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Controlled Production of Monodisperse Double Emulsions by Two-Step Droplet Breakup in Microfluidic Devices
Shingo Okushima, Takasi Nisisako,* Toru Torii, and Toshiro Higuchi
Department of Precision Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan Received August 3, 2004. In Final Form: September 15, 2004
A microfluidic device having both hydrophobic and hydrophilic components is exploited for production of multiple-phase emulsions. For producing water-in-oil-in-water (W/O/W) dispersions, aqueous droplets ruptured at the upstream hydrophobic junction are enclosed within organic droplets formed at the downstream hydrophilic junction. Droplets produced at each junction could have narrow size distributions with coefficients of variation in diameter of less than 3%. Control of the flow conditions produces variations in internal/external droplet sizes and in the internal droplet number. Both W/O/W emulsions (with two types of internal droplets) and oil-in-water-in-oil emulsions were prepared by varying geometry and wettability in microchannels.

Introduction A double emulsion (also referred to as a multiple emulsion) can be defined as a multiple-phase dispersion in which droplets enclosing finer droplets are suspended in a continuous liquid phase. Since double emulsions were first described in 1925,1 both the water-in-oil-in-water (W/ O/W) type and the oil-in-water-in-oil (O/W/O) type have attracted considerable attention because of their potential applications in food science,2 cosmetics,3 and pharmaceutics.4 Many studies have focused on pharmaceutical uses of W/O/W type emulsions, which include encapsulation of water-soluble therapeutic agents for targetable drug delivery,5-7 and preparation of biodegradable microcapsules loaded with bioactive polymers by the solvent evaporation method.8 Other applications include the extraction of hydrocarbons,9 metal ions,10 and organic acids11 across a thin liquid layer mediating between the internal drops and the external continuous phase. Of the techniques explored to date,12-14 two-stage emulsification15 is now mostly used in the practical formulation of double emulsions. This technique exploits the turbulent shear force induced by vigorous mixing so as to rupture the droplets, and the resulting internal and external droplets each have a broad size distribution.
* To whom correspondence should be addressed. Tel: (81) 3 5841 6072. Fax: (81) 3 5841 6072. E-mail: nisisako@intellect.pe.utokyo.ac.jp.
(1) Seifriz, W. J. Phys. Chem. 1925, 29, 738. (2) Matsumoto, S. J. Texture Stud. 1986, 17, 141. (3) Yoshida, K.; Sekine, T.; Matsuzaki, F.; Yanaki, T.; Yamaguchi, M. J. Am. Oil Chem. Soc. 1999, 76, 195. (4) Davis, S. S.; Walker, I. M. Methods Enzymol. 1987, 149, 51. (5) Herbert, W. J. Lancet 1965, 286, 771. (6) Engel, R. H.; Riggi, S. J.; Fahrenbach, M. J. Nature 1968, 219, 856. (7) Gresham, P. A.; Barrett, M.; Smith, S. V.; Schneider, R. Nature 1971, 234, 149. (8) Ogawa, Y.; Yamamoto, M.; Okada, H.; Yashiki, T.; Shimamoto, T. Chem. Pharm. Bull. 1988, 36, 1095. (9) Li, N. N. AIChE J. 1971, 17, 459. (10) Boyadzhiev, L.; Bezenshek, E. J. Membr. Sci. 1983, 14, 13. (11) Cerro, C. D.; Boey, D. Chem. Ind. 1988, 7, 681. (12) Kessler, D. P.; York, J. L. AIChE J. 1970, 16, 369. (13) Dokic, P.; Sherman, P. Colloid Polym. Sci. 1980, 258, 1159. (14) Goubault, C.; Pays, K.; Olea, P.; Bibette, J.; Schmitt, V.; LealCalderon, F. Langmuir 2001, 17, 5184. (15) Matsumoto, S.; Kita, Y.; Yonezawa, D. J. Colloid Interface Sci. 1976, 57, 353.

Highly uniform emulsion droplets can be generated by membrane-emulsification techniques,16-18 and these have been applied to the production of double emulsions. Higashi et al. used a porous glass membrane to prepare monodisperse W/O/W emulsions with a coefficient of variation (CV) below 10% for hepatic arterial chemotherapy.19 Sugiura et al. permeated prehomogenized water-in-oil (W/O) dispersions through arrays of microfabricated nozzles to produce W/O/W emulsions with CVs of 5-19%.20 However, no technology currently exists to control the encapsulation efficiency in double-emulsion droplets with precision. In recent years, there have been intensive studies of microfluidic techniques for producing droplets of highly uniform size.21,22 In particular, droplet preparation using micro T-junctions23-26 offers many advantages. Droplet formation is highly reproducible at low Reynolds numbers, and the resulting drops are accurately uniform in size. Also, their size is easily varied across the channel by controlling the flow conditions. Furthermore, droplet formation can be very fast at higher flow rates; in our previous study, a breakup rate of up to 2.5 103 drops/s was confirmed.27 In view of its flexibility and control capability, this technique has led to various promising applications, including a chemical reactor,28 a screening
(16) Nakashima, T.; Shimizu, M.; Kukizaki, M. Adv. Drug Delivery Rev. 2000, 45, 47. (17) Kawakatsu, T.; Kikuchi, Y.; Nakajima, M. J. Am. Oil Chem. Soc. 1997, 74, 317. (18) Kobayashi, I.; Nakajima, M.; Chun, K.; Kikuchi, Y.; Fujita, H. AIChE J. 2002, 48, 1639. (19) Higashi, S.; Setoguchi, T. Adv. Drug Delivery Rev. 2000, 45, 57. (20) Sugiura, S.; Nakajima, M.; Yamamoto, K.; Iwamoto, S.; Oda, T.; Satake, M.; Seki, M. J. Colloid Interface Sci. 2004, 270, 221. (21) Umbanhowar, P. B.; Prasad, V.; Weitz, D. A. Langmuir 2000, 16, 347. (22) Anna, S. L.; Bontoux, N.; Stone, H. A. Appl. Phys. Lett. 2003, 83, 364. (23) Thorsen, T.; Roberts, R. W.; Arnold, F. H.; Quake, S. R. Phys. Rev. Lett. 2001, 86, 4163. (24) Nisisako, T.; Torii, T.; Higuchi, T. Proceedings of the TAS 2001 Symposium; Kluwer Academic: Dordrecht, 2001; p 137. (25) Dreyfus, R.; Tabeling, P. Willaime, H. Phys. Rev. Lett. 2003, 90, 144505. (26) Tice, J. D.; Song, H.; Lyon, A. D.; Ismagilov, R. F. Langmuir 2003, 19, 9127. (27) Nisisako, T.; Torii, T.; Higuchi, T. Lab Chip 2002, 2, 24. (28) Burns, J. R.; Ramshaw, C. Lab Chip 2001, 1, 10.

10.1021/la0480336 CCC: $27.50 2004 American Chemical Society Published on Web 10/08/2004

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Table 1. T-Shaped Channel Size (Width Depth [M]) first junction one-chip module two-chip module
a

second junction 130 65 180 75 225 100 (130-220) 90

type 1 type 2 type 3

60 25 40 10 85 35a 80 40

Cross-junction.

Figure 1. Basic concept for preparing double emulsions (W/ O/W) using T-shaped microchannels.

chip,29,30 and a microfluidic device for polymeric-bead production.31,32 In this technique, the wetting of the channel has a significant effect on the type of dispersion that can be prepared. Channels fabricated on hydrophobic material are suitable for dispersing water drops in an organic phase, whereas hydrophilic channels are suited to oil-in-water dispersions.33 We report here a novel method for preparing monodisperse double emulsions using a two-step method of droplet formation in microchannel networks. For a W/O/W emulsion (Figure 1), for example, the aqueous drops to be enclosed are formed periodically upstream at the hydrophobic T-junction; then, in a continuing series, organic droplets enclosing the aqueous droplets are formed downstream at the hydrophilic T-junction. Droplets of uniform size are reproducibly formed and their size is easily varied by changing the flow conditions at the formation points. By adjusting the relation between the breakup rates at the two junctions, the number of enclosed drops can be controlled. Of the two device configurations investigated, the number of internal drops is precisely determined in a one-chip module in which the first and the second junctions are both implemented on the same chip. For situations in which flexibility is important, a two-chip module consisting of two separate chips connected serially has been developed, producing internal drops in the first chip and external drops in the second. In addition to simple W/O/W and O/W/O emulsions, a W/O/W emulsion in which the external drops include two differently colored internal drops has been prepared. Experimental Section
Microfluidic Devices. Two configurations are proposed for the production of double emulsions: a one-chip type in which the hydrophobic junction and the hydrophilic junction are on the same plate and a two-chip type in which these junctions are on separate chips. The single-chip type channels were made on Pyrex glass. Each junction was fabricated to a determined size by repeated isotropic etching; the smaller junction was for internal droplet formation and the larger for external droplets. Three types of chips were prepared for testing (Table 1). Either junction on each chip could be modified to be hydrophobic, suitable for the formation of aqueous droplets, using a silane-coupling agent (Fuji Systems Corp., Japan). Channels in the two-chip modules were fabricated on quartz glass using machining tools and on Pyrex glass by isotropic
(29) Zheng, B.; Tice, J. D.; Roach, L. S.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2004, 43, 2508. (30) Martin, K.; Henkel, T.; Baier, V.; Grodrian, A.; Scho n, T.; Roth, M.; Ko hler, J. M.; Metze, J. Lab Chip 2003, 3, 202. (31) Kawai, A.; Futami, T.; Kiriya, H.; Katayama, K.; Nishizawa, K. Proceedings of the TAS 2002 Symposium; Kluwer Academic: Dordrecht, 2002; p 368. (32) Nisisako, T.; Torii, T.; Higuchi, T. Chem. Eng. J. 2004, 101, 23. (33) Nisisako, T.; Torii, T.; Higuchi, T. IEEE Int. Conf. Micro Electro Mech. Syst., 16th 2003, 331.

etching (Table 1). The first chip, with a smaller T-junction, and the second chip, with a larger T-junction, were set up by connecting the outlet hole of the former and the inlet of the latter by a short poly(tetrafluoroethylene) (PTFE) tube (l ) 2.6 mm, 0.5 mm i.d., 1.5 mm o.d.). Either chip could be made hydrophobic for the production of aqueous drops as detailed above. Materials. Deionized water was used as the aqueous phase, and corn oil (dynamic viscosity ) 56.7 mPa s) was used as the organic phase. For the preparation of W/O/W dispersions, lecithin (0.5 wt %, Wako Pure Chemicals Co., Ltd., Japan) or tetraglycerincondensed ricinoleic acid ester (1.0 wt %, CR-310, Sakamoto Yakuhin Kogyo Co., Ltd., Japan), as lipophilic surfactant, was added to the intermediating organic phase to prevent the coalescence of internal aqueous droplets. Sodium dodecyl sulfate (SDS, Wako Pure Chemicals Co., Ltd., Japan) or decaglycerol monostearate (MSW-7S, Sakamoto Yakuhin Kogyo Co., Ltd., Japan), as hydrophilic surfactant, was added to the external aqueous phase (0.5 wt %) to stabilize the external organic droplets against coalescence. For O/W/O droplets, poly(vinyl alcohol) (PVA) and lecithin were dissolved into the intermediating aqueous phase (2.0 wt %) and the external organic phase (0.5 wt %), respectively. Equipment. Glass syringes (1000 series, Hamilton Co., NV) and syringe pumps (KDS200, KD Scientific Inc., PA) were used for sending liquids into microchannels. Droplet pictures were recorded by a high-speed video camera (Fastcam-max, Photron Limited, Japan) mounted on an optical microscope (BX-51, Olympus Optical Co. Ltd., Japan).

Results and Discussion The one-chip module (type 1) successfully produced a precisely determined drop-in-drop structure, as shown in Figure 2. For W/O/W dispersions, aqueous drops of uniform size (diameter ) 52 m, CV ) 2.7%) were periodically formed at the first hydrophobic junction and were then carried to the second hydrophilic junction, maintaining a uniform distance from each other. Each aqueous droplet was then enclosed in an organic droplet (diameter ) 83 m, CV ) 2.8%) at the downstream junction. Under these flow conditions, the breakup rates at both junctions were 22 drops/s, and the encapsulation probability was measured as 100% (n ) 100). Small adjustments in the flow rates could reproducibly produce organic drops containing two aqueous drops (Figure 2d). Small discrepancies in the two breakup rates caused a fluctuation in the number of aqueous drops enclosed in an organic droplet, in the range of 0-2, slightly increasing the polydispersity of organic droplets. Coalescence of internal aqueous drops and the external aqueous phase was also occasionally observed during the formation of organic drops. These instabilities are ascribed to the inhomogeneous viscosity of the W/O mixture supplied to the second junction. For successful encapsulation of internal drops, conditions satisfying the following equation were desirable:

R1 )N R2

(1)

where R1 is the breakup rate at the first junction, R2 is the breakup rate at the second junction, and N is a positive integer (1, 2, ...). The two-chip module could also produce monodisperse W/O/W droplets with CVs below 7%. However, even with

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Figure 2. Formation of W/O/W droplets in a one-chip module: (a) Photomicrograph of droplet formation at two consecutive T-junctions. (b) Microchannel configuration (sizes are quoted as width depth) and flow rate in each channel. Qiw ) 0.005 mL/h (internal aqueous phase), Qo ) 0.02 mL/h (organic phase), Qew ) 1.4 mL/h (external aqueous phase), Qiw/Qo/Qew ) 1:4:280. (c,d) Generated W/O/W drops flowing in the microchannel. (e) Size distributions of internal droplets and external droplets.

eq 1 satisfied, the number of internal drops could not be determined as precisely as for the single-chip module. In the first chip, aqueous drops ruptured with a definite periodicity as they flew through the microchannel in formation. This configuration was then mixed up at the connecting area, where the flow path is enlarged drastically, and the regular spacing between the aqueous drops was not recovered in the second chip. A seamless conjunction between the output channel of the first chip and the input channel of the second chip is therefore important for accurate control of encapsulation.

Figure 3a shows the relation between the organic flow rate, the size of the internal aqueous drop, and the number of enclosed drops in a one-chip module (type 2). With an increase in the organic flow speed for given conditions of internal and external aqueous streams, the size of the aqueous drops sheared at the first junction fell from 20 to 12 m with CVs of from 4.3 to 4.9%. Since the volume flow rate of the internal aqueous phase was fixed, this reduction implies an increase in the breakup rate at the first junction, increasing the average number of aqueous drops per organic drop (from 4.7 to 11.9). Even when eq 1 is nearly satisfied, there is significant variation in the number of internal drops, shown as the large error bars in the figure. We observed that a droplet array having a regular sequence was disordered at the point between the two junctions at which the channel is suddenly enlarged. The ratio of the hydraulic diameters at the junctions in this module is 6.4, which is larger than in the type 1 module (2.4). Thus, even in a one-chip type module, and certainly in two-chip modules, a smoother change in the channel size is important in the precision preparation of a double emulsion. Figure 3b shows the effect of the external aqueous flow rate on the size of organic drops and the number of enclosed aqueous drops (diameter ) 45 m) in a two-chip module. With increasing external aqueous flow speed, the breakup rate of organic drops at the second junction increases, and their diameter fell from 220 to 95 m with CVs of from 2.6 to 7.1%. This causes a decrease in the number of enclosed aqueous drops (from 4.0 to 1.2). The size of W/O/W droplets, both internal droplets and external droplets, can therefore be varied by controlling the flow conditions. The number of internal droplets can also be adjusted by changing the breakup rates at the two junctions, the internal drop size, and the external drop size. Various double emulsions can be produced by changing the wetting and geometrical properties of the microchannels. An example is the production of an O/W/O dispersion by arranging a hydrophilic junction upstream of a hydrophobic junction (Figure 4a). This may be useful for the fabrication of uniform three-dimensional colloidal assemblies packed in aqueous droplets.34 By magnifying the difference in channel sizes of the upstream and the downstream junctions, smaller aqueous drops were densely packed in organic drops for higher encapsulation efficiency (Figure 4b). Furthermore, an interesting double emulsion, in which two differently colored (blue or red) aqueous drops were enclosed in an organic droplet, was prepared by using a cross-junction structure as the upstream junction. Red drops and blue drops of similar size were alternately and periodically formed at the cross-junction (Figure 4c,d); they then flew to the downstream T-junction (Figure 4e) to be jointly encapsulated in organic droplets (Figure 4f). This outcome indicates that multiple components can be confined as discrete vesicles in any desired volume fraction within pico/nanoliter-sized drops. A biological screening in a microfluidic system has recently been performed using pairs of microdrops with different compositions.29 We believe our method may provide a useful way for similar applications. No other technology currently exists for creating such double emulsions, so this microfluidic approach may generate new applications of multiple-phase dispersion systems. The one-chip module and the two-chip module described above each have distinct advantages and drawbacks. Compared to the two-chip module with joint parts, the
(34) Yi, G. R.; Thorsen, T.; Manoharan, V. N.; Hwang, M. J.; Jeon, S. J.; Pine, D. J.; Quake, S. R.; Yang, S. M. Adv. Mater. 2003, 15, 1300.

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Figure 3. Effect of flow conditions on the formation of W/O/W droplets: (a) Effect of the organic phase flow condition on the size (n ) 20) and the number (n ) 20) of internal aqueous droplets in a one-chip module, Dh1 ) Dh2 ) 16 m. (b) Effect of the external aqueous stream on the size of external organic droplets (n ) 30) and the number of internal aqueous droplets (n ) 50) in a two-chip module, Dh3 ) 106 m, Dh4 ) 128 m.

Figure 4. Various double emulsions: (a) Formation of aqueous droplets enclosing organic droplets at a hydrophobic T-junction. The diameters of internal and external droplets were 68 and 170 m, respectively. (b) Formation of organic droplets containing many aqueous droplets. (c,d) Alternate formation of red and blue aqueous drops at a cross-junction. (e) Red and blue drops flowing between the first junction and the second junction. (f) Organic droplets enclosing blue and red aqueous drops. The diameter of the external drop is 175 m.

one-chip module with microfabrication has a smoother connection between the two T-junctions. The one-chip module is therefore better suited to the precise determination of the number of internal vesicles. For the twochip module, strict control of the number of internal drops is difficult because the array of drops to be enclosed that forms in the upstream module tends to be disordered at the connecting area. The two-chip concept nevertheless has significant advantages. Since the chip combination is changed, microchannels having different dimensions, geometries, and wetting scenarios can readily be combined to produce diverse double emulsions on demand. A further advantage over the one-chip device is that surface modification is easier. This is because channels in either chip can be modified extensively, unlike the partial modification in the one-chip module. Also, surface treatment is unnecessary when intrinsically hydrophobic and hydrophilic materials, such as poly(dimethylsiloxisane) (PDMS) and glass are coupled together. These advantages of easy surface modification would be most important in the fabrication of integrated multichannels for scaling up the productivity of emulsions. Conclusion We have demonstrated the production of double emulsions using microfluidic devices having a hydrophobic

junction and a hydrophilic junction positioned serially. Droplet formation at each junction is highly reproducible, and the resulting drops have a very sharp distribution of sizes. The size of the droplets and the number of internal droplets can be varied by changing the flow conditions. Both W/O/W and O/W/O emulsions were prepared by interchanging the hydrophobic and hydrophilic components. Also, double emulsions, including internal droplets of two colors, were prepared using a cross-junction as the upstream component. The one-chip concept is better for precise control of encapsulation, and the two-chip approach gives greater flexibility of configuration for generating diverse double emulsions. We believe that our system has the potential for use in various fields, including the analysis of confined chemical reactions, biological screening, drug delivery systems, and control of particle morphology. It is particularly valuable where accuracy of the volume fraction is important within subnanoliter vesicles.

Acknowledgment. This work was supported by a grant from the Research Association of Micro Chemical Process Technology (MCPT) of Japan.
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