The samples were then moved to storage rooms with a constant temperature and reanalyzed at 24 h, 7 d, and 14 d. To test the reproducibility of the FRAP measurements, four gel samples were chosen in the experimental design where the probe and end temperature were randomly selected. Samples with 2% and 5% gelatin concentration were made, where the probes chosen were either fluorescein at an end temperature of 15 °C or 10 kDa FITC-dextran at an end temperature of 15 °C. The reproducibility of the FRAP measurements was good. Within the same gel sample, the standard deviation was around (10%. The standard deviation between replicates was approximately (20%. Experimental Design. Figure 1 shows the experimental space created by varying the end temperature, gel concentration and probe size. The end temperatures (as well as the storage temperatures) chosen were 15 °C (rapid gel forming), 20 °C (medium paced gel forming), and 25 °C (slow gel forming)5 to achieve different gelatin gelation kinetics. The two gelatin concentrations chosen were 2 and 5%, which would result in a weak gel with slower gelation kinetics and a stronger gel with quicker gelation kinetics, respectively. The probes used were Na2-fluorescein (376 Da), which is one of the smallest FRAP probes available and is thought to be small enough to move through the gelatin relatively unhindered, and 10 kDa FITC-dextran, which is a relatively large probe but is thought to be about the right size to be hindered to some extent but not as much as the 500 kDa FITC-dextran probe, which should be large enough to be hindered but not immobilized. Figure 2 displays a conceptual image of the temperature treatment of the samples. The first three FRAP measurements were made in the gelatin solution, the fourth during quenching and the following measurements were made during and after the gelation of the gel.

Gelatin Gelation Kinetics and Probe Diffusion CLSM-FRAP Protocol. The CLSM system used consists of a Leica SP2 AOBS (Heidelberg, Germany) with a 20×, 0.5 NA water objective, and the following settings: 256 × 256 pixels, zoom factor 4 (with a zoom-in during bleaching), and 800 Hz, yielding a pixel size of 0.73 µm and an image acquisition rate of two images per second. The beam expander was set to 1, which lowered the effective NA to approximately 0.35 and yielded a slightly better bleaching and a more cylindrical bleaching profile and made the bleached disk less influenced by edge effects. The areas bleached were 30 µm large discs at 40 µm into the sample and the setup was to take 20 prebleach images, to bleach with only one image and to follow the recovery for 50 images. One FRAP experiment was carried out each fifth minute. This was chosen as it was the best time resolution that was easy to maintain for the operator of the CLSM; each measurement takes about three minutes to set up and carry out without allowing some margin of error for the setup. Five minutes was considered the minimal time needed between FRAP experiments on the Leica SP2 with the chosen settings. Because the probe is introduced into the water in the system, it will dissolve unhindered and thus distribute itself homogeneously in the sample. The very limited volume in the secure-seal grids made the samples retain a 3D structure (for properties smaller than 120 µm) but at the same time limited the potential flows in the sample to a minimum and sealed the gelatin from the environment during storage. The zoomin during bleaching ensures that the laser beam overlaps enough to bleach a homogeneous disk and not stripes. Because all of the probes at all temperatures and gel strengths diffused sufficiently quickly, all of the initial recovery images had an approximately Gaussian distribution of the intensity in the bleached area. This follows the recommendations described by refs 14 and 15 and made the new FRAP model, called “most likelihood estimation for FRAP data with a Gaussian starting profile”, valid for evaluation of the data. This model is described by Jonasson et al.15 and the evaluation using the model was carried out in Matlab. The most likelihood estimation (MLE) model for FRAP is pixel-based. This means that it takes all the pixels in the picture into account instead of averaging the pixel information in the bleached region, as many other models do. It is very robust and can, with a good initial parameter setting, find the diffusion rate for even a non perfect FRAP experiment. One of the built-in properties of the MLE method is that it can give error estimates of the parameters it calculates. Previous experiments have shown that this model has high accuracy and is reliable for the evaluation of FRAP data. Rheology. The samples were poured into preheated sample cups of a Stresstech rheometer, Rheologica Instruments (Lund, Sweden). A bob and cup system was used (bob diameter 15 mm, following ISO 3219). The stresstech was set to analyze at 1 Hz for 24 h after quenching the sample from 60 °C to the respective end quench temperatures. The stresstech could not quench as rapidly as the temperature stages for the CLSM, but this was not a problem for the 20 and 25 °C samples as they showed no signs of gelation until after the desired end temperature had been reached. For 15 °C, the stresstech could not quench quickly enough to capture gelation at the correct temperature. Those samples were instead poured into a precooled sample cup at 15 °C, and the stresstech was started at the same time as the sample was poured in. All of the samples were covered with a low viscosity paraffin oil to prevent evaporation and drying. The gel point was taken as the G′-G′′ crossover at 1 Hz, even if a more strict definition of the gel point requires that the crossover is independent of frequency.17-19

Results and Discussion The approach taken was to relate the time-dependent evolution of the gel strength and the gel point measured by rheology to the time-dependent evolution of the probe diffusion measured by FRAP. Of particular interest was to see whether it was


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Figure 3. Average free diffusion for the probes in distilled water as a function of temperature: 3, Na2-fluorescein; 0, 10 kDa FITCdextran; O, 500 kDa FITC-dextran. Each mark represents a minimum of five measurements. The error bars are smaller than the notation signs and are not shown. The drawn lines are to guide the eye.

possible to detect the existence of a transient network before the gelatin gelled. The way to achieve this would be to see whether or not the changes in diffusion rate are connected to gel forming. To capture this, the experimental space for the

Figure 4. Two typical FRAP recoveries. (A) Fluorescein probe in a 2% gelatin gel at 25 °C. (B) FITC-dextran probe (500 kDa) in a 5% gelatin gel at 15 °C. There is a clear visual difference in the recoveries and, as expected, the bleached region in (A) is visually recovered much earlier than (B) due to the faster diffusion rates. The CLSM images are taken at t ) 0, 1, 5, and 10 s.

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Figure 5. Rheological curves for the gelatin samples during the first 24 h since quenching: (a-c) 2% gel at 15, 20, and 25 °C. respectively; (d-f) 5% gel at 15, 20, and 25 °C, respectively. The solid line represent G′ and the dotted line represent G′′. Note the large difference in modulus on the y axes.

probes and temperatures were chosen so that the range they cover is relatively large. As there is a contribution of temperature to the diffusion rate from the kinetic energy added to the system, the temperature-dependent part of the changes in diffusion rate was removed. This was done by measuring the probe diffusion rate in pure water for the same temperatures to which the gelatin was exposed. The relative D/D0 was then chosen to present the results. Temperature Dependence of the Probe Diffusion. The probe diffusion rate is dependent on the temperature (see eq 1) because higher temperatures add more kinetic energy to a system. This means that the probe diffusion rate will be altered when the temperature is changed. This effect was removed by relating the probe diffusion rate in the gelatin to the temperaturedependent probe diffusion rate in pure water. The probe diffusion in pure water can be considered an infinitely diluted system, making the Stoke-Einstein relation valid (eq 1).

D)

kBT 6πηrH

(1)

In the Stokes-Einstein equation, D is the diffusion coefficient, kB is Boltzmann’s constant, T is the absolute temperature, η is the solution viscosity, and rH is the hydrodynamic radius of the probe. For a given temperature, the greatest difference in the diffusion rate between probes will be caused by the difference in hydrodynamic radii, as all other factors are the same. Figure 3 shows the diffusion rate in distilled water for the three probes used. The rates range between 370 and 557 µm2 s-1 for fluorescein, 64 and 102 µm2 s-1 for 10 kDa FITCdextran, and between 15 and 24 µm2 s-1 for 500 kDa FITCdextran. It can be seen that the diffusion rate of all three probes varies linearly over the temperature range, which is expected from eq 1. These probe diffusion rates correspond to the following hydrodynamic radii in the temperature interval of 15

Gelatin Gelation Kinetics and Probe Diffusion

to 60 °C: 0.5-0.94 nm for fluorescein, 2.86-5.12 nm for 10 kDa FITC-dextran, and 12.05-21.92 nm for 500 kDa FITCdextran. This was calculated with the Stoke-Einstein equation and the measured diffusion rates. These values correspond well with previously measured hydrodynamic radii.20,21 FRAP Measurements in Gelatin Gels. To illustrate how different diffusion rates can appear in the FRAP measurement, Figure 4 shows two time series of CLSM micrographs of gelatin gels. Figure 4a is fluorescein in a 2% w/w gelatin gel at 25 °C, yielding a diffusion rate of approximately 200 µm2 s-1, and Figure 4b is 500 kDa FITC-dextran in a 5% w/w gelatin gel at 15 °C, which results in a diffusion rate of approximately 2 µm2 s-1. As seen in the micrographs in Figure 4, the large difference in diffusion rates results in two different recovery series. The first image in series a and b, respectively, shows the first image acquired directly after bleaching, while the rest of the images show the intensity recovery as a function of time for 1, 5, and 10 s after the bleaching step. The rate of recovery is proportional to the diffusion rate, thus, the fewer images required to capture a full recovery, the faster the diffusion rate. An almost full recovery is seen only in series a, but the MLE model used for data evaluation can find the most likely parameter values with high precision regardless of whether a full recovery is recorded or not. Effect of Gelatin Concentration and End Temperature on Gel Kinetics. To capture what happens before gelation, the parameters were chosen so that there would be both weak and strong gels as well as both slower and quicker gel kinetics. Figure 5 shows the rheological curves for the different gel concentrations and temperatures. Gel formation is defined as when the G′ curve crosses the G′′ curve.17,18 The 2% gelatin formed a gel within 5 min at 15 °C (a), around 45 min at 20 °C (b), and around 4 h at 25 °C (c). The 5% gelatin formed a gel almost instantaneously at 15 °C (d), after approximately 5 min at 20 °C and after about 10 min at 25 °C (f). The addition of the probe to the gel showed no changes in the development of the modulus curves from the cases with and without the probe, and the addition of a FRAP probe was considered not to affect the gel formation. The results in Figure 5, that the gelation kinetics is faster and yields a stronger gel for higher concentration and lower temperature, coincide well with earlier studies of gelatin gels.5,6 Formation of Transient Networks in Gelatin Gels. The time of the rheological sol/gel transition (i.e., the gel point) can be determined from rheological curves of gelatin. As it is desired to see whether diffusion measurements can identify changes before the gelatin reaches the gel point, the diffusion rate curves were plotted together with the modulus curves as a function of time. To correlate the rheology curves with the FRAP measurements, both curves start when the end temperature is reached; this time is denoted “time since quenching”. Figures 6 and 7 show the diffusion rate and the moduli as a function of time for 2 and 5% gels, respectively, for all the probes. Graphs a, b, and c in both figures show the different curves for the different end temperatures, 15, 20, and 25 °C, respectively. The diffusion curves were normalized to remove the temperature effect caused by the quenching. Note the difference in the y scale for the different moduli. One expected result is that the probes are ordered according to size.22,23 When the diffusion rate curves in Figures 6 and 7 are studied, it can be seen that the order is fluorescein as the quickest probe followed by 10 kDa FITC-dextran and 500 kDa FITC-dextran close together. The normalized values of the larger probes overlap at some points due to experimental variations,


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Figure 6. Diffusion rate and modulus of the gel as a function of time: 2% gelatin gels with fluorescein (triangles), 10 kDa FITCdextran (squares), and 500 kDa FITC-dextran (circles). The dotted line represents G′, the dashed line represents G′′; (a) quenched to 15 °C, (b) quenched to 20 °C, (c) quenched to 25 °C. Note the difference in the y axis scale for the modulus. The solid lines are to guide the eye.

but the 10 kDa FITC-dextran probe in general has larger D/D0 values than the 500 kDa FITC-dextran probe. Depending on end temperature, the time until gel formation for the 2% gelatin gels ranges from 5 min to 4 h (Figure 5a-c). In contrast, at 5% gelatin concentration, the times until gelation at the different end temperatures are shorter than the corresponding gelation times at 2% gelatin concentration. This is most noticeable at 20 and 25 °C. These results indicate that it will be easier to capture the effect of the gelatin gelation on the decrease in D/D0 in 2% gels compared to 5% gels. The behavior of the probe diffusion is similar for 10 and 500 kDa FITC-dextran. The probes start with a relatively high D/D0 in solution, and the D/D0 then rapidly decreases after the quenching until it reaches a plateau. In nearly all cases in Figures 6 and 7, the plateau is reached at more or less the same time regardless of when the gelatin solution gels. If the plateau is reached before the gel point, this would indicate that similar

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Figure 7. Diffusion rate and modulus of the gel as a function of time: 5% gelatin gels with fluorescein (triangles), 10 kDa FITC-dextran (squares) and 500 kDa FITC-dextran (circles). The dotted line represents G′, the dashed line represents G′′; (a) quenched to 15 °C, (b) quenched to 20 °C, (c) quenched to 25 °C. Note the difference in the y axis scale for the modulus. The solid lines are to guide the eye.

length scales between the domains are present in the gelatin gel and in the transient structures after the plateau is reached. The rapid D/D0 decrease also means that the larger FRAP probes can detect structural changes in the gelatin prior to the percolation in the system when the gel formation is sufficiently slow. However, the gelation kinetics shown in Figures 6a and 7a,b is too quick and it is not possible to capture any potential changes in structure before the percolation with FRAP. These structural changes influence the probe diffusion rate of 10 and 500 kDa FITC-dextran. The percolation theory says that there are small areas that aggregate simultaneously all over the solution, and the gel point is reached and the solution changes into a gel when all small aggregates have bound together. It is hypothesized that the aggregations might form relatively rapidly and then dissolve again, forming transient networks in the gelatin before the structure is locked with a system-wide percolation. The rapid decline in D/D0 for the heavier probes in Figures 6 and 7 could therefore be due to the formation of transient networks, as it occurred quickly and regardless of when the system percolated into a gel. This also

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shows that FRAP is a valuable tool for following gel kinetics and determining the effect of gelation on probe diffusion in gels. The fluorescein probe does not show the same trend in 2 and 5% gelatin concentrations, as its diffusion rate is relatively unchanged where the larger probes experience a decrease in diffusion rate. The fluorescein probe showed no visual changes in the diffusion rate over the 75 min the gelling experiment was conducted. The lack of visual changes means that the probe does not observe any great structural differences between the solution and the gel, which should give some indication of the average mesh sizes in the gelatin. Influence of Gelatin Gelation on Probe Diffusion. Effect of the Probe Size. Figure 8 shows the change in the probe diffusion rate between the gelatin solution and the gelatin 1 h after quenching for all probes, temperatures and gel concentrations. A comparison of the bar plots in Figure 8 (a-c for 2% gels and d-f for 5% gels) shows the effect of the probe size. The gray bars show the D/D0 before quenching and the white bars represent D/D0 1 h after quenching. The difference between the gray and white bars illustrates how the D/D0 changes. What can be seen is that there is a clear drop in the diffusion rate for the larger probes. There is hardly any drop in the relative diffusion rate at all for each of the fluorescein probe samples (Figure 8a,d). This is because the fluorescein is too small (hydrodynamic radius 2.8 nm for 10 kDa FITC-dextran and >12 nm for 500 kDa FITC-dextran, display a drop in the relative diffusion rate between the solution and the 1 h point because they are influenced by the structural changes in the gelatin. An alternative explanation for the changes seen in Figure 8 would be that, if the FTIC-dextran reacts with the gelatin, the probe size would be altered and a similar decrease would be visible. However, control FRAP experiments have been conducted where gelatin solutions with a 10 kDa FITC-dextran probe were held at 60 °C for over an hour to determine the stability of the FITC-dextran. No changes in diffusion rate were seen in these experiments. Furthermore, recent experiments in phase separated mixtures of gelatin and maltodextrin showed that the 10 kDa FITCdextran probes prefer to be in the maltodextrin phase.24 This indicates that the interaction between gelatin and FITC-dextran probe is minor. Effect of the Gel Concentration. The effect of the gelatin concentration can be observed in Figure 8 by comparing the 2% gels (a-c) with the 5% gels (d-f). It can be seen that the bar plots keep their shapes as the gel concentration increases. However, in general, the probes observe a slightly greater hindrance and the bars drop in height. The effect is not large, but it is noticeable and expected, as other studies have shown similar results.25,26 Effect of the End Temperature. As previously mentioned, the effect that temperature changes have on the diffusion rate is eliminated when the diffusion rates are related to the free diffusion. It would perhaps be easy to expect that the effect of the end temperature to which the samples are quenched would be removed as well. However, because the moduli of the gels vary greatly with end temperature (see Figure 5), it might be expected that the diffusion rate would go down with increasing modulus as the mesh size of the gel is connected with the modulus.27 It can be seen in Figure 8a,d that the D/D0 for the small fluorescein probe varies somewhat erratically over the different end temperatures, where the larger probes show no significant changes with changing end temperature (see also Figure 7a,c).

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Figure 8. The bar plots show the diffusion rate in the solution and after 1 h. Each plot shows one probe at the three different end temperatures: (a) 2% gelatin with fluorescein probe; (b) 2% gelatin with 10 kDa FITC-dextran probe; (c) 2% gelatin with 500 kDa FITC-dextran probe; (d) 5% gelatin with fluorescein probe; (e) 5% gelatin with 10 kDa FITC-dextran probe; (f) 5% gelatin with 500 kDa FITC-dextran probe.

The variations for the fluorescein probes are most likely caused by small variations in the gel concentration in the different gel preparations rather than the different end temperatures because the variations seen in Figure 8a,d are not seen for the probe in pure water when the temperature is varied. In addition, neither of the larger probes show a similar trend, and the fluorescein probe seems to disregard the fact that the gelatin gels. This indicates that the gel structure that the probes experience is fairly similar independent of end temperature. Again, placing this in contrast to the large variations seen in the elastic moduli after 1 h (Figure 5), it can be noted that there is no simple correlation between the gel strength and the diffusion properties. The detailed gel microstructure is likely very important for the diffusion properties, however, because studies in other gels4,28 show that the microstructure affects the diffusion. Effect of Storage. Gelatin is thought to be a dynamic system because it tends to continue to grow stronger and stronger in modulus over time.5 Figure 9 shows the time evolution of the relative diffusion with time over 1 h, 1 d, 7 d, and 14 d of storage at the end temperature. It can be seen that, with time, the gelatin gel will slowly allow faster diffusion rates. This is seen most prominently for fluorescein, especially in the 2%

gelatin concentration. The increase is significant for a t test at a 95% confidence level. The same trends can be seen for the higher concentration and the larger probes, although these trends are not significant. One possibility is that the gel slowly reforms into a more rigid structure and the gel strands will grow thicker and stronger by opening the channels in between the gel strands.3,5 This rearrangement will allow the larger probes some additional movement but will not be sufficient to increase the diffusion rate significantly. For the small fluorescein probe, the rearrangement of the gelatin strands results in less obstruction and an increase in the diffusion rate. This attribute is quite likely due to the fluorescein probe being smaller than the gel mesh size to begin with, whereas the larger probes are closer in size to the mesh size of the gel. Another explanation could be micro syneresis due to heterogeneity in the gelatin gel. As the gel shrinks and expels water, nano-sized channels of free water can form and the increase in the diffusion rate for the fluorescein probe could be caused by the bleached region containing both open channel structures and gelatin network structures. The diffusion rates measured would then be an average between the two structures, one where the probe moves as in free water and one where the probe becomes

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and FRAP using three different probe sizes. It was found that there is no simple correlation between gel strength and diffusion properties. The gelatin gels were stored at the end temperatures for 14 days and analyzed by FRAP at selected intervals. The diffusion rate increased as a function of time, especially for the smallest diffusion probe (fluorescein). Similar trends were observed for the large diffusion probes. These trends were not significant, however. This study shows that FRAP, with the right parameters and time frame, is capable of capturing dynamic changes in the microstructure of gels. By varying the probe size, FRAP is also able to give indications of the average mesh size of the gel network formed. Acknowledgment. This project is part of the VINN Excellence Centre SuMo Biomaterials (Supermolecular Biomaterialss Structure Dynamics and Properties). The financial support of the Centre is gratefully acknowledged.

References and Notes

Figure 9. Gelatin gels of 2% w/w concentration with fluorescein probe showing an increase in the diffusion rate with time: (a) 15 °C, (b) 20 °C, and (c) 25 °C. All samples were stored in constant atmosphere rooms maintained at their designated temperatures for 1 h, 1 d, 7 d, and 14 d of storage.

obstructed by the gel network. If the amount of channels with free mobility increases with time, the average diffusion rate will increase with time. The larger probes will not notice the micro syneresis if the channels are smaller than their size and will therefore not display any significant increments in the diffusion rate with time.

Conclusions FRAP has proven to be a powerful tool for determining structural changes during gelation and storage of gelatin gels. At relatively slow gel kinetics, FRAP and probe diffusion are capable of determining structural changes in gelatin solution before gelation occurs by comparing the rheological gel point with the time evolution of the probe diffusion rate. There is a strong indication that a transient structure is formed during the gelation process by the decrease in diffusion rate prior to gel formation. This effect was clearly seen for the larger diffusion probes, 10 and 500 kDa FITC-dextrans, but was absent for the small diffusion probe (fluorescein). The D/D0 reaches a plateau more or less at the same time in all samples, regardless of when the gelatin percolates. In those cases in which the gelation kinetics is slow, this plateau is reached ahead of the percolation. This indicates that the transient structures possess similar length scales as in the gel. The gelation of gelatin at two different concentrations and three different end temperatures was determined by rheology

(1) Hermansson, A.-M.; Loreń, N.; Nydeń, M. Water Properties of Food, Pharmaceutical, and Biological Materials; CRC Taylor and Francis, 2006; Chapter 5, pp 79-100. (2) Amsden, B. Macromolecules 1998, 31, 8382–8395. (3) Walther, B.; Loreń, N.; Nydeń, M.; Hermansson, A.-M. Langmuir 2006, 22, 8221–8228. (4) Loreń, N.; Shtykova, L.; Kidman, S.; Jarvoll, P.; Nydeń, M.; Hermansson, A.-M. Biomacromolecules 2009, 10, 275–84. (5) Djabourov, M.; Lechaire, J.-P.; Gaill, F. Biorheology 1993, 30, 191– 205. (6) Djabourov, M. Contemp. Phys. 1988, 29, 273–297. (7) De Gennes, P. G. Scaling Concepts in Polymer Physics; Cornell University Press: New York, 1979. (8) Hermansson, A.-M. Carbohydr. Polym. 1989, 10, 163–181. (9) Gordon, M.; Torkington, J. A. Pure Appl. Chem. 1981, 53, 1461. (10) Borchard, W.; Burg, B. Prog. Colloid Polym. Sci. 1990, 83, 200– 210. (11) Pin˜eiro, Y.; Lo´pez-Quintela, M. A.; Rivas, J.; Leisner, D. Phys. ReV. E 2009, 79, 041409. (12) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055–1069. (13) Blonk, J. C. G.; Don, A.; van Aalst, H.; Birmingham, J. J. J. Microsc. 1993, 169, 363–374. (14) Braeckmans, K.; Peeters, L.; Sanders, N. N.; De Smedt, S. C.; Demeester, J. Biophys. J. 2003, 85, 2240–2252. (15) Jonasson, J.; Loreń, N.; Olofsson, P.; Nydeń, M.; Rudemo, M. J. Microsc. 2008, 232, 260–269. (16) Jonasson, J. K.; Hagman, J.; Loreń, N.; Bernin, D.; Nydeń, M.; Rudemo, M. J. Microsc. 2010, 239, 142–153. (17) Winter, H. H. Polym. Eng. Sci. 1987, 27, 1689–1702. (18) Power, D. J.; Rodd, A. B.; Paterson, L.; Boger, D. V. J. Rheol. 1998, 42, 1021–1037. (19) Forgacs, G.; Newman, S. A.; Hinner, B.; Maier, C. W.; Sackmann, E. Biophys. J. 2003, 84, 1272–1280. (20) Ekani-Nkodo, A.; Fygenson, D. K. Phys. ReV. E 2003, 67, 021909. (21) Hwang, H.; Park, J.-K. Anal. Chem. 2009, 81, 9163–9167. (22) Moussaoui, M.; Benlyas, M.; Wahl, P. J. Chromatogr. 1992, 591, 115– 120. (23) Moussaoui, M.; Benlyas, M.; Whal, P. J. Chromatogr. 1991, 558, 71– 80. (24) Deschout, H.; Hagman, J.; Fransson, S.; Jonasson, J.; Rudemo, M.; Loreń, N.; Braeckmans, K. Opt. Express 2010, in press. (25) De Smedt, S. C.; Meyvis, T. K. L.; Demeester, J.; Van Oostveldt, P.; Blonk, J. C. G.; Hennink, W. E. Macromolecules 1997, 30, 4863– 4870. (26) Ho, A. K.; Bromberg, L. E.; O’Connor, A.; Perera, J. M.; Stevens, G. W.; Hatton, T. A. Langmuir 2001, 17, 3538–3544. (27) Yang, Y.-l.; Leone, L. M.; Kaufman, L. J. Biophys. J. 2009, 97, 2051– 2060. (28) Le Feunteun, S.; Mariette, F. J. Agric. Food Chem. 2007, 55, 10764–72.

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Effect of Gelatin Gelation Kinetics on Probe Diffusion Determined by

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