Monodisperse w/w/w double emulsion induced by

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1 Monodisperse w/w/w double emulsion induced by phase separation Yang Song, Ho Cheung Shum* Department of Mechanical Engineering, the University of Hong Kong, Pokfulam Road, Hong Kong *Corresponding address: KEYWORDS: W/W/W double emulsion, monodisperse, microfluidic, phase separation. ABSTRACT We develop an approach to fabricate monodisperse water-in-water-in-water (w/w/w) double emulsion in microfluidic devices. A jet of aqueous solution containing two incompatible solutes, dextran and polyethylene glycol (PEG), is periodically perturbed into water-in-water (w/w) droplets. By extracting water out of the w/w droplet, the solute concentrations in the droplet phase increase; when the concentrations exceed the miscibility limit, the droplet phase separates into two immiscible phases. Consequently, PEG-rich droplets are formed within the single emulsion templates. These PEG-rich droplets subsequently coalesce with each other, resulting in transiently stable w/w/w double emulsions with a high degree of size uniformity. These double emulsions are free of organic solvents and thus are ideal for use as droplet-vessels in protein 1

2 purification, as microreactors for biochemical reactions, and as templates for fabrication of biomaterials. 1 INTRODUCTION Double emulsions, which are suspensions of droplets containing smaller droplets of an immiscible phase within them, are important templates for synthesizing core-shell structured materials, [1] such as vesicles, capsules and particles. [2-4] In conventional double emulsions, organic solvents are frequently used as at least one of the emulsion phases. [5, 6] As concerns over the safety and biocompatibility of these emulsions are increasing, a route of producing allaqueous double emulsion without an organic solvent phase is desired. Meanwhile, when these emulsions are used as liquid vessels for proteins delivery, or microreactors for enzyme reactions, the bioactivity of these biomolecules may be impaired due to denaturation by harmful organic solvents. In addition, hydrogel particles that are templated by water-in-oil (w/o) emulsion droplets often need to be washed repeatedly to remove any remaining oil phase before use with biological tissues and cells. This tedious step severely limits the attractiveness of microfluidic approaches for fabricating hydrogel particles for biomedical applications. An organic-solventfree approach to fabricate droplets is needed, not only for biomedical applications, but also for food industries, cosmetics formulation, and bio-inspired studies. Formation of all-aqueous emulsions requires two immiscible aqueous phases, which are commonly known as aqueous two-phase systems (ATPS). The two immiscible aqueous phases are formed by dissolving two incompatible solutes in water above a critical concentration of phase separation. Dextran and polyethylene glycol (PEG) are one such example of incompatible solutes; a solution with high concentrations of dextran and PEG spontaneously separates into a 2

3 dextran-rich phase and a PEG-rich phase. The equilibrium compositions of these two phases have been measured and presented in phase diagrams. [7] This particular ATPS has been widely used for preparing microspheres, [8] as well as for separating proteins, cells and particles. [9-11] Recently, a microfluidic approach has been introduced to produce all-aqueous emulsions using ATPS. [12, 13] At typical flow rates of µl/h, a dispersed phase forms a jet surrounded in an immiscible continuous phase in microfluidic devices. By applying oscillating pressure, the jet can be perturbed to break up into droplets. Using similar approaches, double emulsion has been successfully fabricated by sheathing the inner jet inside a middle jet and subsequently breaking up the compound jet. [12] These double emulsion droplets typically have a polydispersity of around 10%. Further reduction in the polydispersity of the all-aqueous emulsion droplets is desired, especially for biomedical applications that are sensitive to the droplet size; these include quantitative analysis of biomolecules and screening of enzyme bioactivity in biochemical reactions. In this paper, we demonstrate the generation of w/w/w double emulsions with an excellent polydispersity of less than 4%. A single-phase solution of 5% dextran/ 1% PEG/ 94% water is injected as the dispersed phase, which is surrounded by a concentrated PEG solution in a microchannel. When the driving pressure of the dispersed phase varies periodically, the jet breaks up into droplets. We investigate the polydispersity of the resultant w/w droplets as a function of the perturbation frequency and flow rates. Droplets that are formed directly at the nozzle show superior monodispersity when compared with those formed due to the breakup of jets at some distance downstream from the nozzle. To form w/w/w double emulsion, we use monodisperse w/w droplets as templates, and gradually extract water out of these droplets. This raises the 3

4 concentrations of both PEG and dextran in the droplets, thus triggering a spontaneous phase separation. Using this approach, the size uniformity of the single emulsion templates is preserved. To the best of our knowledge, the resultant w/w/w double emulsion attains the highest size uniformity so far. 2 EXPERIMENTAL SECTION 2.1 Emulsion preparation An aqueous solution with 5 wt % dextran (Mw=500,000Da, Shanghai Kayon Biological Technology Co.) and 1 wt % PEG (Mw=8000Da, Aldrich) was chosen as the inner fluid (disperse phase). This fluid remains a single phase at room temperature and atmospheric pressure, according to the phase diagram in fig.1a. The outer fluid, which extracts water from the inner fluid upon mixing, was an aqueous phase with 8 wt % PEG and 20 wt % glycerol. Glycerol increases the viscosity and density of the outer fluid. 2.2 Fabrication of the glass capillary microfluidic device We used a capillary microfluidic device to generate all-aqueous emulsions. [14] Briefly, a cylindrical glass capillary with inner and outer diameters of 0.86 mm and 1.0 mm respectively, was pulled to achieve a tapered tip geometry using a pipette puller (SUTTER INSTRUMENT CO., U.S.A,). Then we polished the tips of two capillaries on an abrasive sand paper until their inner diameters reach about 30 µm and 125 µm. Subsequently, the two capillaries were coaxially positioned inside another square capillary with an inner diameter of 1.0 mm, as shown by the schematic in fig.1b. The inner and outer fluids were separately injected into the device through 4

5 two inlets, each of which was connected through a plastic tubing to a syringe driven using a syringe pump (Longer pump, model LSP01-2A). Figure 1. (a) Phase diagram of dextran (M w = 500,000 Da)-PEG (Mw = 8000 Da)-H2O at room temperature (22 ), redrawn from ref.(7), and the blue dashed lines are tie lines. Compositions of the outer and inner fluids used to form w/w/w double emulsion are denoted by points A and B, respectively. The line BD represents the change in emulsion compositions upon mixing of the two fluids. The line AE is a tie line estimated by drawing a line parallel to the measured tie lines. (b) Schematic of a capillary device used for preparing w/w/w double emulsions. The configuration of the capillaries inside the device is magnified in the schematic at the bottom half of fig.1b. 2.3 Perturbation-induced droplet formation and phase separation within the droplets. We tuned the velocity of the inner fluid Q in from 10 to 200 µl/h, and fixed the velocity of the outer fluid Q out at 1000 µl/h. The resultant jets were observed under an optical microscope (DM 5

6 180, Motic Inc.). Then we perturbed the jet using a mechanical vibrator (PASCO scientific, SF- 9324), which was controlled by a sinusoidal-wave generator (RIGOL, DG1012). The inner tubing is firmly inserted between a tubing holder and the vibrator. This vibrator oscillates under the control of the generator, thus the inner plastic tubing is squeezed and relaxed accordingly. As a result, the instantaneous flow rate of the inner fluid also changes periodically. The input voltage of vibrator was fixed at 10 volts, and the perturbation frequency f p was varied from 2 Hz to 30 Hz. The vibrator produces periodical pressure fluctuation in the inner tubing, leading to an oscillatory flow of the inner fluid. Consequently, the jet was forced to break up at certain frequencies. After the jet was broken up into droplets, the diameters of the fabricated droplets were measured using an open-source image-processing software, ImageJ. The uncertainty of the measured diameter was about ± 1 µm. The polydispersity was obtained by measuring the diameters of at least 100 droplets. To observe the dynamics of phase separation, we monitored the shapes of droplets inside the microchannel using a camera connected to the optical microscope. The droplets were dyed with wt % methylene blue to improve image contrast. 3 RESULTS AND DISCUSSION 3.1 Perturbation-induced formation of monodisperse droplets The inner fluid forms a jet surrounded by the outer fluid upon injection inside the microfluidic device. In the absence of perturbation, the jet spontaneously breaks up into polydisperse w/w droplets with a broad size distribution. To control the droplet sizes and improve the droplet size 6

7 distribution, the jet is periodically perturbed using a vibrator and forced to break up into [13, 15, 16] droplets. As the instantaneous flow rate of the inner fluid changes sinusoidally, the resultant jet diameter varies and forms a corrugated interface. This instantaneous deviation of the jet diameter from its average diameter is defined as the perturbation amplitude of the system. With an increase in the input voltage of perturbation U p, the perturbation amplitude of the jet also rises. [17] When this perturbation amplitude reaches a length scale comparable with the jet radius, the jet breaks up into droplets immediately at the nozzle [17] and no satellite drops are observed. The resultant droplets are highly uniform in size. By contrast, with a smaller perturbation amplitude obtained at lower U p, the jet breaks up into droplets at some distance from the nozzle of the receiving tube. Satellites drops are occasionally observed along with the perturbed jet; this raises the polydispersity of the droplets downstream. The resultant droplets have a larger polydispersity (around 5 %) than those formed directly at the nozzle (< 3 %). Figure 2. A state diagram showing the polydispersity of droplets produced at different Q in and perturbation frequency f p. Generally, droplets produced directly at the nozzle show the lowest polydispersity, except for the data points indicated by the blue triangles at the lower right corner 7

8 in the plot, where the originally monodisperse droplets coalesce with their neighboring droplets due to the small inter-droplet separation distance under the prescribed flow conditions. The input voltage of perturbation is 10 V and Q out = 1000 µl/h. The frequency of perturbation f p applied to the inner tubing also significantly affects the polydispersity of the droplets (fig.2). At high perturbation frequency f p, a jet randomly breaks up into droplets without a defined droplet generation rate, suggesting uncontrolled formation of droplets. In this regime, droplets generated have a broad size distribution, with a polydispersity of over 10%. When the frequency f p decreases below a certain critical frequency f p *, the resultant droplets are generated at the nozzle and the droplet generation rate is the same as the perturbation frequency, confirming the controlled generation of droplets. These droplets are monodisperse, with a polydispersity of less than 3%. The critical frequency, f p *, increases as the flow rate of the inner fluid Q in decreases, as shown by the plot in fig.3a. To obtain controlled droplets, the wavelength of the perturbed jet, L p, should be larger than the circumference of the jet. [18] L p = u/f p = 4Q in /(πd jet 2 ) > πd jet, where u is the average velocity of the inner fluid, and d jet is the diameter of the inner jet. Thus, the critical frequency f p * can be expressed as: f p * = 4Q in /(π 2 d 3 jet ). Decreasing Q in reduces 3 the jet diameter (fig.3b), but the ratio of Q in and d jet increases. Consequently, a jet with a thinner diameter can be perturbed into droplets at a higher f p. Our experimental results support this relationship (fig.3a). 8

9 Figure 3. (a) A state diagram of the flow regimes under which droplets forms after perturbation. At low perturbation frequency, f p and inner fluid flow rate Q in, as indicated by the area under the curve in Fig. 3a, droplets form at the nozzle of the injection capillary; at high perturbation frequency and inner fluid flow rates, droplets form at the end of a jet. The boundary of the flow regimes can be estimated using Plateau s criterion, [18] shown by the red line. (b) A plot of the diameter of the inner jet as a function of the inner fluid flow rates, Q in, which ranges from 10 µl/h to 190 µl/h. The input voltage of perturbation is 10 V and the Q out = 1000 µl/h. The polydispersity of the w/w droplets depends on the manner in which the jet breaks up under perturbation. By adjusting the amplitude, U p, and frequency, f p, of perturbation, as well as the inner fluid flow rate, Q in, the inner jet can be perturbed into droplets. The w/w droplets with the best monodispersity are produced only if the inner jet breaks up at the nozzle of the injection tube. In this regime, the initial size of the droplets can be predicted by the relation: D = (6 Q in /πf p ) 1/3. Experimentally, after optimizing the flow rates and perturbation frequency, we obtain monodisperse w/w single emulsion droplets (<3 %) with diameters ranging from 60 µm to 200 µm (fig.4). 9

10 Figure 4. (a) An optical microscope image showing the formation of a w/w droplet in the dripping-under-perturbation regime; Optical microscope images showing monodisperse w/w droplets with diameters of (b) 60 µm, (c) 120 µm, and (d) 200 µm, produced under the drippingunder-perturbation regime. All scale bars are 200 µm. 3.2 Formation of monodisperse w/w/w double emulsion With the monodisperse emulsion droplets as templates, the w/w/w double emulsion can be fabricated by inducing phase separation inside them. This approach is illustrated by a schematic diagram and microscopic images in fig.5. The original compositions of the single emulsion droplets are 5% dextran, 1% PEG and water (point B in fig.1a), and the compositions of the continuous phase are 8% PEG solution (point A in fig.1a). The initial diameter of the single emulsion droplet is estimated by D = (6Q in /πf p ) 1/3, as shown by the solid square in fig.6. According to the phase diagram, the continuous phase at point A is in equilibrium with a concentrated dextran phase, which is composed of 15% dextran, 0.9% PEG and water (point D in fig.1a). As the single emulsion droplets move downstream, water is extracted from the droplets to the continuous phase, so the single emulsion droplets shrink in size. Meanwhile, the 10

11 concentrations of both PEG and dextran in the emulsion droplets increase. As soon as the emulsion concentrations reach the critical concentrations of phase separation (point C in fig.1a), small PEG-rich droplets start to form inside the single emulsion templates (fig.5a). As water is continuously extracted from the droplets, the phase-separated PEG-rich droplets grow bigger in size and coalesce with each other (fig.5b). Eventually, all of the phase-separated PEG-rich droplets coalesce into a single droplet, so the resultant w/w/w double emulsion has a single PEGrich core droplet inside (fig.5c). After phase separation, each double emulsion droplet is stable in size and the dextran-rich shell phase is in equilibrium with the continuous and the innermost PEG-rich phases. These double emulsions are stable for at least tens of seconds, until the innermost PEG-rich phase coalesces with the continuous phase, or the double emulsion drops coalesce with each other. Compared with the initial diameter of single emulsions, the diameter of the final double emulsion is reduced by more than 30% after phase separation (fig.6). The diameter ratio of the PEG-rich core droplets to the final double emulsion droplets is around 60%, which can be predicted by mass conservation based on the phase diagram (see video in AVI format). The polydispersity of the resultant double emulsion droplets is less than 4% (shown by a video in the Supporting Information), which is close to the polydispersity of the single emulsion templates. Before phase separation, the volume ratio of the emulsion phase and the continuous phase are controlled by the relative fluid flow rates specified by the syringe pumps. During phase separation, the concentrated dextran phase becomes highly viscous, thus preventing coalescence of the innermost PEG-rich droplets with the continuous PEG-rich phase. After phase separation, the dextran shell phase is in equilibrium with both PEG-rich core phase and PEG-rich continuous phase, thus net extraction of water from the dispersed phases is zero. This ensures the 11

12 monodispersity and size stability of the final w/w/w double emulsion. Unlike conventional methods for fabricating double emulsion, which require two steps of droplets generation, our approach does not require synchronized generation of the innermost droplets and the surrounding middle droplets. Our approach requires only one step of droplet generation inside the device; therefore, the polydispersity of the final double emulsion mainly depends on the polydispersity of the single emulsion templates, which can be generated with very high size uniformity using [19, 20] microfluidic techniques. Figure 5. Formation of w/w/w double emulsion is induced by phase separation inside the w/w single emulsion templates. (a1,a2) Small PEG droplets form inside the w/w single emulsion template; (b1,b2) the PEG droplets grow in size and coalesce with one another; (c1,c2) the monodisperse w/w/w double emulsion droplets with single cores are formed downstream. Images are captured at (a2) 2 cm; (b2) 3.5 cm; (c2) 5 cm downstream from the nozzle. Q in = 10 µl/h, Q out = 1000 µl/h, f p = 5 Hz, and the scale bars are 100 µm. 12

13 Figure 6. Compared with the diameter of the w/w single emulsion templates, the diameter of the w/w/w double emulsion droplets reduce after phase separation. As the perturbation frequency increases, the w/w emulsion drops decrease in size; accordingly, the size of the w/w/w double emulsion drops also decreases with the size of the single emulsion templates after phase separation. The diameters of the all-aqueous emulsion droplets are measured at equilibrium concentrations. The flow rates for generating the droplets are as follows: Q in = 10 µl/h, Q out = 1000 µl/h. 4 CONCLUSIONS We fabricate monodisperse w/w/w double emulsion by inducing phase separation inside single emulsion droplets. For this purpose, a single-phase aqueous solution containing dextran and PEG is selected as the dispersed phase. Jets formed by the dispersed phase are then forced to break up into w/w single emulsions by mechanical perturbation. By varying the operating flow rates, perturbation amplitude and perturbation frequency, the polydispersity of the resultant single emulsion droplets can be tuned. Our results suggest that the polydispersity of droplets is significantly affected by the way the jet breaks up into droplets. Droplets formed from the 13

14 immediate breakup of the jet at the nozzle of the injection capillary achieve the best uniformity in size. By utilizing these single emulsions as templates, w/w/w/ double emulsions are fabricated upon extraction of water from the single emulsions. The concentrations of dextran and PEG in the emulsion phase are increased, leading to spontaneous phase separation. The concentrated dextran phase becomes viscous, which prevents coalescence between the inner PEG-rich droplets and the continuous PEG-rich phase. Extraction of water from the emulsion phase proceeds until phase equilibrium between the inner and continuous phases is achieved. The final diameter of the double emulsion is controlled by that of the single emulsion templates; as a result, the size uniformity of the double emulsion is also determined by that of the single emulsion templates, which can be easily manipulated in microfluidic devices. Apart from the droplet size, the structure of the double emulsion can also be controlled by varying the compositions of the dispersed phase. For instance, when the viscosity of the middle phase is raised by increasing the concentration of dextran, coalescence of the small inner droplets within the shell phase is significantly slowed down. Thus, the resultant double emulsion, within reasonable time scales, contains multiple inner droplets, rather than a single inner core. Our approach offers a new route to produce highly monodisperse all-aqueous emulsions without the use of organic solvents. This promises more biocompatible and eco-friendly formulations than most oil-based emulsions. Further challenges remain as to how these all-aqueous emulsions are stabilized, before their potentials in food production, cosmetic industry, and biomedical engineering can be fully realized. 14

15 Supporting Information Estimation on the ratio of the diameter of the inner PEG core to that of the entire w/w/w double emulsion drops. This material is available free of charge via the Internet at ACKNOWLEDGMENT This research was supported by the Seed Funding Programme for Basic Research ( ) and Small Project Funding ( ) from the University of Hong Kong. REFERENCES (1) Park, J. I.; Saffari, A.; Kumar, S.; Günther, A.; Kumacheva, E. Microfluidic Synthesis of Polymer and Inorganic Particulate Materials. Annu. Rev. Mater. Res. 2010, 40, (2) Shum, H. C.; Lee, D. I.; Yoon, K. T.; Weitz, D. A. Double Emulsion Templated Monodisperse Phospholipid Vesicles. Langmuir 2008, 24, (3) Gao, F.; Su, Z. G.; Wang, P.; Ma, G. H. Double Emulsion Templated Microcapsules with Single Hollow Cavities and Thickness-Controllable Shells. Langmuir 2009, 25, (4) Chen, C.H.; Shah, R. K.; Abate, A. R.; Weitz, D. A. Janus Particles Templated from Double 15

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17 (13) Sauret, A.; Shum, H. C. Forced generation of simple and double emulsions in all-aqueous systems. Appl. Phys. Lett. 2012, 100, , DOI: / (14) Zhao, Y.; Shum, H. C.; Chen, H.; Adams, L. L. A.; Gu Z.; Weitz, D. A. Microfluidic Generation of Multifunctional Quantum Dot Barcode Particles. J. Amer. Chem. Soc. 2011, 133, (15) Shum, H.C.; Varnell, J.; Weitz, D. A. Microfluidic fabrication of water-in-water (w/w) jets and emulsions. Biomicrofluidics 2012, 6, , DOI: / (16) Sauret, A.; Shum, H. C. Beating the jetting regime. Int. J. Nonlinear Sci. Numer. Simul. 2012, DOI: /ijnsns (17) Geschiere, S. D.; Ziemecka, I.; Steijn, V. V.; Koper, G. J. M.; Esch, J. H.; Kreutzer, M. T. Slow growth of the Rayleigh-Plateau instability in aqueous two phase systems. Biomicrofluidics 2012, 6, , DOI: / (18) Utada, A. S.; Fernandez-Nieves, A.; Gordillo, J. M.; Weitz, D. A. Absolute instability of a liquid jet in a coflowing stream. Phys. Rev. Lett. 2008, 100, (19) Umbanhowar, P. B.; Prasad, V.; Weitz, D. A. Monodisperse Emulsion Generation via Drop Break Off in a Coflowing Stream. Langmuir 2000, 16, (20) Utada, A. S.; Lorenceau, E.; Link, D. R.; Kaplan, P. D.; Stone, H. A.; Weitz, D. A. Monodisperse Double Emulsions Generated from a Microcapillary Device. Science 2005, 308,

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