Continuously Electrotriggered Core Coalescence of Double-Emulsion

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Continuously electrotriggered core coalescence of double-emulsion drops for microreactions Likai Hou, Yukun Ren, Yankai Jia, Xiaokang Deng, Weiyu Liu, Xiangsong Feng, and Hongyuan Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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ACS Applied Materials & Interfaces

Continuously electrotriggered core coalescence of double-emulsion drops for microreactions †

Likai Hou, Yukun Ren,*

† ‡

†

†

†

†

Yankai Jia, Xiaokang Deng, Weiyu Liu, Xiangsong Feng, and Hongyuan Jiang*

† ‡

†School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, PR China. ‡State Key Laboratory of Robotics and System, Harbin Institute of Technology, Harbin 150001, PR China. KEYWORDS: electrocoalescence, double emulsion, droplet, microreactor, microfluidics

ABSTRACT: Microfluidically-generated double emulsions are promising templates for microreactions, which protect the reaction from external disturbance and enable in vitro analyses with large-scale samples. Controlled combination of their inner droplets in a continuous manner is an essential requirement toward truly applications. Here, we first generate dual-cored doubleemulsion drops with different inner encapsulants using a capillary microfluidic device; then, we transfer the emulsion drops into another electrode integrated PDMS microfluidic device and utilize external AC electric field to continuously trigger the coalescence of inner cores inside

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these emulsion drops in continuous flow. Hundreds of thousands of monodisperse microreactions with nanoliter-scale reagents can be conducted using this approach. The performance of core coalescence is investigated as a function of flow rate, applied electrical signal and core conductivity. The coalescence efficiency can reach up to 95%. We demonstrate the utility of this technology for accommodating microreactions by analyzing an enzyme catalyzed reaction and by fabricating cell-laden hydrogel particles. The presented method can be readily used for controlled triggering of microreactions with high flexibility for a wide range of applications, especially for continuous chemical or cell assays.

INTRODUCTION Double emulsions are functional soft materials in which dispersed drops contain smaller inner droplets, which are widely utilized in various applications, including foods1-2, cosmetics3-4, and drug delivery5. Microfluidic technology has emerged as a promising tool for generating and manipulating emulsion templates6-7. Since microfluidic approaches provide exquisite control over flows, they can produce highly monodispersed emulsion drops ranging from dozens to hundreds of microns. Moreover, one outstanding feature of double emulsions is that the inner reagents are isolated from the external environment through the protection of the outer shell. Therefore, emulsion drops have served as excellent platforms to accommodate microreactions, such as syntheses of microparticles8, cell assays9-10 and test of new drugs11. These promising applications always require to trigger large amount of microreactions precisely and continuously. However, this requirement is barred by the paucity of methods to consecutively trigger the coalescence of the inner cores in continuous flow, upon which microreactions inside doubleemulsion drops can be made possible by mixing the enclosed reagents.


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Several methods have been reported to fuse the internal cores of double emulsions. For instance, reactions in double emulsion are activated by coalescence of the inner cores through precise control of the flow rate12. Another scheme utilized capillary force, resulting from a nonspherical envelope of a thin shell, to trigger the coalescence of two inner cores13. Moreover, coalescence of encapsulated droplets can also be initiated by osmotic pressure, due to the discrepancy of salt concentration between the inner cores and the suspending medium14. These methods depend either on the strict control of flows in microfluidic chips, or on an extended amount of time for coalescence. Therefore, new technologies that are able to fuse encapsulated droplets continuously with convenience and flexibility, need to be further investigated. Electric fields are highly suitable for manipulation of micro objects and flows in microfluidics systems15-19. Manipulations of droplets using electric field at micrometer scale provide benefits of voltage-based control and dominance over other stochastic forces20. In electric field, the interfacial electrical stress induces droplet deformation and dynamic flows both inside and outside of droplet, owing to the discrepancy of the electric conductivity and dielectric properties between the droplets and the suspending medium21-22. Using this mechanism, electric fields have been utilized to trigger the coalescence of single-emulsion homogenous droplets in continuous flow23-24. However, this method requires strictly controlled synchronization of coalescing droplets, therefore undesirable multiple coalescence sometimes occurs12. Besides, since the reagents in single-emulsion droplets contact directly with the continuous phase, liquid diffusion between reagents and suspending medium may cause cross contamination. For paired droplets encapsulated in double-emulsion drops, electric field can also penetrate the outer shell to trigger the coalescence of the inner droplets in static manner25. But, for most in situ chemical or biological applications, the analyses such as cell assays need to be conducted in large-scale


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group (even up to millions)9. Therefore, there is a crucial requirement that incorporating the advantages of double-emulsion microreactions with continuous-flow microfluidic networks, where chemical or biological analysis can be performed continuously, meanwhile protecting the reactions by the outer shell. In this work, we describe a robust approach for initiating reactions by fusing the inner cores inside dual-cored double-emulsion drops in continuous flow. Firstly, the dual-cored doubleemulsion drops with two different inner encapsulants are generated using a capillary microfluidic device. Then, the emulsion drops are transferred into another PDMS microfluidic device in a continuous flow with controlled flow rates. The core coalescence is induced by external AC electric field applied across the suspending medium through an electrode pair. The coalescing performance as function of flow rate, electrical signal and conductivity discrepancy is investigated. Finally, to prove the utility of this method for accommodating microreactions, we demonstrate two applications of this technology. We use this method to trigger the enzyme catalyzed reaction that is widely used for determine of glucose concentration. Moreover, we fabricate cell-laden hydrogel particles by using electrocoalescence in continuous flow. This approach for initializing nanoliter-scale microreactions not only enables reactions with largescale samples in a continuous manner, but also protects the encapsulated reagents from crosscontaminations by oil shells. MATERIALS AND METHODS Materials. Polyvinyl alcohol (PVA, 87–89% hydrolyzed, average Mw = 13 000–23 000), glucose, glucose oxidase (from Aspergillus niger, G7141), methylene blue, peroxidase (P8125), 4-amino antipyrine (A4382, 4-AAP), N-ethyl-N-sulfopropyl-m-toluidine (E8506, TOPS) are purchased from Sigma-Aldrich. As the outer phase (Wo), an aqueous solution of 2 wt % PVA is


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used. As the middle oil phase (O), a mixture of polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) and silicone oil (50cSt, PMX-200, Dow Corning) with the volume ratio of 3:7 is employed. Depending on different experiments, solutions of potassium chloride (KCl), calcium chloride solution, sodium alginate solution, glucose, glucose oxidase, 4-AAP, and TOPS are used as the innermost aqueous phases (W1 and W2). PVA of 0.5 wt% is added to each of the innermost phases in order to avoid the spontaneous core-coalescence after generation of the emulsion drops. In some experiments, methylene blue is dissolved into one of the inner phases for a dye to distinguish different cores. All water used in this work is deionized water unless otherwise noted.

Figure 1. Microfluidic platform for core coalescence of double-emulsion drops. (a) Schematic of the glass capillary device (device #1) for generating double emulsion drops. The emulsion drops are collected in a syringe filled with a suspending medium with low salt concentration. (b) Schematic of the microfluidic chip (device #2) for core coalescence. (c), (d) and (e) are the optical microscopy snapshots for generation, suspending and core coalescence of the doubleemulsion drops in the chips, respectively. The scale bars are 200 µm. Strategy for Continuous Core coalescence. The strategy for the continuous core coalescence of double emulsions are illustrated in Figure 1. We demonstrate this approach by using a water-


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in-oil-in-water (W/O/W) double emulsion. The monodisperse dual-cored double-emulsion drops are fabricated in a capillary microfluidic device (Figure 1a and c, Device #1), as shown in Supporting Information. Each drop contains a PDMS shell and two inner aqueous cores. The two cores are compartmentalized by a PDMS film, breaking of which would result in coalescence of the two cores. By controlling the flow rates QW1: QW 2 : QO : QWo = 130 : 130 : 450 : 5000 µL/h, the diameters of shell and cores of generated emulsion drop are approximately 330 µm and 170 µm in diameter, respectively. The volume of each inner core is around 2.5 nL. The generated emulsion drop is a slightly squeezed sphere — a prolate ellipse (Figure 1d and e). The newly formed W/O/W emulsion drops are collected into a syringe that is filled with lowconcentration KCl solution (conductivity of 20 mS/m). The KCl solution serves as the suspending medium for the emulsion drops, as shown in Figure 1d. The medium with emulsion drops is then reinjected by a pump into a PDMS microfluidic device (Device #2) after a controlled delay, as shown in Figure 1b. The device is composed of a PDMS slab with a channel, and a glass slide with two patterned ITO electrodes at the bottom of the channel, as shown in Supporting Information. The electrodes are linked to an alternating current signal source, therefore providing an AC field between the electrodes in the medium flow in the channel. As the emulsion drops flow through the channel, an appropriate electric field would orientate the emulsion drops, push the cores against each other, thin and break the film, and finally fuse the cores together (Figure 1e). For the experiments, the coalescence efficiency is computed as

η = (1 − Nloss N A ) ×100% , where N A is the number of double-emulsion drops (per 3 minutes) flowing through the electrode area, and Nloss is the number of double-emulsion drops (per 3 minutes) flowing through the area without core coalescence. All the drop numbers are counted


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for 5 times with 3 minutes each time. The coalescence efficiency is averaged over the efficiencies for the 5 times after application of the AC field. RESULTS AND DISCUSSION Mechanism of Electrotriggered Core Coalescence. It is well studied that droplet coalescence is resulted from film thinning26-27. When the double-emulsion drops are formed, there exists a Laplace capillary pressure jump Pσ at the core/shell interfaces. On the PDMS film between the two cores, the pressure balance gives Ps + Pσ = Pc with negligible viscous flow, where the subscripts s and c represent the shell phase and core phase, respectively. As the PDMS film thins, there arises on the film another pressure, disjoining pressure, Π =Pvdw + Pr , where Pvdw represents the attractive van der Vaal stress, and Pr is the repulsive component of the disjoining pressure. The disjoining pressure starts to act below film thickness of ca. 100 nm27. Therefore, the pressure on the film is Pf = Pc − Pσ + Π , where the subscript f represents the PDMS film between the two inner cores. The driving pressure for film thinning, ∆P , is determined by ∆P = Pc − Pf = Pσ − Π . The driving pressure ∆P directly determines the rate of film drainage,

which can be presented as28-29:

VRe = −

dh 2 h 3 = ∆P dt 3η r 2

(1)

where h is the film thickness, t is time, η is the dynamic viscosity of the film, and r is the film radius. If at a certain film thickness, the capillary pressure balances the disjoining pressure, the driving pressure is zero, leading to the formation of an equilibrium film. This is evidenced by our observation that the stable double-emulsion drops, without external perturbing, can last for more than 20 days. It is noteworthy that surfactant adsorption at the core/shell interfaces plays a vital


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role for emulsion stabilization. Surfactant would affect the elasticity and surface viscosity of the film, and therefore provides the film with enhanced resist to local deformations and rupture of the film27, 30. To understand the coalescence mechanism, we focus our analysis on the PDMS film between the two cores (Figure S1 in Supporting Information). When an electric field is applied to the emulsion drops of finite permittivity ε and conductivity σ , free surface charges σ f are allowed to accumulate at the core/shell interfaces, a phenomenon termed Maxwell-Wagner interfacial polarization. The structural polarization is most significant on the film between the cores because of the concentrated field in the film25. The interfacial polarization gives rise to an electrical

sr 2 surface pressure T E ⋅ n , at the core/film interfaces, in the direction of film thinning. Besides, 1

another electric stress would compress the ellipsoid drop at the equatorial plane, making the core move toward each other. These two electric pressures both thin the film between the cores, which is indicated by a general name, Pe , as shown by the blue arrows in Figure S1. Then, the driving pressure for film thinning becomes ∆P = Pe + Pσ − Π . Therefore, fluid physics in the presence of an electric field is more complicated in that both the electrokinetic effect and viscous diffusion have to be included in the interfacial stress balance at the drop/shell interface

sr sr 2 2 T E ⋅ n + ( p2 − p1 ) ⋅ n + Π + T vis c ous ⋅ n =γ n∇s ⋅ n 1

where

2 1

1

(2)

denotes the jump value of the variable across the liquid/liquid interface (external-

sr sr 1   internal). T E = ε  EE − E 2 I  and T viscous = µ ( ∇u + ∇uT ) are the Maxwell and viscous stress 2  

sr 2 tensors, respectively. The electric pressure T E ⋅ n greatly accelerates the rate of film thinning 1


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by driving local viscous fluid flow toward the direction of film thinning. When the film thickness reduced below a critical thickness, coalescence of cores occurs27, 31.

Effect of Flow Rate on Coalescing Voltage. Before investigating core coalescence under various conditions, we first focus on the coalescing process of the emulsion drops, as shown in Figure 2. A coalescence zone (dashed blue box) is established between the electrode pair by the electric field, in which the emulsion drops would undergo three steps of transition before coalescence occurs (Figure 2a). First, the randomly distributed drops would rotate, so as to orient the cores parallel to the background field lines. The rotation results from the alignment of the induced core dipoles along the applied field. Second, the cores squeeze against each other under the effect of the electric compression, thinning the film between them, as described in Equation (1). Finally, when the film is thinned to a critical thickness, slight external disturbances such as thermal shocks, vibration and particles would cause the rupture of the film and therefore the coalescence of the cores. A snapshot series for the electrotriggered coalescence process of four emulsion drops in continuous flow is shown in Figure 2b-e. The flow rate for this experiment is 3 mL/h, while the magnitude of the AC signal is 49.6 V at 100 KHz. The coalescence process of a cluster of double-emulsion drops under this experimental condition is also shown in Movie S1 of Supporting Information.


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Figure 2. (a) Schematic of the coalescence process for a double-emulsion drop. (b)-(e) Sequential snapshots for the coalescence process of four emulsion drops (numbered 1, 2, 3, 4) at a flow rate of 3 mL/h, while a square wave signal (100 KHz, 49.6 V) is applied between the ITO electrode pair. The conductivity of suspending medium, blue inner aqueous core and transparent inner aqueous core is 20 mS/m, 1300 mS/m and 168 mS/m, respectively. The distance of the two electrodes is 2 mm. For continuous electrocoalescence to occur, the flow rate of the suspending medium plays an important role for the coalescence performance. Here, the coalescence performance in response to different flow rates is investigated as shown in Figure 3. The field frequency is fixed at 100 KHz. For a given flow rate, when increasing the field magnitude, the coalescence efficiency


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increases initially and then declines, indicating the existence of an optimal voltage spectrum in which the efficiency is above 90%. The optimal voltage spectrum is shown by the red strip in each pillar in Figure 3. Below the optimal voltage spectrum, some of the drops tends to rotate but no core coalescence occurs. This is because the combined effect of the electric pressure Pe and the Laplace pressure is not sufficient enough to suppress the disjoining pressure (

∆P = Pe + Pσ − Π ≤ 0 ). The balance of the three pressures ensures the stability of the emulsion drops. When the applied voltage is above the optimal range, the shells of the drops are likely to break. This can be explained that the strong electric pressure ( Pe + Pσ >> Π ) caused by the high electric magnitude not only ruptures the PDMS film between the cores but also decomposes the thin shell between the cores and the suspending medium. For example, when the flow rate is 3 mL/h, the optimal voltage spectrum is from 46.8 V to 50 V. Either increasing the voltage to above 50 V or decreasing the voltage to below 46.8 V would lessen the efficiency as shown by the other strips in the pillar.

Figure 3. Coalescence efficiency for different flow rates at varied voltages. The efficiency is presented by the color variant as shown in the color bar. The flow rates experimented are 2, 3, 4, 5, 6 mL/h from left to right, respectively. The field frequency is fixed at 100 KHz. The


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conductivity of suspending medium and two inner aqueous cores is 20 mS/m, 1300 mS/m and 168 mS/m, respectively. We vary the flow rate in a wide range from 2 mL/h to 6 mL/h to investigate the effect of flow rate on coalescence performance. Generally, the coalescing voltage increases with the increasing flow rate. This could be explained by the fact that a fast flow shortens the duration time of the emulsion drops in the electric field and therefore a stronger electric compression Pe is required to reduce the time needed for film thinning as indicated in Equation 2. The electrical force acting on the droplet is approximately FE = ε E 2 S with S denoting the drop surface area, and the viscous shear force exerted by the continuous flow medium is given by the Stokes law

Fviscous = ηuR , the ratio of the two force components is dictated by the nondimensional number A = ε E 2 R ηu . For a large A , electrical stress stipulates the droplet motion behavior, and coalescence is easy to occur as in static flow condition. For a small A , the final droplet shape is mainly determined by the viscous effect, e.g. if the flow rate is sufficiently large in dynamic flow condition, the droplet keeps the original spherical shape, with a failure in the coalescing process. It is noteworthy that increasing the continuous phase flow rate leads to a raise in both the flow velocity and hydrodynamic shear stress, so based on a force balance analysis, E 2 has be to enhanced accordingly to render the electrical force effective. In addition, our observation indicates that the optimal voltage spectrum is shortened as the flow speeds up (red strip in Figure 3). This can be explained that the strong electric pressure ( Pe ) needed for a high flow rate is much bigger than the disjoining pressure ( Π ). Hence, this thinning process is very susceptible to external disturbance such as vibration and flow shocks, increasing the possibility of rupture of


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the film between the cores and the suspending medium. Therefore, the optimal voltage spectrum (with coalescing efficiency of > 90%) shrinks as the electric field increases.

Effect of Core Conductivity on Coalescing Performance. Besides the dependence on the flow rate, the coalescing performance also changes sharply with the electrical conductivity of the cores (Figure 4). In our experiments, the conductivity is adjusted by changing the salt concentration of the cores. We define core 1 and core 2 as the cores with relatively higher and lower conductivities, respectively. We fix the conductivity of one core at 168 mS/m, and vary the conductivity of the other core at 8.6 mS/m, 38.7 mS/m, 168 mS/m and 1300 mS/m, as shown in the inset table in Figure 4. The coalescence efficiency is examined in response to these four conductivity combinations under different field frequencies. For a specific frequency, the coalescence efficiency is counted under the voltage where the efficiency is the highest, which means the efficiency is tested at varied voltage in corresponding to field frequency. For a specific conductivity combination (e.g. 168 mS/m and 38.7 mS/m shown by the blue curve in Figure 4), upon an increase in the field frequency, the coalescence efficiency increases significantly initially. However, as the field frequency increases to a certain value, the efficiency reaches a climax, and then the efficiency declines rapidly with further increase of the field frequency. At relatively low field frequency, the electric pressure Pe is affected presumably by polarization at the multiple interfaces, and increases along with the increasing frequency. When the field frequency excesses a critical value, the electric pressure declines and the coalescence efficiency declines accordingly25.


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Figure 4. coalescence efficiency as a function of field frequency for different conductivity combinations. The unit of the conductivity σ c is mS/m. The coalescence frequency is found to be determined by the core 1 with higher conductivity. The coalescence frequency also increases with an increase of the higher core conductivity. When the conductivity in core 1 is 168 mS/m, the highest efficiencies manifest at 50 KHz for all the combinations indicated by the pink, blue and red curves. However, when the conductivity in core 1 is 1300 mS/m, the coalescence frequency for the highest efficiency increases to 100 KHz as shown by the black curve. This indicates that the coalescing field frequency increases with the ion concentration of the cores. This phenomenon implies that the surface charge experiences a time-varying process ∂σ f ∂t + n ⋅ J

2 1

= 0 ( J being the conduction current) at the interfacial

region, and the relaxation frequency has a strong dependence on the maxima conductivity of the paired induced dipoles. In addition, judging from the pink, blue and red curves, the coalescence efficiency increases with an increasing conductivity of core 2. This is probably because that the increasing ion concentration increases the polarizability of the cores. The electric pressure Pe increases accordingly due to the increased polarizability difference between the cores and the


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surrounding PDMS shell. Therefore, a stronger electric pressure shortens the time for film thinning as described in Equation 1, and improves the efficiency of core coalescence.

Core coalescence for enzyme catalyzed reaction. To prove the feasibility of this method for microreaction, we demonstrate an enzyme catalyzed reaction for detection of glucose in a continuous-flow manner. Enzymes catalyzed reactions, mimicking the synthetic strategies used by the living organisms, are of great importance for synthesis of compounds and detection of biomarkers32. However, some of the enzymes are precious for reactions and massively large amount of individual reactions (e.g. cell assays) need to be analyzed. Droplet-based microfluidics can be applied to these circumstances. Here, we use the core coalescence approach to accommodate the reaction for glucose detection based on the Trinder’s reaction33. Glucose solution is encapsulated in one inner droplet at varied concentration, while the other inner droplet is the reagent mixture containing 6 U/ml glucose oxidase, 6 U/ml peroxidase, 6 mM 4-AAP, and 10 mM TOPS in 0.01 M phosphate buffered saline (PBS). After core coalescence, glucose is oxidized to glucose acid and hydrogen peroxide in the presence of glucose oxidase. Meanwhile, the generated hydrogen peroxide reacts with 4AAP and TOPS in the presence of peroxidase, leading to the formation of violet colored quinoneimine, as illustrated in Figure 5a. By fine adjustment of the flow rate and applied electrical signal, a large amount of monodisperse dual-cored double-emulsion drops containing glucose and reagent can be triggered to coalesce and react at a high efficiency. In this case, the flow rate of the suspending medium is 3 mL/h; the voltage is 50 V; the field frequency is 100 KHz; the conductivity of suspending medium is 20 mS/m. After core coalescence, the transparent solution changes to a violet color and the color saturates after 2 mins. As shown in Figure 5b, the resulting emulsion drops are


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monodisperse in size, where the glucose concentration is 400 mg/dL. The color intensity in the central of the reacted emulsion drops are uniform with coefficient of variation of 1.2 %, as shown in the color distribution curve in Figure 5c. In addition, color intensity against various glucose concentrations is investigated as illustrated in Figure 5d, which indicates that the color intensity increases linearly with the glucose concentration. The experiment proves that the corecoalescence approach can be readily used as a microreactor for enzyme catalyzed reactions. In addition, hundreds of thousands of the monodisperse microreactions triggered by our continuous manner can be used to analyze the uniformity of reactions under required conditions.

Figure 5. (a) Optical microscope images showing the double-emulsion drops before and after enzymes catalyzed reaction caused by electrocoalescence. (b) Optical microscope images showing the monodisperse emulsion drops after continuous coalescence and reaction. (c) Distribution of the violet color in the reacted emulsion drops. (d) Color intensity against different glucose concentrations. The scale bars are 200 µm.

Core coalescence for microgel generation and cell immobilization. Moreover, we demonstrate the capability of the electrocoalescence approach to fabricate alginate microgel, and finally to immobilize yeast cells in the microgel, as shown in Figure 6. Alginate gels are of high


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mechanical stability and biocompatibility, imparting them with great potential to be served as matrix for entrapment of cells13,

34-36

. Microgels have been fabricated using droplet-based

microfluidics, including the diffusion-caused gelation and the fusing of aqueous droplets in emulsions12,

37-38

. Here, we use the design in Figure 1a to generate monodisperse dual-core

double-emulsion drops, where phase W1 is 0.45 wt% sodium alginate solution and phase W2 is 0.1 wt% calcium chloride solution.

Figure 6. (a) Schematic illustration showing the electrocoalescence in dual-cored doubleemulsion drops and subsequent gelation of alginate microgel in the fused core. (b) Time series of optical microscope images showing electrocoalescence and gelation processes. (c) Time series of optical microscope images showing gelation of alginate microgel and immobilization of yeast cells in it. (d) Optical microscope images showing proliferation process of yeast cells embedded in microgels, which are cultivated at 40 °C for 12 h. The boundary of microgel is denoted with arrows and dashed circles in (b-d). The scale bars are 200 µm.


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The schematic and experimental snapshots for the coalescence process are shown in Figure 6a and b, respectively. Similar to the previous coalescence process of aqueous cores, the emulsion would rotate initially because of electric polarization induced orientation, and then the cores fuse together after film thinning, as shown in Movie S2 of Supporting Information. In this demonstration, the flow rate of the suspending medium is 3 mL/h; the voltage is 50 V; the field frequency is 100 KHz; the conductivity of suspending medium is 20 mS/m. The time interval between application of electric field and core coalescence is observed to be 0.47 s. Upon core coalescence, the two cores contact, leading to the immediate gelation of alginate. At 1.41 s, as shown in Figure 6b, a boundary line is identified between the two reagents. As the calcium ions continues diffusing into the alginate phase, microgel is generated in the coalesced core after 2.35 s. A spherical microgel is fully formed after 9.4 s. Through this approach, up to 210 microgels can be produced per minute, with a coalescence efficiency up to 95%. Compared to the generation rate of the emulsion drops in device #1, the throughput is relatively low, which is limited by the duration time of the drops in the effective electric field. However, the throughput can be greatly improved by incorporating more microchannels in device #2. This is feasible because both the microchannel and the ITO electrodes are easy to parallel using the soft lithography fabricating method. The newly generated microgels are encapsulated in a PDMS shell, and this provides protection for the microgel from possible external contamination. On demand, the shell can be removed by rinsing the drops with DI water and hexane in turn. Furthermore, to prove biocompatibility and usability of the technique for cell culture, immobilization of yeast cells in the microgels and their subsequent viability are investigated. To prepare yeast cell samples, 20 mg of baker’s yeast cells (Saccharomyces cerevisiae) are reactivated in 2 mL of 0.01 M PBS for 1 h. The reactivated cells are washed 3 times with the


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same solution. And then, we disperse the cells in the sodium alginate solution (W1) with a cell concentration of about 2.5 × 107 cells/mL. The phase W2 consists of 0.1 wt% CaCl2 solution, 0.5 wt% potato extract and 2 wt% dextrose, as shown in Figure 6c. As expected, after gelation the yeast cells are entrapped in the formed microgel, with no yeast cells observed in the surrounding medium. Besides, we further investigate yeast immobilization in emulsion drop with asymmetric cores by adjusting the flow rate of phase W1 and W2, as shown in Figure S2 in Supporting Information. The size of the generated microgels are almost the same as that of the sodium alginate core. This indicates that mocroreactions in given proportion can be achieved precisely by using this electrocoalescence approach. To study the cell viability, we place the fused emulsion drops in a medium that of the same composition with phase W2 at 40 °C for 12 h. Our observations show that the cells start multiplying in 8 h and continuous to proliferate in following 4 h, as shown in Figure 6d. This indicates that the effect of electric field on the viability of yeast cells is benign, presumably owing to the protection of the oil shell, and the immobilized cells remain metabolically active with supply of nutrients.

CONCLUSIONS In summary, we present a technology to trigger the coalescence of paired droplets encapsulated in double-emulsion drops in continuous flow by using an AC electric field. The robustness of our method by inducing core coalescence is demonstrated in a wide range of flow rates, as well as core conductivities. The experimental results show a coalescing efficiency up to 95% in a continuous manner. To illustrate possible applications, we demonstrate that the core-coalescence approach can be used as a microreactor for enzyme catalyzed reactions. We further use this approach to fabricate alginate microgels and to immobilize yeast cells in the microgels. The microreactions are triggered inside the double-emulsion drops, which can protect the


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microreactions from external disturbance, and also avoid cross contamination. Besides, hundreds of thousands of monodisperse microreactions with nanoliter-scale reagents can be conducted using this approach in a continuous manner. Furthermore, since the coalescence of cores is triggered by AC electric field, the timing of coalescence and subsequence microreactions can be controlled precisely. We envisage that our proposed technique for inducing core coalescence has the potential to realize controlled picoliter- and nanoliter-scale reactors for various biological and chemical analyses in continuous-flow manner, such as synthesis of particles39-40, drug screening and cell assays11, 41-42.

ASSOCIATED CONTENT

Supporting Information. The following files are available free of charge. Fabrication and geometry of the microfluidic device #1 and #2; Generation of monodisperse dual-cored W/O/W double-emulsion drops; Schematic for the coalescence mechanism of a double-emulsion drop; Generated emulsion drops with varied size of inner cores. (PDF) Continuous electrocoalescence of paired droplets encapsulated in double-emulsion drops in device #2. (WMV) Synthesis of alginate microgels using electrocoalescence method in continuous flow. (WMV)

AUTHOR INFORMATION

Corresponding Author


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

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources National Natural Science Foundation of China (No. 11672095 and 11372093); Self-Planned Task of State Key Laboratory of Robotics and System (HIT) (No. 201510B and SKLRS201606C). ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (No. 11672095 and 11372093), and Self-Planned Task (No. 201510B and SKLRS201606C) of State Key Laboratory of Robotics and System (HIT). The authors wish to acknowledge Prof. Lei Lei’s group (Harbin Medical University) for their assistance with the Micropipette Puller. REFERENCES 1.

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Continuously Electrotriggered Core Coalescence of Double-Emulsion

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