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Jul 26, 2017 - This work demonstrates that the solute concentration inside 100 micrometer-sized aqueous microdroplets can be controlled by adjusting t...
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Kinetic Switching of the Concentration/Separation Behavior of Microdroplets Mao Fukuyama, Akihide Hibara, Yumi Yoshida, and Kohji Maeda Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02062 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 28, 2017

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Analytical Chemistry

Kinetic Switching of the Concentration/Separation Behavior of Microdroplets Mao Fukuyama†‡*, Akihide Hibara§, Yumi Yoshida†, and Kohji Maeda† †Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, 1 Hashigami-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan ‡PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan §Institute of Multidisciplinary Research for Advanced Materials, Tohoku Univesity, 2-1-1 Katahira, Aoba-ku Sendai 980-8577, Japan Abstract: This work demonstrates that the solute concentration inside 100 µm-sized aqueous microdroplets can be controlled by adjusting the time required for the aqueous nanometer-sized droplets (nanodroplet) or reverse micelles to pass over the surface of the microdroplet. The kinetics of molecular transport between the microdroplets and nanodroplets was investigated by utilizing a microdroplet array, and based on these results, a control over the concentration selectivity of the contents of the microdroplet was achieved. This method is operationally simple and can be potentially applied as a pretreatment method for microanalytical systems that require high-density microdroplet arrays. This method can also be utilized for parallel small sample analyses such as single cell analysis.

Aqueous droplets dispersed in an immiscible phase have been widely utilized as a platform for the miniaturization of bioassays and material syntheses.1−3 By compartmentalizing the reagents and samples in a micrometer-sized droplet (microdroplet), operations such as chemical reaction and component mixing can be isolated in the droplet. Droplet microfluidics involves the formation of monodispersed microdroplets of volumes ranging from a picoliter to a nanoliter,4 and high throughput and precise manipulations such as fusion, fission, mixing, and sorting of single microdroplets are made feasible using this technique.5 The rapid development of droplet microfluidics has resulted in the miniaturization of the massive and parallel biochemical analyses to ultimately trace amount targets, such as a single cell6,7 and single molecules.8,9 Owing to the small volume and short optical path length of a microdroplet, the detection of its contents is one of bottlenecks in their study. In order to broaden the choices of the analytical methods applicable to droplet microfluidics, the integration of versatile pretreatment methods such as concentration10−14 and separation15−17 of microdroplet contents is indispensable. Thus far, most of the concentration/separation processes have been conducted by employing liquid-liquid extraction between the aqueous and organic phases. In this regard, the separation of water-soluble molecules from the microdroplet by conventional extraction methods is not well established yet, even though it is an important pretreatment step in biochemical analyses. Recently, several groups have reported on the separation of water soluble molecules from the microdroplet by utilizing the partitioning between the

aqueous droplet and ionic liquid.18 reversed micelles19,20 or nanodroplets,21 and its application to protein analyses.21,22 These separation techniques seemingly rely on the partition equilibrium. Therefore, in order to modify the selectivity of either concentration or separation for a solute with similar partition characteristics, an adjustment of equilibrium parameters is required and can be accomplished by either changing the extractants23 and/or via pH control.24,25 In droplet microfluidics, however, rather complicated hydrodynamic operations such as fusion or fission of droplets are necessary for such adjustments of the chemical composition. Such hydrodynamic operations are especially difficult to realize for a droplet array. Therefore, this work proposes that if partition kinetics is precisely controlled, various operations such as the switching of the concentration/separation behavior of the microdroplet solutes can be realized without modifying the chemical composition of the microdroplet. In order to demonstrate this concept, the kinetics of water and solute transport from the microdroplets to the reverse micelles has been investigated in a selective enrichment method that is based on spontaneous emulsification.20 While the transport of water and the solute from the aqueous phase to the reverse micelle has been individually studied26−28 in the context of reverse micelle extraction,29 the simultaneous measurement of water and solute transport has not been discussed to date. Owing to the difficulty of exerting precise control over the interfacial conditions during the extraction process, it is difficult to clearly follow the partition kinetics and the transport of water and solute molecules. Microfluidic devices provide a means to

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precisely and reproducibly control fluids 30. Therefore, it might be possible to analyze and even control the partition kinetics by using such devices. In this study, the transport of water and solutes between the microdroplet and the nanodroplet (or water-swelled reverse micelles) has been investigated by using a microfluidic droplet system, and a kinetic concentration/separation control technique was subsequently developed.

Figure 1. Micro- and nanodroplet formation in a microdevice. (a) A sketch of the microdevice. (b)–(d) Micrographs of the droplet formation in the microdevice. (b) The microdevice was initially filled with the aqueous phase. (c) On introduction of hexadecane, microdroplets are formed in the microwells. (d) Spontaneous nanodroplet formation (spontaneous emulsification) occurs at the interfaces of the microdroplets when the hexadecane solution of Span 80 is passed through the device. The solution of Span 80 flowed from left to right.

EXPERIMENTAL SECTION

RESULTS AND DISCUSSION

Microdevice: A PDMS microfluidic device with 75 microwells was used to observe microdroplets (Figure 1a). In this device, microwells of size 20 µm × 210 µm (depth × width) were placed on the ceiling of a wide channel that had dimensions of 44 µm × 3 mm (depth × width). Chemicals: Rhodamine 123, Rhodamine 6G, and hexadecane were obtained from Wako Pure Chemical Industries (Osaka, Japan). Span 80 was purchased from Sigma Aldrich KK (Tokyo, Japan) while cascade Blue was obtained from Thermo Fisher Scientific KK (Yokohama, Japan). These reagents were used as received without further purification. Experimental procedure: Initially, the microwells and the microchannel were filled with the aqueous solutions of fluorescent dyes (Figure 1b). These dye solutions were isolated in each microchamber and transformed into microdroplets by introducing hexadecane into the microchannel (Figure 1c). After the formation of microdroplets, a hexadecane solution of 30–100 mM Span 80, a nonionic surfactant known to induce spontaneous nanodroplet formation (spontaneous emulsification),31 was introduced in the microchannel by using a syringe pump (Model KDS210, KD Scientific Inc., USA). The microdroplets were observed by an inverted microscope (IX-73, Olympus Corporation, Tokyo, Japan) and their fluorescence micrographs were captured by using CCD camera (Moticam 5.0, Shimadzu Co. Japan).

The partitioning of the water-soluble solute and water from a microdroplet to nanodroplets and reverse micelles was investigated by using aqueous microdroplets containing 50–200 µM Rhodamine 123 (R123). A spontaneous emulsification was started at the interface of the microdroplets by introducing the hexadecane solution of Span 80. The size and the fluorescence intensity of the microdroplets changed with the partitioning of water and R123 to the reverse micelles (or nanodroplets) of Span 80 (Figure 2a). The droplet areas and fluorescence intensities were measured over the course of time for the three droplets on the left, upstream side (Figure 2b) and converted into the microdroplet volume (V, see SI for detail) and concentration of R123 (Cmicro),20 respectively (Figure 2c). While the microdroplet volume V decreased monotonically, Cmicro decreased initially and then started to increase after several minutes. This initial declination corresponded to the partitioning of R123 at the interface into the Span 80 micelles. The reduction in the value of Cmicro stopped within several minutes. After introducing the flow of Span 80, Span 80 started to adsorb on the interface and its adsorption eventually reached a steady state. Analysis of this initial stage transport dynamics is complicated because the adsorption dynamics of Span 80 would need to be considered in addition to water and solute transport. Therefore, in order to simplify the analysis, only the partitioning that took place after the attainment of adsorption steady state was considered. In addition, when the diameter of the droplet became smaller, it was observed to have deformed slightly and thus, the volume of the droplet was overestimated. Therefore, the data pertaining to droplets of diameter smaller than 90 µm was discarded. From a series of micrographs, it was possible to calculate the fluxes of water (Fw) and R123 (FR123) from inside to outside of the microdroplets (see Supporting Information (SI) for details).

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Analytical Chemistry

Figure 2. Study of the transport of water and R123 from the microdroplets during spontaneous emulsification. (a) Fluorescence micrographs of the microdroplets during spontaneous emulsification. (b) The measured microdroplet area (A, red) and the fluorescence intensity (I, green) were converted into (c) the microdroplet volume (V, red) and R123 concentration (Cmicro, green), and subsequently, (d) the fluxes of water (Fw, red) and R123 (FR123, green) from the microdroplet were calculated (see SI for details).

Figure 3. The flux of water (FW, a) and R123 (FR123, b) from microdroplets to nanodroplets (or reverse micelles) depending on the concentration of R123 (Cmicro). The concentration of R123 in the nanodroplets (Cnano = FR123/FW, c) was found to be identical under all CSpan 80 concentrations and the value of Kapp (Cmicro/Cnano) was 0.37. The flow rate (Q) was 5 µL min−1.

In this experiment, the amount of the micelles supplied to a microdroplet interface in unit time and the time required for a micelle to pass on the microdroplet interface (τ) could be changed by adjusting the concentration of Span 80 (CSpan 80) and the flow rate of the hexadecane solution of Span 80 (Q), respectively. The value of τ was calculated as τ = w/v, where w and v refer to the width of the wells and flow velocity of Span 80 solution, respectively. In order to evaluate the dependence of the partitioning of water and R123 from the microdroplet to nanodroplets on the amount of the micelles supplied to a microdroplet interface in unit time, Fw and FR123 were investigated with different values of CSpan 80 (Figure 3a and b) at the constant flow rate (Q = 5 µL min−1). Fw was found to be independent of Cmicro under all tested CSpan 80 values (Figure 3a). This indicates that the partitioning of water was unaffected by the presence of R123 in the tested concentration range. In addition,

Fw was found to increase proportionally with increasing values of CSpan 80, a trend that is similar to a previous study.22 On the other hand, FR123 varied depending on both Cmicro and CSpan 80 (Figure 3b). In order to determine the apparent partition coefficient between the micro and nanodroplets (Kapp), the concentration of R123 in a nanodroplet (Cnano) was calculated as Cnano = FR123/Fw. As a result, Cnano was found to be identical under all CSpan 80 values and Kapp was determined to be 0.37 (Figure 3c). In these experiments, while the amount of the micelles supplied to a microdroplet interface in unit time increased with increasing CSpan 80, τ remained constant because of the same value of Q (5 µL min−1). Since τ is the same under these CSpan 80 conditions, the amount of water and R123 transported from the microdroplets to each single nanodroplet showed the same Kapp in spite of the different CSpan 80.

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Figure 4. Switching of the concentration/separation behavior of R123 in the microdroplets. (a, b, and c) R123 prefers rather concentrated microdroplets instead of partitioning to nanodroplets at a higher flow rate. R123 is partitioned to nanodroplets at a lower Q. (d) Owing to the differences in the kinetic behavior of water and R123, Kapp (Cmicro/Cnano) increased with τ (w/v).

Next, the control of concentration/separation behavior was studied by varying τ. At a higher Q, R123 preferably remained in the microdroplets instead of partitioning to the nanodroplets. However, it partitioned to the nanodroplets at a lower value of Q (Figure 4 a, b, and c). While studying water transport, it was found that the amount of water in a single micelle decreased with Q as the value of Fw was decreased (see SI for details). This result implies that the transport of water from the microdroplet to the micelle is affected by the degree of hydration of the micelle; the amount of water transported was lowered when the degree of hydration of the micelle was higher. Furthermore, a study of R123 transport showed that the value of FR123 increased with τ (see SI for details). This trend is observed because that the transport of R123 is also affected by the various properties such as the degree of hydration or curvature of the micelles.27,28 Due to the differences in the kinetic behaviors between water and R123, the value of Kapp typically increased with τ. The dependence of Kapp on τ is dictated by the molecular properties. Therefore, it was expected that the selectivity towards either concentration or partition of solutes in the microdroplets could be controlled simply by changing the flow rate, Q, as presented in this work. In order to demonstrate this concept, the concentration/separation behaviors of three fluorescent dyes with different hydrophobicity, Cascade Blue (CB, Figure 5a left), R123 (Figure 5a center) and Rhodamine 6G (R6G, Figure 5a right) were investigated. While CB and R123 were concentrated at Q = 10 µL min−1 (Figure 5b), only CB was concentrated at Q = 1 µL min−1 (Figure 5c). This result shows that depending on the hydrophobicity of the solute, its selectivity towards either concentration or partition can be controlled simply by altering the value of Q, without changing the composition of the water/organic phases.

Figure 5. The switching of concentration selectivity of the contents of a microdroplet depends on the flow rate. (a) Structures of Cascade Blue (CB, left), R123 (center), and Rhodamine 6G (R6G, right). (b) CB (blue) and R123 (green) were concentrated in the microdroplet and R6G (red) partitioned at Q = 10 µL min−1. (c) CB was concentrated in the microdroplet and both R123 and R6G partitioned at Q = 1 µL min−1.

In conclusion, the partition kinetics of molecules during spontaneous emulsification was investigated in this work. It has also been demonstrated that it is possible to control the concentration/separation behavior of the contents of the microdroplet simply by changing the flow rate of the organic phase. This simple method may be employed as a pretreatment method for high-density microdroplet arrays,32 in which it is difficult to integrate the microstructures for adjustment of chemical composition of each microdroplet. Furthermore, it can be expected that this method will be useful for parallel small sample analyses such as single cell analysis.

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Calculation methods of droplet volume and interface area and fluxes of water and R123 from microdroplets to nanodroplets. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Present Addresses †

(M. F.) Institute of Multidisciplinary Research for Advanced Materials, Tohoku Univesity, 2-1-1 Katahira, Aoba-ku Sendai 980-8577, Japan

ACKNOWLEDGMENT This work was financially supported by JST/PRESTO Grant Number JPMJPR15F9, Japan. MF appreciates the technical support provided by Ms. Miyagi and Ms. Sone.

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