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Crystallization of Femtoliter Surface Droplet Arrays Revealed by Synchrotron Small-Angle X‑ray Scattering Brendan Dyett,† Lisa Zychowski,‡,§ Lei Bao,† Thomas G. Meikle,‡ Shuhua Peng,† Haitao Yu,† Miaosi Li,† Jamie Strachan,‡ Nigel Kirby,∥ Amy Logan,*,§ Charlotte E. Conn,*,‡ and Xuehua Zhang*,†,⊥

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†

Soft Matter & Interfaces Group, School of Engineering and ‡Molecular Assembly Laboratory, School of Science, RMIT University, Melbourne, Victoria 3001, Australia § CSIRO Agriculture and Food, Werribee, Victoria 3030, Australia ∥ Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3169, Australia ⊥ Department of Chemical and Materials Engineering, University of Alberta, Edmonton, T6G1H9 Alberta, Canada S Supporting Information *

ABSTRACT: The crystallization of oil droplets is critical in the processing and storage of lipid-based food and pharmaceutical products. Arrays of femtoliter droplets on a surface offer a unique opportunity to study surfactant-free colloidlike systems. In this work, the crystal growth process in these confined droplets was followed by cooling a model lipid (trimyristin) from a liquid state utilizing synchrotron small-angle X-ray scattering (SAXS). The measurements by SAXS demonstrated a reduced crystallization rate and a greater degree of supercooling required to trigger lipid crystallization in droplets compared to those of bulk lipids. These results suggest that surface droplets crystallize in a stochastic manner. Interestingly, the crystallization rate is slower for larger femtoliter droplets, which may be explained by the onset of crystallization from the three-phase contact line. The larger surface nanodroplets exhibit a smaller ratio of droplet volume to the length of three-phase contact line and hence a slower crystallization rate.

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INTRODUCTION Droplet arrays offer the unique possibility to study droplets within a closed system and are frequently utilized within microfluidics to study a broad range of physical and biological processes.1−3 One such process is the crystallization of and within droplets, which is a burgeoning area with applications in food science,4 pharmaceuticals,5 and high-throughput screening of protein crystallization.6,7 Within food science, the crystallization of emulsion droplets has been studied owing to its influence on physical properties which impact the flavor, stability, texture, and appearance of foods. Given that many bioactive components are lipophilic, food-grade lipids have also attracted interest for their potential as nanoparticles for the delivery of active ingredients.8,9 Triacylglycerol (TAG) nanoparticles, in particular, are being explored owing to their ability to protect bioactives from degradation and prevent bioactive crystallization, which can significantly lower their bioavailability.10,11 The performance of the lipid carriers is also critically dependent on the crystallization properties of the lipid and its active component.12−14 Significant differences exist between the physical properties of liquid droplets and those of their bulk counterparts, largely attributed to increased surface effects. The crystallization temperature of emulsified droplets has been demonstrated to be lower than that of the bulk material, increasingly so as the droplet size decreases.15,16 The difference in crystallization © XXXX American Chemical Society

temperature is commonly referred to as under- or supercooling and is attributed to the decrease in catalytic impurities found within the lipid droplet phase. Depending on the droplet size and the number of impurities in the system, it is possible that some droplets may be impurity-free, thereby relying on secondary nucleation events through collision with other droplets before nucleation can occur.17 While dispersions of lipid droplets have been characterized for some time, the crystallization of lipid nanodroplets on surfaces is yet to be explored. Note that the term surface nanodroplet broadly denotes droplets situated on a solid substrate with at least one dimension, usually height, of less than 1 μm. Typically such droplets are tens to hundreds of nanometers in height and several hundred nanometers to several micrometers in lateral diameter. These dimensions yield femtoliter volumes and are often prepared in contact with an immiscible liquid phase.18 In contrast to emulsified dispersions, surface droplets demonstrate long-term stability in the absence of surfactants or other stabilizers. The immobilization of surface nanodroplets also removes any influence of interdroplet effects such as collisions or coalescence and allows for the examination of isolated Received: April 16, 2018 Revised: June 11, 2018 Published: July 18, 2018 A

DOI: 10.1021/acs.langmuir.8b01252 Langmuir XXXX, XXX, XXX−XXX

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allow for transmission through the thin lipid film, the plane of the sample was aligned to yield a nominal incident angle of 0°. The alignment was achieved by performing individual exposures on the silicon wafer. The wafer was elevated to block half of the beam. By tilting the stage and the corresponding change intensity, the orientation of the wafer could be inferred. An incident angle of 0° was achieved when tilting in either direction resulted in a decrease in beam intensity (by increasing the portion of wafer blocking the beam). The cell was then heated to 60 °C, above the melting point of the lipid (56 °C). After the complete melting of the lipid, the cell was cooled from 60 to 4 °C at 1 °C/min and subsequently held for 5 min. SAXS images (gapless mode, which uses three individual exposures to assemble a complete 2-D image free from the gaps between detector modules; 1 s individual exposures) were taken before and after cooling. In addition, SAXS images were recorded during the cooling process. Because of issues with radiation damage, images were only obtained close to the known temperature of crystallization for each lipid. After 5 min of equilibration at 4 °C, the stage was reequilibrated at room temperature before loading the next sample. Baseline correction was performed using the Australian Synchrotron SAXS/WAXS software (ScatterBrain, V2.71, Australia). However, because of slight variations in the sample alignment, the total subtraction of the polyimide film (windows of the cell) was not possible. SAXS data were calibrated using a known standard (silver behenate, d = 58.38 Å) and analyzed in terms of the maximum intensity and the full width at half-maximum (fwhm), fitting a Gaussian peak function according to eq 1 using MatLab (Math Works Inc., Matlab R2014b, USA)

homogeneous nucleation-induced crystallization. To investigate the crystallization properties of surface nanodroplets, trimyristin (C45) was selected as a model system. Trimyristin is a common fat whose crystallization temperatures and parameters have been established in earlier research.19−21 Synchrotron sources have well-established advantages for studying crystallization and have previously been used to investigate the crystallization of milk fat and palm- and rapeseed-based lipids as oil-in-water emulsions.13,22−25 For droplet arrays, the immobilization to a substrate facilitates the system to be studied as a thin film, yet efforts to measure crystallization within these systems can be challenging due to the presence of a solid substrate. The development of synchrotron SAXS, however, has enabled the determination of structure for thin films on a surface.26 This study utilizes the transmission SAXS through a thin film to study crystallization within a lipid droplet array and quantify the effect of droplet size on the crystallization process of confined droplets.

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EXPERIMENTAL METHODS

Materials. Trimyristin (≥99%; C45), ethanol (≥99%), and octadecyltrimethylchlorosilane (>90%; OTS) were used as received from Sigma-Aldrich. Acetone and isopropyl alcohol of AR grade were used as received from Chem-Supply Pty Ltd. (Gillman, Australia). Silicon wafers were received from University Wafer Inc. (Boston, MA). The photoresist (AZ1512HS) and developer (AZ 400 K) were received from MicroChemicals GmbH (Ulm, Germany). Preparation of Droplet Array. A chemically patterned substrate was prepared by utilizing standard photolithography techniques.27 This substrate was then mounted within a custom-designed, temperature-controlled fluid cell for performing solvent exchange.28 The dimensions of the fluid cell were 17 mm in width, 60 mm in length, and 0.68 mm in depth. In general, solvent exchange denotes the process where a good solvent is displaced by a poor solvent. During this process, an oversaturation of the relevant solute occurs, leading to the nucleation and growth of surface nanodroplets.29 Here, the first solution, termed Solution A, consisted of lipid (0.2 g, C45) dissolved in ethanol (10 mL), while the second solution, termed Solution B, was water. Solvent exchange was performed at 60 °C to ensure that the lipid was in a molten state. All syringes and solutions were preheated accordingly. After heating, Solution A was injected into the fluid cell. The temperature of the fluid cell and Solution A was allowed to equilibrate for 10 min before the contents of the fluid cell were exchanged with preheated Solution B. The droplet size was controlled by the flow rate of solvent exchange.29 Small droplets were produced at 200 μL/min while large droplets were produced at 500 μL/min. Upon completion of the solvent exchange, an array of surface lipid droplets was achieved. Imaging of the nanodroplets was carried out before and after solvent exchange using an optical microscope in reflection mode (Huvitz HRM-300, Gunpo, South Korea). The morphology of the nanodroplets was characterized by AFM (MFP3D, Asylum Research) in tapping mode in air using an AC240TS-R3 tip (k = 2 N/m, nominal tip radius = 7 nm). Synchrotron X-ray Analysis. Small-angle X-ray scattering (SAXS) measurements were carried out at the SAXS beamline at the Australian Synchrotron30 using a monochromatized X-ray beam of wavelength λ= 0.827 Å (15 keV) with a beam size of 250 × 25 μm2 at the sample position. A Pilatus2 1 M detector was used with a sampleto-detector distance of 0.96 m and an observable q range of 0.03−0.6 Å−1. In order to study droplet array crystallization, a custom-made cell fitted with a temperature controller was designed and fabricated at the Australian Synchrotron. The cell, shown in Figure S1, was equipped with a 4 × 1 × 11 mm3 well to hold the silicon wafer samples in place and can be used for both transmission SAXS through a thin film (as used here) and GISAXS analysis. Silicon wafers coated by the lipid droplets or bulk lipid sample were inserted into the cell and covered with degassed Milli-Q water. To

2

f (x) = ae−((x − b) /2c

2

)

(1)

where a is the height of the diffracted peak, b is the peak center, and c is the fwhm intensity. Differential Scanning Calorimetry Measurements. Thermal analysis of the bulk sample was conducted using a DSC 1 STARe System (Mettler Toledo, Port Melbourne, Australia). The data were analyzed by the instrument software (version 14.0). The lipid sample was melted, and quantities of ∼20 mg were weighed into 40 μL aluminum pans (Mettler Toledo, part number ME-27331). The samples were then stored at room temperature for over 48 h before analysis. After storage, the pans were heated in the DSC chamber to 60 °C and held for 5 min. DSC analysis was performed as the sample was cooled to 0 °C at 1 °C/min. After cooling, the sample was held at 0 °C for 10 min before being reheated to 60 °C. The DSC STARe software calculated the temperature onset of each peak (Tonset) and endset (Tendset) and the maximum values for each peak.

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RESULTS AND DISCUSSION SAXS of Bulk Lipid. Bulk lipid samples were monitored using synchrotron SAXS, cooling from 60−4 °C at 1 °C/min. SAXS patterns recorded progressively during the cooling process are shown in Figure 1A,B as stacked plots of 1-D intensity versus q encompassing the temperature range over which crystallization occurred. No Bragg peaks were initially observed in the SAXS patterns, characteristic of fully melted fluid lipid samples. With decreasing temperature, diffraction peaks gradually appeared, corresponding to a crystalline lamellar phase formed by the triacylglycerol (TAG) molecules.31 This was consistent with the expected 2L structure, corresponding to a double chain length arrangement of the TAG molecules.25,31 With decreasing temperature, diffraction peaks corresponding to the longitudinal spacing of a crystalline lamellar phase formed. Between 36 and 37 °C, peaks corresponding to the (001) and (003) peaks of a 2L structure with a lattice parameter of 36.6 Å were initially observed. The intensity of these peaks increased quickly with decreasing temperature, and two orders of reflection (001) and (003) were observed. A B

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technique. Note that the (002) reflection was not observed, which is consistent with previous studies on emulsified milk fat where the (001) and (003) reflections of the 2L lamellar phase are typically the most intense.32 Stability of Surface Nanodroplets with Temperature. Typically, droplet systems are characterized as dispersed systems, such as oil/water emulsions.9,15,33 In this study, an immobilized droplet array was utilized where lipid droplets were adsorbed onto a silicon wafer surface. Depending on the liquid properties, the production of droplet arrays can be challenging. Solvent exchange, however, has been demonstrated to be a versatile technique for the preparation of a broad range of liquids;28,29 through the utilization of chemical surface patterns, lipid droplets can be deposited with controlled size and spacing,27 as shown in Figure 2. The substrate exhibited an array of hydrophobic circular domains and hydrophilic surroundings. The hydrophobic lipid preferentially grew within the hydrophobic domains, without spreading to the hydrophilic regions. Two groups of C45 droplets were produced by controlling the flow rate. The large droplets were 5.28 ± 0.42 μm in the lateral diameter while the small droplets were 4.05 ± 0.15 μm. As described in previous work,34 the droplets first grew within the hydrophobic domains, maintaining a constant contact angle. Once the droplets filled the hydrophobic domains, the droplets then grew in a constant contact area mode, increasing the contact angle of the droplets. Hence, the lateral size did not appear

Figure 1. (A) Stacked plot of 1-D SAXS patterns (intensity vs q) for the C45 bulk lipid sample recorded during cooling from 60 to 4 °C. As the temperature is reduced, a number of peaks appear and increase in height. The peaks labeled as q = 0.172 and 0.519 Å−1 correspond to the (001) and (003) reflections of a 2L lamellar phase, with lattice parameter d = 36.6 Å. (B) Magnified plot for the 2L(001) peak at q = 0.172 Å−1.

magnified plot of 1-D spectra of intensity versus q for the (001) peak is shown in Figure 1B with a sharp increase in the intensity of this peak between 36.0 and 34.8 °C. The peak corresponds to the stable β polymorph of trimyristin.19 The lattice parameter of the 2L phase did not vary with decreasing temperature, indicating that the TAG molecules crystallized immediately into the final structure with no lipid rearrangements during the process of crystal growth. Importantly, the onset temperatures for crystallization determined by SAXS measurements correspond well to the DSC measurements (Table S1, Figure S2), indicating the reliability of the

Figure 2. (A) Schematic of droplet array formation by solvent exchange and typical droplet morphology, described by a spherical cap. The lipid solution, termed Solution A, is displaced by a relatively poor solvent, termed Solution B. The mixing of two solutions affords a temporal oversaturation pulse which drives the nucleation and growth of surface nanodroplets. For oil droplets, the position of the formed droplets can be tailored by a chemical pattern of hydrophobic domains. (B) Optical images and size distribution for C45 droplets utilized within this study. The blue and red highlights correspond to the small (d = 4.05 ± 0.15 μm) and large (d = 5.28 ± 0.42 μm) droplet sizes, respectively. Scale bar = 5 μm. (C) AFM height (h) profile and single-line section for individual small and large droplets. From the lateral and height parameters, the contact angle is determined by a spherical cap model. C

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Figure 3. (A) Series of optical micrographs during the cooling cycle utilized in crystallization experiments for the large droplets. The temperatures at T1, T2, and T3 were 60, 30, and 10 °C, respectively. Scale bar = 10 μm. (B) Plot of the average droplet diameter vs time during the cooling cycle.

different although the droplet volume increased with the flow rate. As the array is immobilized, the droplet spacing and thus interactions can be controlled. In addition, in this system the lipid−water interface was pristine (or could be doped), which is important to distinguish any external influence of contaminates, including surfactants which can induce heterogeneous nucleation.35 To determine the feasibility of using droplet arrays within crystallization studies, the droplet arrays were first exposed to a temperature cycle, initially heating to ∼60 °C before cooling to ∼10 °C. Optical images taken in situ and the corresponding average diameter are shown in Figure 3. The negligible change in droplet morphology demonstrates the stability of the droplet array to both heating and cooling over this temperature range. This result reinforces the suitability of the droplet array for studying lipid crystallization. In addition, the minimal change in lateral diameter indicates that the droplet contact line remains pinned throughout the cooling process. Crystallization of Surface Nanodroplets. The same synchrotron protocol was utilized to characterize the crystallization of two nanodroplet arrays, as shown in Figure 2. The two droplet samples are described as small and large. In both cases, the droplet center-to-center spacing was 10 μm, and the number of droplets probed by the beam was estimated to be ∼10 000. Using a spherical cap model and AFM measurements, the contact angle of the small and large droplets was determined to be 17.6 ± 2.2 and 26.4 ± 1.7°. The average values of the surface area (SA) in contact with water (in contact with substrate) of the small and large droplets were ∼13.2 and ∼23.1 μm2 (∼12.8 and ∼21.8 μm2), respectively. The average values of the volume (V) of the small and large droplets were ∼2 and ∼7 femtoliters, yielding sample density per unit surface areas of ∼20 and ∼70 attoliters/μm2, respectively. The evolution of the 1-D SAXS patterns on cooling the large droplet sample (C45) from 60 to 4 °C is presented in Figure 4. Peaks corresponding to the (001) peak of a 2L structure with a lattice parameter of 35.2 Å were initially observed at ∼30 °C, again corresponding to the stable β polymorph of trimyristin.19 While the intensity of the (001) peak subsequently increased as the temperature was lowered, we note that the overall diffraction intensity was markedly reduced compared to the that of the bulk sample as expected for droplets. As a consequence, higher-order reflections seen for the bulk samples were not observed for droplet samples. Similarly, Figure 5 shows a stacked plot of 1-D SAXS patterns for small droplets (C45). Peaks corresponding to the (001) peak of a 2L structure with a lattice parameter of 36.0 Å were initially observed at ∼27 °C. A secondary peak was also observed around 40.7 Å indicating polymorphism within the crystalline structure, specifically the presence of the unstable α

Figure 4. (A) Stacked plot of 1-D SAXS patterns for the C45 large droplet sample recorded during cooling from 60 to 4 °C. As the temperature was reduced, the peak corresponding to the lamellar structure, labeled 0.178 Å−1 (d = 35.2 Å) (2L)(001), increased in height. (B) Magnified plot for peak 0.178 Å−1 (2L)(001).

Figure 5. (A) Stacked plot of 1-D SAXS patterns for the C45 small droplet sample recorded during cooling from 60 to 4 °C. As the temperature was reduced, the peak corresponding to the lamellar structure, labeled 0.175 Å−1 (d = 36 Å) (2L)(001), increased in height. (B) Magnified plot for peaks 0.175 Å−1 (d = 36.0 Å) and 0.154 Å−1 (d = 40.7 Å) (2L)(001).

polymorph of trimyristin.19 Both polymorphs appeared stable over the time scale of the experiment (∼60 min). This is consistent with previous research demonstrating that the α polymorph, once formed, can be stable for a period of days.36 It is possible that the unstable α polymorph may ultimately transition into the stable β polymorph;37 however, it was not possible to keep the wafer in the sample holder to verify this. For both peaks the intensity increased with decreasing temperature. Again, the reduction in overall scattering intensity meant that higher-order reflections are not observed for the droplet sample. The overall reduction in scattering intensity is also evident in Figure 6, which shows representative 2-D SAXS patterns for each sample. For the bulk sample, rings appear to be homogeneous, indicating that crystal growth was not occurring on a preferential direction. However for droplet samples, scattering intensity is highly inhomogeneous and indicates the alignment of crystal growth with the substrate surface. D

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crystallization rate with decreasing droplet size was also observed for miniemulsion droplets where it was speculated that increased heat transport facilitates a more rapid crystallization in smaller droplets.15 In emulsions, secondary nucleation may also occur where crystals protruding through a droplet surface may catalyze nucleation in a colliding droplet, which is influenced by droplet number and proximity. However, in this study, droplet attachment to the silicon wafer surface eliminates interdroplet collision events or enhanced heat transport (i.e, a negligible volume of droplets compared to the cooled bulk). In contrast to dispersed droplets in emulsions where the crystallization process is closer to homogeneous nucleation, we relate the nucleation rate of the surface droplets to their morphology, with a reduction in size exhibiting lower crystallization temperatures. The 2-D scattering in Figure 6 suggests that the onset of crystallization may occur from the interface of the droplets or the three-phase contact line. Indeed, the freezing of water droplets with ∼20 μm and ∼1 mm in diameters in air has been shown to initiate from the liquid−solid interface.40,41 If crystallization is initiated at the interface, the ratio of ksmall/klarge may be expected to be proportional to the respective surface area-to-volume ratios. In this case ksmall/klarge ∼2.42 while (SA/V)small/(SA/V)large ∼1.99. If crystallization is expected to depend on the three-phase contact line (CL), we arrive at (CL/V)small/(CL/V)large ∼2.60. This higher ratio showed the closest correlation with the experimental results and may indicate that surface droplet crystallization nucleates at the solid−lipid−water boundary. In addition to the contrast in the crystallization rate, the lattice parameters of the crystallized trimyristin (C45) for both the small (36.0 Å) and large (35.2 Å) droplet samples were slightly lower than that observed in the bulk sample (36.6 ± 0.11 Å). The lower lattice parameter and the polymorphism observed in smaller droplets indicate that confinement may influence the molecular packing in crystallized droplets. Recently, Seddon et al.42 demonstrated that the lipid crystallization and surfactant crystallization within droplets ∼1 mm in diameter were sensitive to the interface and demonstrated interfacial orientation. Given the high surface area-to-volume ratio of the surface droplets, it is plausible that the interface may also influence the polymorphs formed. Conversely, it has also been demonstrated that the more rapid crystallization of milk fat leads to the formation of less stable polymorphs.36 As the smaller droplets also exhibited a faster polymerization rate, it is difficult to determine whether the presence of the α polymorph was driven by the interface or the rate of crystallization. The influence of the interface may be further supported by the absence of the α polymorph in the bulk sample, which crystallized significantly faster than did the small droplet sample.

Figure 6. Representative 2-D SAXS patterns for the C45 bulk large and small droplet samples.

The onset temperature for crystallization decreased with sample size from bulk to large and small droplets (summarized in Table S2). This is demonstrated by a plot of intensity versus temperature in Figure 7A. Here the intensity has been

Figure 7. (A) Plot of normalized intensity at respective 2L(001) peaks vs temperature (°C) for the C45 bulk (green), small (blue), and large (red) droplets. (B) Plot of normalized intensity (by maximum recorded intensity) at respective 2L(001) peaks vs time (s) for the C45 bulk (green), small (blue), and large (red) droplets, corresponding to the fits to I = 1 − e−kT.

normalized against the highest recorded peak for each sample. The separation between the data sets highlights the contrast in the onset temperature. The undercooling required for the droplet samples and the reduced rate of crystallization indicates a stochastic crystallization process whereby each droplet nucleates and crystallizes independently. The increase in the normalized scattering intensity of the (001) peak with decreasing temperature was also markedly more rapid for the bulk sample in comparison to that of the droplet samples. From the correlation between the crystallization temperatures obtained by DSC and SAXS measurements for the bulk sample, it is expected that any sample heating from the X-ray beam was negligible during the crystallization process. Cheng and Caffrey have demonstrated that the localized heating from similar exposures was limited to ∼0.02 °C.38 Moreover, following the approach recently outlined by Hopkins and Thorne,39 the estimated temperature increase here is ∼0.064 °C. (A detailed calculation is included within the Supporting Information.) The crystallization rate can be described as the crystallized volume of the sample (Φ) against time. The crystallization rate is then expected to be proportional to the volume of liquid droplets, dΦ/dT = k(1− Φ). The volume fraction of solidified droplets with time is then described as Φ = 1 − e−kT. By assuming intensity (I) = Φ, the plot of normalized intensity against time (s) shown in Figure 7B was used to estimate the rates of crystallization; ksmall = (2.9 ± 0.6) × 10−3 and klarge = (1.2 ± 0.3) × 10−3. Interestingly, the results show that the smaller droplets crystallized faster than did the larger droplets. The difference in the nucleation rate cannot be attributed to the number of droplets given that in each case the number of droplets was the same for the small and large droplets, which was predetermined by the array design. The trend of increasing

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CONCLUSIONS The synchrotron SAXS protocol utilized is capable of revealing the crystallization of femtoliter lipid nanodroplets immobilized on a surface. Our experimental measurements show that the onset temperature of droplet crystallization is lower than that of the bulk lipids. The smaller droplets were shown to crystallize faster than their larger counterpart, which was attributed to the droplet morphology. The difference in crystallization rates is in good agreement with the hypothesis that surface droplets first crystallize at the triple-phase line. This synchrotron SAXS protocol may offer the potential to E

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Nanotechnology; Hernández-Sánchez, H., Gutiérrez-López, G. F., Eds.; Springer International Publishing, 2015; pp 99−143. (11) Aditya, N. P.; Macedo, A. S.; Doktorovova, S.; Souto, E. B.; Kim, S.; Chang, P.-S.; Ko, S. Development and Evaluation of Lipid Nanocarriers for Quercetin Delivery: A Comparative Study of Solid Lipid Nanoparticles (SLN), Nanostructured Lipid Carriers (NLC), and Lipid Nanoemulsions (LNE). LWT - Food Sci. Technol. 2014, 59 (1), 115−121. (12) Ribeiro, H. S.; Gupta, R.; Smith, K. W.; van Malssen, K. F.; Popp, A. K.; Velikov, K. P. Super-Cooled and Amorphous Lipid-Based Colloidal Dispersions for the Delivery of Phytosterols. Soft Matter 2016, 12 (27), 5835−5846. (13) Zychowski, L. M.; Logan, A.; Augustin, M. A.; Kelly, A. L.; Zabara, A.; O’Mahony, J. A.; Conn, C. E.; Auty, M. A. E. Effect of Phytosterols on the Crystallization Behavior of Oil-in-Water Milk Fat Emulsions. J. Agric. Food Chem. 2016, 64 (34), 6546−6554. (14) te Velde, A. A.; Brüll, F.; Heinsbroek, S. E. M.; Meijer, S. L.; Lütjohann, D.; Vreugdenhil, A.; Plat, J. Effects of Dietary Plant Sterols and Stanol Esters with Low- and High-Fat Diets in Chronic and Acute Models for Experimental Colitis. Nutrients 2015, 7 (10), 8518−8531. (15) Montenegro, R.; Antonietti, M.; Mastai, Y.; Landfester, K. Crystallization in Miniemulsion Droplets. J. Phys. Chem. B 2003, 107 (21), 5088−5094. (16) Coupland, J. N. Crystallization in Emulsions. Curr. Opin. Colloid Interface Sci. 2002, 7 (5), 445−450. (17) Walstra, P.; Kloek, W.; Van Vliet, T. Fat Crystal Networks. In Crystallization Processes in Fats and Lipids; Garti, N., Sato, K., Eds.; Marcel Dekker, 2001; pp 289−328. (18) Lohse, D.; Zhang, X. Surface Nanobubbles and Nanodroplets. Rev. Mod. Phys. 2015, 87 (3), 981−1035. (19) Takeuchi, M.; Ueno, S.; Sato, K. Synchrotron Radiation SAXS/ WAXS Study of Polymorph-Dependent Phase Behavior of Binary Mixtures of Saturated Monoacid Triacylglycerols. Cryst. Growth Des. 2003, 3 (3), 369−374. (20) Markiewicz-Kęszycka, M.; Czyżak-Runowska, G.; Lipińska, P.; Wójtowski, J. Fatty Acid Profile of Milk-a Review. Bull. Vet. Inst. Pulawy 2013, 57 (2), 135−139. (21) Jensen, R. G. The Composition of Bovine Milk Lipids: January 1995 to December 2000. J. Dairy Sci. 2002, 85 (2), 295−350. (22) Verstringe, S.; Dewettinck, K.; Ueno, S.; Sato, K. Triacylglycerol Crystal Growth: Templating Effects of Partial Glycerols Studied with Synchrotron Radiation Microbeam X-Ray Diffraction. Cryst. Growth Des. 2014, 14 (10), 5219−5226. (23) Wassell, P.; Okamura, A.; Young, N. W. G.; Bonwick, G.; Smith, C.; Sato, K.; Ueno, S. Synchrotron Radiation Macrobeam and Microbeam X-Ray Diffraction Studies of Interfacial Crystallization of Fats in Water-in-Oil Emulsions. Langmuir 2012, 28 (13), 5539−5547. (24) Arima, S.; Ueno, S.; Ogawa, A.; Sato, K. Scanning Microbeam Small-Angle X-Ray Diffraction Study of Interfacial Heterogeneous Crystallization of Fat Crystals in Oil-in-Water Emulsion Droplets. Langmuir 2009, 25 (17), 9777−9784. (25) Lopez, C.; Bourgaux, C.; Lesieur, P.; Ollivon, M. Coupling of Time-Resolved Synchrotron X-Ray Diffraction and DSC to Elucidate the Crystallisation Properties and Polymorphism of Triglycerides in Milk Fat Globules. Lait 2007, 87 (4−5), 459−480. (26) Renaud, G.; Lazzari, R.; Leroy, F. Probing Surface and Interface Morphology with Grazing Incidence Small Angle X-Ray Scattering. Surf. Sci. Rep. 2009, 64 (8), 255−380. (27) Bao, L.; Rezk, A. R.; Yeo, L. Y.; Zhang, X. Highly Ordered Arrays of Femtoliter Surface Droplets. Small 2015, 11 (37), 4850− 4855. (28) Dyett, B.; Yu, H.; Zhang, X. Formation of Surface Nanodroplets of Viscous Liquids by Solvent Exchange. Eur. Phys. J. E: Soft Matter Biol. Phys. 2017, 40, 1−6. (29) Zhang, X.; Lu, Z.; Tan, H.; Bao, L.; He, Y.; Sun, C.; Lohse, D. Formation of Surface Nanodroplets under Controlled Flow Conditions. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (30), 9253−9257. (30) Kirby, N. M.; Mudie, S. T.; Hawley, A. M.; Cookson, D. J.; Mertens, H. D. T.; Cowieson, N.; Samardzic-Boban, V. A Low-

study the influence of external media such as surfactants, droplet geometry, and droplet spacing on crystallization in future studies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b01252. Photograph of the GIXAS fluid cell, DSC thermographs, table of DSC thermal parameters, table of SAXS thermal parameters, and sample heating calculation (DOCX)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Brendan Dyett: 0000-0002-8417-2736 Lei Bao: 0000-0002-8243-2872 Xuehua Zhang: 0000-0001-6093-5324 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS X.Z. acknowledges the support from the Australian Research Council (FT120100473, LP140100594). C.C. is the recipient of an Australian Research Council DECRA Fellowship, DE160101281. L.B. and M.L. acknowledge support from the RMIT Vice-Chancellor’s Postdoctoral Research Fellowship. We acknowledge the use of the SAXS/WAXS beamline and the associated technical support at the Australian Synchrotron. We also acknowledge the RMIT MicroNano Research Facility for providing access to equipment and resources.

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REFERENCES

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DOI: 10.1021/acs.langmuir.8b01252 Langmuir XXXX, XXX, XXX−XXX