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W) double emulsion preparation
Sensors and Actuators B 215 (2015) 330–336
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Spatial wettability patterning of glass microchips for water-in-oil-in-water (W/O/W) double emulsion preparation Zeqing Bai, Bo Wang, Hengwu Chen, Min Wang ∗ Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou 310058, China
a r t i c l e
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Article history: Received 17 October 2014 Received in revised form 15 March 2015 Accepted 30 March 2015 Available online 9 April 2015 Keywords: Double emulsions Wettability patterning Hydrophilic Hydrophobic Glass microchips
a b s t r a c t Double emulsions are of great potential in many applications, including cosmetics, foods, and drug delivery. However, current preparation methods for double emulsions are not able to satisfactorily control emulsion size and structure. Microfluidic techniques open up new opportunities for this field but challenges still remain. This article presents a partially hydrophilic and partially hydrophobic glass microchip for generating water-in-oil-in-water (W/O/W) double emulsions. The device was first hydrophobized by forming an octadecyltrichlorosilane layer through self-assembly on the entire microchannel surface. Then, a hydrophilic surface was obtained by spatially photodegrading the hydrophobic organic layer with a deep UV-light source and a photomask. Thus, a distinctive wettability contrast was achieved on different parts of the microchannel surface. By optimizing the UV irradiation time, W/O/W double emulsions were successfully created on the microchip. Double emulsions prepared by this device were monodisperse and had narrow size distribution with coefficient of variation values less than 8%. Moreover, both the size and the number of inner droplets could be controlled by adjusting the flow rates. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Double emulsions, or “emulsions within emulsions”, are emulsions with smaller droplets encapsulated in larger droplets. Water-in-oil-in-water (W/O/W, water droplets in oil organic shell dispersed in a continuous aqueous phase) and oil-in-water-in-oil (O/W/O, oil drops in an aqueous phase shell suspended in a continuous oil phase) are two typical examples of double emulsions. Here the intermediate phase acts as a barrier that separates the inner fluid from the outer fluid, which makes double emulsions of great potential for controlled release of substances. Active substances such as drugs [1], foods [2] and cosmetics [3] have been encapsulated in the inner fluid for sustained release or targetable delivery. Other applications of double emulsions include chemical separations [4], preparation of microcapsules [5] and formation of liposomes [6]. The two-step emulsification method [7] and membraneemulsification technique [8] are conventional ways of making double emulsion in bulk solutions. They suffer from either producing polydisperse emulsions or losing control of the volume and
∗ Corresponding author at: Zhejiang University, Institute of Microanalytical Systems, Department of Chemistry, 102 Chem Lab Center, Hangzhou 310058, China. Tel.: +86 571 88206772; fax: +86 571 88273572. E-mail address: [email protected] (M. Wang). http://dx.doi.org/10.1016/j.snb.2015.03.072 0925-4005/© 2015 Elsevier B.V. All rights reserved.
the number of embedded droplets. Microfluidic techniques offer an alternative way to overcome these shortages. A microcapillary device consisted of two cylindrical glass capillary tubes nested within a square glass tube was designed by Weitz’s group [9–11] to produce double emulsions with high monodispersity, narrow size distribution and well controlled number of the inner droplets. However, fabrication of the glass device requires precise alignment of three microcapillary tubes to form a coaxial geometry, which is labor intensive and experience dependent. Microfluidic chips are favored by researchers in preparing double emulsions. To form a double emulsion in microfluidic chips, microchannels consist of serial hydrophilic and hydrophobic sections are necessary. Two strategies can be applied to realize it. One is two-chip module [12–14], where two chips with microchannels possessing different wettabilities are combined together by an external connector. For this strategy, flow disturbances might happen at the connecting area which would result in uncertain number of embedded inner droplets [15]. The other strategy is one-chip module, where the channels of one chip were differently wettability patterned with appropriate chemistry. Double emulsions made by this strategy share the characteristics of high monodispersity, narrow size distribution and well controlled morphology. Nevertheless, there has been limited number of reports about this method, mainly due to difficulties in spatial patterning of micro channel wettability. The flow confinement method is a good option to overcome these difficulties. Nisisako et al. [16] first
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hydrophobized the entire glass channels via the formation of a silane hydrophobic layer. Then a sodium hydroxide solution (2 mol L−1 ) was infused from an inlet to remove the hydrophobic layer while an immiscible inert solution was introduced from other inlets to protect the confined sections from being hydrophilized. In Abate et al.’s works [17,18], a laminar flow consisted of an inert solution and a reactive solution containing poly(acrylic acid) (PAA) was formed in a cross-shaped PDMS channels which were previously coated with fluorosilane functionalized sol–gel. Under UV irradiation the PAA was grafted to the surface via polymerization, turning the reactive solution occupied area hydrophilic while keeping other area hydrophobic. Corona discharge was applied by Davies et al. [19] to spatially hydrophilize the surface of PDMS microchannels. An oil-blocking technique was employed by them to selectively prevent the corona arc from hydrophilizing the entire channels. Using similar strategy, Bauer et al. [20] and Garstecki et al. [21] also successfully obtained partially hydrophilic and partially hydrophobic microfluidic devices for the generation of W/O/W double emulsions. The flow confinement method is versatile considering that various surface treatments can be used, but challenges arise when more complicated channel networks are required. Besides, it is difficult to keep the interface between the reactive solution and inert solution steady in a microchannel. Another approach for spatially patterning wettability is using polymerization reaction initiated by UV light through photomask. The resolution can be controlled by a spatial UV-transmitting photomask. Abate et al. [22] coated PDMS devices with a sol–gel layer that functionalized with fluorinate and photoreactive silanes. Upon UV irradiation, PAA was grafted to UV-transmitting area. Hu et al. [23] employed benzophenone as photoinitiator to directly graft PAA onto PDMS channels with a spatial resolution of about 100 m. Their method was later improved by Schneider et al. [24] to spatially pattern the wettability of PDMS microchannels for producing W/O/W double emulsions, but the polymerization reaction was strongly influenced by a lot of parameters according to their report. Besides, the above PDMS chips’ incompatibility with some organic solvents is another problem to be considered. Localized surface modification of glass microchips for double emulsification by silanization and UV photodegradation through a mask was reported by Torii’s group [25,26]. According to their work, the UV photodegradation efficiency was related to surface temperature of microchannel. Therefore, a heater was employed to reduce irradiation time for degradation. In this paper, a simple method for preparing double emulsions in glass microchips is presented. The glass microchannels were first hydrophobized via formation of a self-assembly layer of octadecyltrichlorosilane, followed by spatial photodegradation of the layer by exposing to UV/O3 with a photomask. With the obtained partially hydrophilic and partially hydrophobic device, presenting high wettability contrast, monodisperse W/O/W double emulsions were successfully prepared with easy control of the size and the number of the inner droplets as well.
2. Experimental
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phase. Span 80, as surfactant was added to the oil phase to stabilize the emulsions. 2.2. Apparatus A PL16-110 photo surface processor (Sen Lights Corporation, Osaka, Japan) equipped with a built-in UV lamp (SUV110GS-36 Synthetic Quartz Lamp), which generates 185 nm and 254 nm light, was employed to photodegrade the octadecyltrichlorosilane selfassembly layer. The 185 nm light can transform oxygen into ozone. Syringe pumps (KDS100, KD Scientific Inc., PA) were used for pumping liquids into microchannels. Droplet images were recorded by a CCD camera mounted on an optical microscope (XTL20C, Puda Optical Instrument Co., Ltd, Shanghai, China). 2.3. Device fabrication The microchannel design is shown in Fig. 1. Standard UV photolithography, wet chemical etching [27] and room temperature bonding [28] techniques were employed to fabricate the glass microchip. Briefly, the layout of the channel network was created on a PET transparency using a high resolution laserprinter, and subsequently transferred onto a chromium and AZ1805 photoresist-precoated soda-lime glass plate (20 mm × 60 mm × 1.6 mm, Shaoguang Microeletronics Corp., Changsha, China) by UV exposure. The microchannels were etched into the plate in a well-stirred, dilute HF-NH4 F bath kept at 40 ◦ C for about 35 min. Four 1-mm-diameter access holes were drilled on the etched plate at the sites as illustrated in Fig. 1 using an emery drill-bit. A quartz glass plate (20 mm × 60 mm × 2 mm, Shanghai Xinhaotian Optical & Electric Materials Co., Shanghai, China) was used as the cover to bond with the etched soda-lime glass plate. The two plates were aligned and brought into close contact under a stream of running tap-water flowing between them. Afterwards, the combined plates were left at room temperature for 2 days to be firmly bonded together. 2.4. Wettability patterning Wettability patterning of the microchannels was carried out according to an alkylsilane self-assembling and UVphotodegrading process which was previously reported for paper-based microfluidic devices and hybrid soda-lime/quartz glass chips [29,30]. Briefly, all the channels of the glass chip in Fig. 2a were filled with a 0.5% (v/v) octadecyltrichlorosilane solution in n-hexane for 30 min. Then ethanol was introduced to remove the solvent and unreacted reagent. Nitrogen from a nitrogen gas cylinder was infused into the microchip via a piece of plastic tube (2 m × 5 mm o.d. × 3 mm i.d.) to dry the hydrophobic channels (Fig. 2b). Afterwards, a photomask was placed on the quartz-glass side of the microchip, as shown in Fig. 2c. The device was put into the chamber of the photo surface processor, 3 cm underneath the UV-lamp where the intensity of the UV light was 35 mW cm−2 measured at 254 nm. The processor was then turned on and different irradiation times were studied.
2.1. Chemicals and reagents 2.5. Preparation of W/O/W double emulsions All chemicals used were of analytical grade unless otherwise stated and deionized water from a Milli-Q water purification system (Millipore, Bedford, MA) was used throughout. Octadecyltrichlorosilane (OTS, 95%) was purchased from Acros Organics (Springfield, NJ, USA), n-hexane, span 80 and mineral oil were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). For preparing W/O/W double emulsions, deionized water was used as the inner and outer phases, while mineral oil the intermediate
The above wettability patterned microchip was used to prepare W/O/W double emulsions. The inner aqueous phase, intermediate mineral oil and outer aqueous phase were introduced from Inlet 1, Inlet 2 and Inlet 3, respectively. These three fluids were loaded in three 1-mL PVC disposable syringes and driven with three syringe pumps. Three pieces of 15 cm × 1 mm o.d. × 0.3 mm i.d. PTFE tubing were used to connect the disposable syringes and microchip inlets.
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Fig. 1. Schematic diagram of the microchannel network (not to scale). The T-junction was the first droplet maker and the decussation was the second droplet maker. Inlet 1, Inlet 2 and Inlet 3 were for introducing the inner phase, intermediate phase and outer phase, each. All the channels had a depth of about 50 m. The narrow channels had a top-width of about 150 m and other channels had a top width of about 300 m.
Flow rates were adjusted and optimized to control the formation of double emulsions.
3. Results and discussion 3.1. Spatial wettability patterning of microchip channel Microfluidic two-phase system has attracted great interests from microfluidic community in the last decade. To the downscale of tens of micrometers, the wettability of channel surface has a significant effect on the behavior of two immiscible fluids. For example, when two immiscible phases are introduced into a hydrophobic microchannel through a T-junction, the oil phase would wet the hydrophobic surface and segment the water phase, resulting in forming water in oil (W/O) droplets. Therefore, hydrophobic microchannels are suitable for preparing W/O droplets, whereas hydrophilic microchannels are suited to O/W droplets [31]. If the W/O droplets are again dispersed in another aqueous phase, W/O/W double emulsions will be formed. This requires that the hydrophobic channel be followed by a hydrophilic channel. The present work was intended to prepare W/O/W double emulsions in a glass microchip. Thus, wettability patterning of the device to be partially hydrophobic and partially hydrophilic is a must. The designed microchannel network was shown in Fig. 1 and it was purposefully wettability patterned to the status as shown in Fig. 2d. Alkylsilane self-assembling and UV-photodegrading strategies were combined to achieve this objective. Although this method has been utilized to pattern the wettability of paper surfaces and glass surfaces [29,30], it was not for double emulsion preparation.
In this work, a 0.5% (v/v) octadecyltrichlorosilane solution in nhexane was first introduced into the glass channels to form a hydrophobic self-assembly layer (with long alkyl chain) on the entire surface. Then, the hydrophobic microchip (with the quartz side on the top) was spatially covered by a photomask as shown in Fig. 2c, with the cross-shaped microchannel and photomask aligned (Fig. S1), followed by exposure to UV/O3 without being heated. The deep UV light combined with ozone degraded the hydrophobic self-assembly layer to form polar oxygen-containing moieties such as ketones, aldehydes and carboxylates [32], turning the surface wettability from hydrophobic to hydrophilic. Therefore, the channels covered by the photomask remained hydrophobic whereas the UV light exposed channels turned hydrophilic. The wettability patterned effect is later demonstrated in the double emulsion formation process.
3.2. Preparation of W/O/W double emulsions With such a wettability patterned microchip, attempts were made to prepare W/O/W double emulsions. The red color aqueous solution (containing 2 wt% food dye) and transparent mineral oil (containing 5 wt% Span 80) were introduced into the channel network from Inlet 1 and Inlet 2 respectively. As shown in Fig. 3a, the oil phase wetted the channel surface and cut the aqueous phase into W/O segments (with a length of about 600 m), indicating that this section of channel is hydrophobic. When the W/O segments moved to the enlarged channel (Fig. 3b), the red color segments turned into oval (with a size of about 400 m), which facilitated the W/O droplets to be enclosed by the outer aqueous solution. Meanwhile, a green color aqueous solution (containing 2 wt% food
Fig. 2. Schematic diagrams of the wettability patterning processes. (a) The native glass microchip (60 mm × 20 mm × 3.7 mm), (b) a hydrophobic self-assembly layer of octadecyltrichlorosilane formed on the channel surface, (c) the T-junction covered with a photomask, (d) after the channel network shown in (c) was exposed to UV/O3 for 60 min. The black color and gray color represent hydrophilic region and hydrophobic region, respectively.
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Fig. 3. Photomicrographs of W/O/W double emulsion forming process in the microchannel. (a) columnar W/O segments formed at the T-junction, (b) the columnar W/O segments turn into oval after entering the enlarged channel, (c) the oval W/O droplets was about to be enclosed by the outer phase and (d) the generated W/O/W double emulsions. Three fluids involved here were mineral oil containing 5 wt% Span 80 (transparent), aqueous red dye solution (red color) and aqueous green dye solution (green color). (e) Size distributions of inner water droplets (n = 50) and oil droplets (n = 50). (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)
dye) was introduced from Inlet 3. After reaching the decussation, it wetted the downstream channel and cut the continuous oil phase containing oval aqueous segments into segments (Fig. 3c), resulting in the formation of W/O/W double emulsions (Fig. 3d). It indicates that the decussation downstream channel surface was hydrophilic as well. The double emulsions forming process can be found in Supporting Information, Video S1. The inner water droplets tended to be leaned at the left side of the oil droplets, because they were carried by the oil phase to move forward. Their size distributions are shown in Fig. 3e, the low values of CVs indicated that the droplets are uniform in the channel. 3.3. Optimization of UV/O3 exposing time The wettability difference between the two sections of channel strongly influences the formation of double emulsions. It is still unknown how large the wettability difference should be to produce double emulsions. According to our previous report [30], water contact angle of a planar glass surface after octadecyltrichlorosilane treatment was about 108◦ and it decreased to about 60◦ , 30◦ and 15◦ after exposing to UV/O3 for 30 min, 60 min and 90 min, each. Four microchips prepared as described in Section 2.4 were exposed to UV/O3 for 0 min, 30 min, 60 min and 90 min to study the wettability contrast on the forming of double emulsions. For irradiation times of 0 min and 30 min, the outer aqueous did not wet the decussation downstream channel surface, resulting in the failure of preparing double emulsions. More details are described in Fig. S2. When the irradiation times increased to 60 min and 90 min, W/O/W double emulsions were formed as described in Fig. 3. This indicates that a wettability contrast of about 78◦ (water contact angle) is enough for
double emulsions formation. To speed up the wettability patterning process, 60 min exposure to UV/O3 was adopted in later experiments. Additionally, the wettability patterned device was durable. After being filled with water for 1 month it still performed well in making W/O/W double emulsions. 3.4. Optimization of surfactant The formation of W/O/W double emulsions also was affected by the concentration of added surfactant. Span 80 was added to mineral oil with concentration of 0 wt%, 0.5 wt%, 1 wt%, 2 wt% and 5 wt% to find the appropriate condition for producing double emulsions. It was found that Span 80 with concentration less than 5 wt% was not enough to stabilize the water in oil droplet at the channel decussation, resulting in a leakage of the water phase. With Span 80 concentration being increased to 5 wt%, no fracture was observed, and the W/O droplets were cut by the outer water phase for the second emulsification. The generated W/O/W double emulsions were quite stable in the microchannel, keeping their shape well when moving downstream, but tended to be coalesced after leaving the microchannel (Supporting Information, Video S2). It is reported [33] that the stability of double emulsions has a close relationship with interfacial tension, which can be adjusted by adding surfactant. Considering the coalescence happened between the oil phase, Tween 20 was added to the outer water phase to stabilize the W/O/W double emulsion. The coalescence of oil phase still happened when the concentration of Tween 20 was increased from 0 wt%, 0.5 wt%, 1 wt%, 2 wt% up to 5 wt%. Attempts have also been made to replace the mineral oil by fluorocarbon oil, n-octanol, oleic acid and liquid paraffin as oil phase, but all failed in producing
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Fig. 4. Photomicrographs of the prepared W/O/W double emulsions with different inner droplet sizes. The flow rate of the inner phase (Q1 ) was: (a) 0.2 L min−1 , (b) 0.3 L min−1 , (c) 0.4 L min−1 , (d) 0.5 L min−1 , (e) 0.6 L min−1 and (f) 0.7 L min−1 . The flow rates of intermediate phase (Q2 ) and outer phase (Q3 ) were fixed at 0.5 L min−1 and 5.0 L min−1 in each photomicrograph. (g) Effect of Q1 on the sizes and cutting frequencies of inner and outer droplets (n = 50).
W/O/W double emulsion off-chip. More efforts need to be carried out to increase the stability of the off chip double emulsion in future work. 3.5. The controllability of inner droplets Controlling the size and the number of the inner droplets are both important in applications of double emulsions. For example, when the inner droplets are used for cell culture, their size determines the number of cells that can be enclosed. And the number of inner droplets is critical for structuring 3D colloidal assemblies having spherical shape [34]. To realize such controls, adjusting the inner, middle and outer fluid flow rates (Q1 , Q2 , and Q3 , respectively) is a good way, because the W/O/W double emulsions are consisted of these fluids. 3.5.1. Size control The sizes of the inner droplets could be controlled by adjusting Q1 . In the experiment, the values of Q2 and Q3 were fixed at 0.5 L min−1 and 5.0 L min−1 with Q1 increased from 0.2 L min−1 to 0.7 L min−1 . The obtained W/O/W double emulsions under different flow rate conditions were shown in Fig. 4 (a–f), and the size information of inner droplets (measured after the second junction) was listed in Table 1. It can be seen that the sizes of the inner droplets increased with the increase of Q1 , and all the CVs were less than 8%, which demonstrating that they are uniform in each case. The controllable inner droplets size in the current device ranges
from about 250 m to 630 m. The data of Q1 vs. sizes of inner and outer droplets in Fig. 4 (a–f) is summarized in Fig. 4g. According to the black scatters, the increase of Q1 drastically increased the sizes of inner droplets but did not show great impact on the sizes of outer droplets. This observation implies an increase in the cutting frequencies of outer droplets. Therefore, the cutting frequencies of inner and outer droplets were measured and also shown in Fig. 4g. According to the red scatters, as the flow rate of the inner phase was increased, the cutting frequencies of inner and outer droplets both increased. The results agreed well with our inference. 3.5.2. Number control In such a system, the length of the intermediate oil phase was determined by the cutting frequency of the outer aqueous phase. Therefore, longer oil segments could be obtained when the outer phase flow rate was decreased. The longer the oil segments, the more inner droplets they could enclose. Here the number of the Table 1 Size distribution of inner droplets produced at various Q1 , with Q2 and Q3 fixed at 0.5 L min−1 and 5.0 L min−1 . Q1 (L min−1 )
Average size (m)
CV
0.2 0.3 0.4 0.5 0.6 0.7
243 302 376 453 558 632
7.5% 7.6% 6.7% 5.5% 5.8% 7.6%
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Fig. 5. Photomicrographs of the prepared W/O/W double emulsions with different inner droplet numbers. The flow rate of the outer phase (Q3 ) was: (a) 4.0 L min−1 , (b) 2.5 L min−1 and (c) 1.0 L min−1 . Q1 and Q2 were both fixed at 0.5 L min−1 in each photomicrograph.
inner droplets was controlled by adjusting Q3 . With Q1 and Q2 values fixed at 0.5 L min−1 and 0.5 L min−1 , Q3 was decreased from 5.0 L min−1 to 4.0 L min−1 , 2.5 L min−1 and 1.0 L min−1 . In Fig. 5a, Q3 was adjusted to 4.0 L min−1 , resulting in increasing the length of the oil segments by about 30% compared with that in Fig. 4d (Q3 = 5.0 L min−1 ). But there was just one inner droplet in the double emulsions. Fig. 5b and c display the obtained two inner droplets and three inner droplets containing W/O/W double emulsions. When Q3 = 2.5 L min−1 , the length of the intermediate phase was about twice of that in Fig. 4d. Two inner droplets were enclosed by the intermediate oil phase under this condition. When Q3 = 1.0 L min−1 , the length of the intermediate phase was about triple of that in Fig. 4d and the number of the inner droplets increased to three. It was difficult to prepare W/O/W double emulsions containing more than three inner droplets in such a device, because as Q3 decreases to values lower than 1.0 L min−1 the outer aqueous phase could not cut the intermediate oil phase into segments any more. 4. Conclusions We have demonstrated preparation of highly controllable W/O/W double emulsions by using glass microchips that was wettability patterned to be partially hydrophobic and partially hydrophilic. The method was simple and straightforward compared with other reported strategies. The wettability contrast needed to produce double emulsions in the microchip was about 78◦ when expressed by water contact angle. The size of the inner droplets could be controlled to some extent and the number of the inner droplets that could be produced was no more than 3. The high degree of controllability provided by the device makes it promising in biomedical science. For example, by changing the intermediate oil phase, the prepared W/O/W double emulsions can be used for constructing lipid vesicles, simulating cellular microenvironment and producing microcapsules. Acknowledgments This project is sponsored by Zhejiang Provincial Natural Science Foundation under Grant No. Z4110019, and Natural Science Foundation of China under Grant No. 21435004. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.03.072 References [1] D. Vasiljevic, J. Parojcic, M. Primorac, G. Vuleta, An investigation into the characteristics and drug release properties of multiple W/O/W emulsion systems containing low concentration of lipophilic polymeric emulsifier, Int. J. Pharm. 309 (2006) 171–177. [2] J. Weiss, I. Scherze, G. Muschiolik, Polysaccharide gel with multiple emulsion, Food Hydrocolloids 19 (2005) 605–615.
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Biographies Zeqing Bai is a Ph.D. student in the Department of Chemistry at Zhejiang University. He received his B.S. in chemistry from Xiamen University in 2010. His research interests include microfluidics and wormometry-on-a-chip. Bo Wang is an M.S. student in the Department of Chemistry at Zhejiang University. She received her B.S. in Chemistry from Zhejiang Normal University in 2012. Her research interests include electrochemical cell analysis and paper-based microfluidics. Hengwu Chen received Ph.D. in analytical chemistry from Shenyang Pharmaceutical University, China in 1998. Presently, he is a Professor working in the Department of Chemistry, Zhejiang University, China. His current research interest mainly includes flow-injection analysis, microfluidic chips with electrochemical or fluorescent detection for bio-analysis and pharmaceutical analysis. Min Wang is currently a Professor in the Department of Chemistry at Zhejiang University. She received her B.S. in chemistry from Peking University in 1997, and her Ph.D. in chemistry from the Ohio State University in 2002. She worked as a postdoctoral research associate in the Department of Biomolecular and Chemical Engineering in the University of Illinois at Urbana-Champaign 2002–2003. She is a visiting scholar in the Department of Chemistry and Biochemistry of University of California at San Diego 2014–2015. Her research interests include nanomaterials, microfluidics, and chemical sensors.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Spatial wettability patterning of glass microchips for water-in-oil-in-water (W/O/W) double emulsion preparation Zeqing Bai, Bo Wang, Hengwu Chen, Min Wang ∗ Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou 310058, China
a r t i c l e
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Article history: Received 17 October 2014 Received in revised form 15 March 2015 Accepted 30 March 2015 Available online 9 April 2015 Keywords: Double emulsions Wettability patterning Hydrophilic Hydrophobic Glass microchips
a b s t r a c t Double emulsions are of great potential in many applications, including cosmetics, foods, and drug delivery. However, current preparation methods for double emulsions are not able to satisfactorily control emulsion size and structure. Microfluidic techniques open up new opportunities for this field but challenges still remain. This article presents a partially hydrophilic and partially hydrophobic glass microchip for generating water-in-oil-in-water (W/O/W) double emulsions. The device was first hydrophobized by forming an octadecyltrichlorosilane layer through self-assembly on the entire microchannel surface. Then, a hydrophilic surface was obtained by spatially photodegrading the hydrophobic organic layer with a deep UV-light source and a photomask. Thus, a distinctive wettability contrast was achieved on different parts of the microchannel surface. By optimizing the UV irradiation time, W/O/W double emulsions were successfully created on the microchip. Double emulsions prepared by this device were monodisperse and had narrow size distribution with coefficient of variation values less than 8%. Moreover, both the size and the number of inner droplets could be controlled by adjusting the flow rates. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Double emulsions, or “emulsions within emulsions”, are emulsions with smaller droplets encapsulated in larger droplets. Water-in-oil-in-water (W/O/W, water droplets in oil organic shell dispersed in a continuous aqueous phase) and oil-in-water-in-oil (O/W/O, oil drops in an aqueous phase shell suspended in a continuous oil phase) are two typical examples of double emulsions. Here the intermediate phase acts as a barrier that separates the inner fluid from the outer fluid, which makes double emulsions of great potential for controlled release of substances. Active substances such as drugs [1], foods [2] and cosmetics [3] have been encapsulated in the inner fluid for sustained release or targetable delivery. Other applications of double emulsions include chemical separations [4], preparation of microcapsules [5] and formation of liposomes [6]. The two-step emulsification method [7] and membraneemulsification technique [8] are conventional ways of making double emulsion in bulk solutions. They suffer from either producing polydisperse emulsions or losing control of the volume and
∗ Corresponding author at: Zhejiang University, Institute of Microanalytical Systems, Department of Chemistry, 102 Chem Lab Center, Hangzhou 310058, China. Tel.: +86 571 88206772; fax: +86 571 88273572. E-mail address: [email protected] (M. Wang). http://dx.doi.org/10.1016/j.snb.2015.03.072 0925-4005/© 2015 Elsevier B.V. All rights reserved.
the number of embedded droplets. Microfluidic techniques offer an alternative way to overcome these shortages. A microcapillary device consisted of two cylindrical glass capillary tubes nested within a square glass tube was designed by Weitz’s group [9–11] to produce double emulsions with high monodispersity, narrow size distribution and well controlled number of the inner droplets. However, fabrication of the glass device requires precise alignment of three microcapillary tubes to form a coaxial geometry, which is labor intensive and experience dependent. Microfluidic chips are favored by researchers in preparing double emulsions. To form a double emulsion in microfluidic chips, microchannels consist of serial hydrophilic and hydrophobic sections are necessary. Two strategies can be applied to realize it. One is two-chip module [12–14], where two chips with microchannels possessing different wettabilities are combined together by an external connector. For this strategy, flow disturbances might happen at the connecting area which would result in uncertain number of embedded inner droplets [15]. The other strategy is one-chip module, where the channels of one chip were differently wettability patterned with appropriate chemistry. Double emulsions made by this strategy share the characteristics of high monodispersity, narrow size distribution and well controlled morphology. Nevertheless, there has been limited number of reports about this method, mainly due to difficulties in spatial patterning of micro channel wettability. The flow confinement method is a good option to overcome these difficulties. Nisisako et al. [16] first
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hydrophobized the entire glass channels via the formation of a silane hydrophobic layer. Then a sodium hydroxide solution (2 mol L−1 ) was infused from an inlet to remove the hydrophobic layer while an immiscible inert solution was introduced from other inlets to protect the confined sections from being hydrophilized. In Abate et al.’s works [17,18], a laminar flow consisted of an inert solution and a reactive solution containing poly(acrylic acid) (PAA) was formed in a cross-shaped PDMS channels which were previously coated with fluorosilane functionalized sol–gel. Under UV irradiation the PAA was grafted to the surface via polymerization, turning the reactive solution occupied area hydrophilic while keeping other area hydrophobic. Corona discharge was applied by Davies et al. [19] to spatially hydrophilize the surface of PDMS microchannels. An oil-blocking technique was employed by them to selectively prevent the corona arc from hydrophilizing the entire channels. Using similar strategy, Bauer et al. [20] and Garstecki et al. [21] also successfully obtained partially hydrophilic and partially hydrophobic microfluidic devices for the generation of W/O/W double emulsions. The flow confinement method is versatile considering that various surface treatments can be used, but challenges arise when more complicated channel networks are required. Besides, it is difficult to keep the interface between the reactive solution and inert solution steady in a microchannel. Another approach for spatially patterning wettability is using polymerization reaction initiated by UV light through photomask. The resolution can be controlled by a spatial UV-transmitting photomask. Abate et al. [22] coated PDMS devices with a sol–gel layer that functionalized with fluorinate and photoreactive silanes. Upon UV irradiation, PAA was grafted to UV-transmitting area. Hu et al. [23] employed benzophenone as photoinitiator to directly graft PAA onto PDMS channels with a spatial resolution of about 100 m. Their method was later improved by Schneider et al. [24] to spatially pattern the wettability of PDMS microchannels for producing W/O/W double emulsions, but the polymerization reaction was strongly influenced by a lot of parameters according to their report. Besides, the above PDMS chips’ incompatibility with some organic solvents is another problem to be considered. Localized surface modification of glass microchips for double emulsification by silanization and UV photodegradation through a mask was reported by Torii’s group [25,26]. According to their work, the UV photodegradation efficiency was related to surface temperature of microchannel. Therefore, a heater was employed to reduce irradiation time for degradation. In this paper, a simple method for preparing double emulsions in glass microchips is presented. The glass microchannels were first hydrophobized via formation of a self-assembly layer of octadecyltrichlorosilane, followed by spatial photodegradation of the layer by exposing to UV/O3 with a photomask. With the obtained partially hydrophilic and partially hydrophobic device, presenting high wettability contrast, monodisperse W/O/W double emulsions were successfully prepared with easy control of the size and the number of the inner droplets as well.
2. Experimental
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phase. Span 80, as surfactant was added to the oil phase to stabilize the emulsions. 2.2. Apparatus A PL16-110 photo surface processor (Sen Lights Corporation, Osaka, Japan) equipped with a built-in UV lamp (SUV110GS-36 Synthetic Quartz Lamp), which generates 185 nm and 254 nm light, was employed to photodegrade the octadecyltrichlorosilane selfassembly layer. The 185 nm light can transform oxygen into ozone. Syringe pumps (KDS100, KD Scientific Inc., PA) were used for pumping liquids into microchannels. Droplet images were recorded by a CCD camera mounted on an optical microscope (XTL20C, Puda Optical Instrument Co., Ltd, Shanghai, China). 2.3. Device fabrication The microchannel design is shown in Fig. 1. Standard UV photolithography, wet chemical etching [27] and room temperature bonding [28] techniques were employed to fabricate the glass microchip. Briefly, the layout of the channel network was created on a PET transparency using a high resolution laserprinter, and subsequently transferred onto a chromium and AZ1805 photoresist-precoated soda-lime glass plate (20 mm × 60 mm × 1.6 mm, Shaoguang Microeletronics Corp., Changsha, China) by UV exposure. The microchannels were etched into the plate in a well-stirred, dilute HF-NH4 F bath kept at 40 ◦ C for about 35 min. Four 1-mm-diameter access holes were drilled on the etched plate at the sites as illustrated in Fig. 1 using an emery drill-bit. A quartz glass plate (20 mm × 60 mm × 2 mm, Shanghai Xinhaotian Optical & Electric Materials Co., Shanghai, China) was used as the cover to bond with the etched soda-lime glass plate. The two plates were aligned and brought into close contact under a stream of running tap-water flowing between them. Afterwards, the combined plates were left at room temperature for 2 days to be firmly bonded together. 2.4. Wettability patterning Wettability patterning of the microchannels was carried out according to an alkylsilane self-assembling and UVphotodegrading process which was previously reported for paper-based microfluidic devices and hybrid soda-lime/quartz glass chips [29,30]. Briefly, all the channels of the glass chip in Fig. 2a were filled with a 0.5% (v/v) octadecyltrichlorosilane solution in n-hexane for 30 min. Then ethanol was introduced to remove the solvent and unreacted reagent. Nitrogen from a nitrogen gas cylinder was infused into the microchip via a piece of plastic tube (2 m × 5 mm o.d. × 3 mm i.d.) to dry the hydrophobic channels (Fig. 2b). Afterwards, a photomask was placed on the quartz-glass side of the microchip, as shown in Fig. 2c. The device was put into the chamber of the photo surface processor, 3 cm underneath the UV-lamp where the intensity of the UV light was 35 mW cm−2 measured at 254 nm. The processor was then turned on and different irradiation times were studied.
2.1. Chemicals and reagents 2.5. Preparation of W/O/W double emulsions All chemicals used were of analytical grade unless otherwise stated and deionized water from a Milli-Q water purification system (Millipore, Bedford, MA) was used throughout. Octadecyltrichlorosilane (OTS, 95%) was purchased from Acros Organics (Springfield, NJ, USA), n-hexane, span 80 and mineral oil were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). For preparing W/O/W double emulsions, deionized water was used as the inner and outer phases, while mineral oil the intermediate
The above wettability patterned microchip was used to prepare W/O/W double emulsions. The inner aqueous phase, intermediate mineral oil and outer aqueous phase were introduced from Inlet 1, Inlet 2 and Inlet 3, respectively. These three fluids were loaded in three 1-mL PVC disposable syringes and driven with three syringe pumps. Three pieces of 15 cm × 1 mm o.d. × 0.3 mm i.d. PTFE tubing were used to connect the disposable syringes and microchip inlets.
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Fig. 1. Schematic diagram of the microchannel network (not to scale). The T-junction was the first droplet maker and the decussation was the second droplet maker. Inlet 1, Inlet 2 and Inlet 3 were for introducing the inner phase, intermediate phase and outer phase, each. All the channels had a depth of about 50 m. The narrow channels had a top-width of about 150 m and other channels had a top width of about 300 m.
Flow rates were adjusted and optimized to control the formation of double emulsions.
3. Results and discussion 3.1. Spatial wettability patterning of microchip channel Microfluidic two-phase system has attracted great interests from microfluidic community in the last decade. To the downscale of tens of micrometers, the wettability of channel surface has a significant effect on the behavior of two immiscible fluids. For example, when two immiscible phases are introduced into a hydrophobic microchannel through a T-junction, the oil phase would wet the hydrophobic surface and segment the water phase, resulting in forming water in oil (W/O) droplets. Therefore, hydrophobic microchannels are suitable for preparing W/O droplets, whereas hydrophilic microchannels are suited to O/W droplets [31]. If the W/O droplets are again dispersed in another aqueous phase, W/O/W double emulsions will be formed. This requires that the hydrophobic channel be followed by a hydrophilic channel. The present work was intended to prepare W/O/W double emulsions in a glass microchip. Thus, wettability patterning of the device to be partially hydrophobic and partially hydrophilic is a must. The designed microchannel network was shown in Fig. 1 and it was purposefully wettability patterned to the status as shown in Fig. 2d. Alkylsilane self-assembling and UV-photodegrading strategies were combined to achieve this objective. Although this method has been utilized to pattern the wettability of paper surfaces and glass surfaces [29,30], it was not for double emulsion preparation.
In this work, a 0.5% (v/v) octadecyltrichlorosilane solution in nhexane was first introduced into the glass channels to form a hydrophobic self-assembly layer (with long alkyl chain) on the entire surface. Then, the hydrophobic microchip (with the quartz side on the top) was spatially covered by a photomask as shown in Fig. 2c, with the cross-shaped microchannel and photomask aligned (Fig. S1), followed by exposure to UV/O3 without being heated. The deep UV light combined with ozone degraded the hydrophobic self-assembly layer to form polar oxygen-containing moieties such as ketones, aldehydes and carboxylates [32], turning the surface wettability from hydrophobic to hydrophilic. Therefore, the channels covered by the photomask remained hydrophobic whereas the UV light exposed channels turned hydrophilic. The wettability patterned effect is later demonstrated in the double emulsion formation process.
3.2. Preparation of W/O/W double emulsions With such a wettability patterned microchip, attempts were made to prepare W/O/W double emulsions. The red color aqueous solution (containing 2 wt% food dye) and transparent mineral oil (containing 5 wt% Span 80) were introduced into the channel network from Inlet 1 and Inlet 2 respectively. As shown in Fig. 3a, the oil phase wetted the channel surface and cut the aqueous phase into W/O segments (with a length of about 600 m), indicating that this section of channel is hydrophobic. When the W/O segments moved to the enlarged channel (Fig. 3b), the red color segments turned into oval (with a size of about 400 m), which facilitated the W/O droplets to be enclosed by the outer aqueous solution. Meanwhile, a green color aqueous solution (containing 2 wt% food
Fig. 2. Schematic diagrams of the wettability patterning processes. (a) The native glass microchip (60 mm × 20 mm × 3.7 mm), (b) a hydrophobic self-assembly layer of octadecyltrichlorosilane formed on the channel surface, (c) the T-junction covered with a photomask, (d) after the channel network shown in (c) was exposed to UV/O3 for 60 min. The black color and gray color represent hydrophilic region and hydrophobic region, respectively.
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Fig. 3. Photomicrographs of W/O/W double emulsion forming process in the microchannel. (a) columnar W/O segments formed at the T-junction, (b) the columnar W/O segments turn into oval after entering the enlarged channel, (c) the oval W/O droplets was about to be enclosed by the outer phase and (d) the generated W/O/W double emulsions. Three fluids involved here were mineral oil containing 5 wt% Span 80 (transparent), aqueous red dye solution (red color) and aqueous green dye solution (green color). (e) Size distributions of inner water droplets (n = 50) and oil droplets (n = 50). (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.)
dye) was introduced from Inlet 3. After reaching the decussation, it wetted the downstream channel and cut the continuous oil phase containing oval aqueous segments into segments (Fig. 3c), resulting in the formation of W/O/W double emulsions (Fig. 3d). It indicates that the decussation downstream channel surface was hydrophilic as well. The double emulsions forming process can be found in Supporting Information, Video S1. The inner water droplets tended to be leaned at the left side of the oil droplets, because they were carried by the oil phase to move forward. Their size distributions are shown in Fig. 3e, the low values of CVs indicated that the droplets are uniform in the channel. 3.3. Optimization of UV/O3 exposing time The wettability difference between the two sections of channel strongly influences the formation of double emulsions. It is still unknown how large the wettability difference should be to produce double emulsions. According to our previous report [30], water contact angle of a planar glass surface after octadecyltrichlorosilane treatment was about 108◦ and it decreased to about 60◦ , 30◦ and 15◦ after exposing to UV/O3 for 30 min, 60 min and 90 min, each. Four microchips prepared as described in Section 2.4 were exposed to UV/O3 for 0 min, 30 min, 60 min and 90 min to study the wettability contrast on the forming of double emulsions. For irradiation times of 0 min and 30 min, the outer aqueous did not wet the decussation downstream channel surface, resulting in the failure of preparing double emulsions. More details are described in Fig. S2. When the irradiation times increased to 60 min and 90 min, W/O/W double emulsions were formed as described in Fig. 3. This indicates that a wettability contrast of about 78◦ (water contact angle) is enough for
double emulsions formation. To speed up the wettability patterning process, 60 min exposure to UV/O3 was adopted in later experiments. Additionally, the wettability patterned device was durable. After being filled with water for 1 month it still performed well in making W/O/W double emulsions. 3.4. Optimization of surfactant The formation of W/O/W double emulsions also was affected by the concentration of added surfactant. Span 80 was added to mineral oil with concentration of 0 wt%, 0.5 wt%, 1 wt%, 2 wt% and 5 wt% to find the appropriate condition for producing double emulsions. It was found that Span 80 with concentration less than 5 wt% was not enough to stabilize the water in oil droplet at the channel decussation, resulting in a leakage of the water phase. With Span 80 concentration being increased to 5 wt%, no fracture was observed, and the W/O droplets were cut by the outer water phase for the second emulsification. The generated W/O/W double emulsions were quite stable in the microchannel, keeping their shape well when moving downstream, but tended to be coalesced after leaving the microchannel (Supporting Information, Video S2). It is reported [33] that the stability of double emulsions has a close relationship with interfacial tension, which can be adjusted by adding surfactant. Considering the coalescence happened between the oil phase, Tween 20 was added to the outer water phase to stabilize the W/O/W double emulsion. The coalescence of oil phase still happened when the concentration of Tween 20 was increased from 0 wt%, 0.5 wt%, 1 wt%, 2 wt% up to 5 wt%. Attempts have also been made to replace the mineral oil by fluorocarbon oil, n-octanol, oleic acid and liquid paraffin as oil phase, but all failed in producing
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Fig. 4. Photomicrographs of the prepared W/O/W double emulsions with different inner droplet sizes. The flow rate of the inner phase (Q1 ) was: (a) 0.2 L min−1 , (b) 0.3 L min−1 , (c) 0.4 L min−1 , (d) 0.5 L min−1 , (e) 0.6 L min−1 and (f) 0.7 L min−1 . The flow rates of intermediate phase (Q2 ) and outer phase (Q3 ) were fixed at 0.5 L min−1 and 5.0 L min−1 in each photomicrograph. (g) Effect of Q1 on the sizes and cutting frequencies of inner and outer droplets (n = 50).
W/O/W double emulsion off-chip. More efforts need to be carried out to increase the stability of the off chip double emulsion in future work. 3.5. The controllability of inner droplets Controlling the size and the number of the inner droplets are both important in applications of double emulsions. For example, when the inner droplets are used for cell culture, their size determines the number of cells that can be enclosed. And the number of inner droplets is critical for structuring 3D colloidal assemblies having spherical shape [34]. To realize such controls, adjusting the inner, middle and outer fluid flow rates (Q1 , Q2 , and Q3 , respectively) is a good way, because the W/O/W double emulsions are consisted of these fluids. 3.5.1. Size control The sizes of the inner droplets could be controlled by adjusting Q1 . In the experiment, the values of Q2 and Q3 were fixed at 0.5 L min−1 and 5.0 L min−1 with Q1 increased from 0.2 L min−1 to 0.7 L min−1 . The obtained W/O/W double emulsions under different flow rate conditions were shown in Fig. 4 (a–f), and the size information of inner droplets (measured after the second junction) was listed in Table 1. It can be seen that the sizes of the inner droplets increased with the increase of Q1 , and all the CVs were less than 8%, which demonstrating that they are uniform in each case. The controllable inner droplets size in the current device ranges
from about 250 m to 630 m. The data of Q1 vs. sizes of inner and outer droplets in Fig. 4 (a–f) is summarized in Fig. 4g. According to the black scatters, the increase of Q1 drastically increased the sizes of inner droplets but did not show great impact on the sizes of outer droplets. This observation implies an increase in the cutting frequencies of outer droplets. Therefore, the cutting frequencies of inner and outer droplets were measured and also shown in Fig. 4g. According to the red scatters, as the flow rate of the inner phase was increased, the cutting frequencies of inner and outer droplets both increased. The results agreed well with our inference. 3.5.2. Number control In such a system, the length of the intermediate oil phase was determined by the cutting frequency of the outer aqueous phase. Therefore, longer oil segments could be obtained when the outer phase flow rate was decreased. The longer the oil segments, the more inner droplets they could enclose. Here the number of the Table 1 Size distribution of inner droplets produced at various Q1 , with Q2 and Q3 fixed at 0.5 L min−1 and 5.0 L min−1 . Q1 (L min−1 )
Average size (m)
CV
0.2 0.3 0.4 0.5 0.6 0.7
243 302 376 453 558 632
7.5% 7.6% 6.7% 5.5% 5.8% 7.6%
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Fig. 5. Photomicrographs of the prepared W/O/W double emulsions with different inner droplet numbers. The flow rate of the outer phase (Q3 ) was: (a) 4.0 L min−1 , (b) 2.5 L min−1 and (c) 1.0 L min−1 . Q1 and Q2 were both fixed at 0.5 L min−1 in each photomicrograph.
inner droplets was controlled by adjusting Q3 . With Q1 and Q2 values fixed at 0.5 L min−1 and 0.5 L min−1 , Q3 was decreased from 5.0 L min−1 to 4.0 L min−1 , 2.5 L min−1 and 1.0 L min−1 . In Fig. 5a, Q3 was adjusted to 4.0 L min−1 , resulting in increasing the length of the oil segments by about 30% compared with that in Fig. 4d (Q3 = 5.0 L min−1 ). But there was just one inner droplet in the double emulsions. Fig. 5b and c display the obtained two inner droplets and three inner droplets containing W/O/W double emulsions. When Q3 = 2.5 L min−1 , the length of the intermediate phase was about twice of that in Fig. 4d. Two inner droplets were enclosed by the intermediate oil phase under this condition. When Q3 = 1.0 L min−1 , the length of the intermediate phase was about triple of that in Fig. 4d and the number of the inner droplets increased to three. It was difficult to prepare W/O/W double emulsions containing more than three inner droplets in such a device, because as Q3 decreases to values lower than 1.0 L min−1 the outer aqueous phase could not cut the intermediate oil phase into segments any more. 4. Conclusions We have demonstrated preparation of highly controllable W/O/W double emulsions by using glass microchips that was wettability patterned to be partially hydrophobic and partially hydrophilic. The method was simple and straightforward compared with other reported strategies. The wettability contrast needed to produce double emulsions in the microchip was about 78◦ when expressed by water contact angle. The size of the inner droplets could be controlled to some extent and the number of the inner droplets that could be produced was no more than 3. The high degree of controllability provided by the device makes it promising in biomedical science. For example, by changing the intermediate oil phase, the prepared W/O/W double emulsions can be used for constructing lipid vesicles, simulating cellular microenvironment and producing microcapsules. Acknowledgments This project is sponsored by Zhejiang Provincial Natural Science Foundation under Grant No. Z4110019, and Natural Science Foundation of China under Grant No. 21435004. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.03.072 References [1] D. Vasiljevic, J. Parojcic, M. Primorac, G. Vuleta, An investigation into the characteristics and drug release properties of multiple W/O/W emulsion systems containing low concentration of lipophilic polymeric emulsifier, Int. J. Pharm. 309 (2006) 171–177. [2] J. Weiss, I. Scherze, G. Muschiolik, Polysaccharide gel with multiple emulsion, Food Hydrocolloids 19 (2005) 605–615.
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Biographies Zeqing Bai is a Ph.D. student in the Department of Chemistry at Zhejiang University. He received his B.S. in chemistry from Xiamen University in 2010. His research interests include microfluidics and wormometry-on-a-chip. Bo Wang is an M.S. student in the Department of Chemistry at Zhejiang University. She received her B.S. in Chemistry from Zhejiang Normal University in 2012. Her research interests include electrochemical cell analysis and paper-based microfluidics. Hengwu Chen received Ph.D. in analytical chemistry from Shenyang Pharmaceutical University, China in 1998. Presently, he is a Professor working in the Department of Chemistry, Zhejiang University, China. His current research interest mainly includes flow-injection analysis, microfluidic chips with electrochemical or fluorescent detection for bio-analysis and pharmaceutical analysis. Min Wang is currently a Professor in the Department of Chemistry at Zhejiang University. She received her B.S. in chemistry from Peking University in 1997, and her Ph.D. in chemistry from the Ohio State University in 2002. She worked as a postdoctoral research associate in the Department of Biomolecular and Chemical Engineering in the University of Illinois at Urbana-Champaign 2002–2003. She is a visiting scholar in the Department of Chemistry and Biochemistry of University of California at San Diego 2014–2015. Her research interests include nanomaterials, microfluidics, and chemical sensors.