pNIPAAm Microgels

Jan 13, 2015 - glycerol/barium acetate water solutions. By changing the initial droplet size and glycerol concentration of the collecting solution, th...
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Fabrication of shape controllable Janus alginate/pNIPAAm microgels via microfluidics technique and off-chip ionic crosslinking Yuandu Hu, Shibo Wang, Alireza Abbaspourrad, and Arezoo Ardekani Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504422j • Publication Date (Web): 13 Jan 2015 Downloaded from http://pubs.acs.org on January 24, 2015

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Fabrication of shape controllable Janus alginate/pNIPAAm microgels

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via microfluidics technique and off-chip ionic crosslinking

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Yuandu Hu1, Shibo Wang1, Alireza Abbaspourrad3, Arezoo M. Ardekani*1,2

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1 Department of Aerospace and Mechanical Engineering, University of Notre Dame, Notre

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Dame, Indiana 46556, USA

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2 School of Mechanical Engineering, Purdue University, 585 Purdue Mall, West Lafayette,

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Indiana 47907, USA

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3 School of Engineering and Applied Sciences, Harvard University, Cambridge,

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Massachusetts 02138, USA

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*Corresponding Author

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E-mail: [email protected]

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ABSTRACT: A novel method to fabricate shape controllable alginate/pNIPAAm

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complex microgels is reported. Monodisperse alginate/pNIPAAm droplets are created

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via microfluidics and crosslinked in different concentrations of hot glycerol/barium

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acetate water solutions. By changing the initial droplet size and glycerol concentration

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of the collecting solution, the resultant microgel shape and surface details can be

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systematically tuned. High-speed imaging is used to visualize and explain the

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microgel formation process.

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INTRODUCTION

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Anisotropic and multicomponent microparticles have been widely investigated in the

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past decades for various applications.1-4 For example, the two distinguishable surface

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areas of Janus particles make them ideal candidates for displays,5-6 target recognition,7

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interfacial stabilizers,8 encoding,9-10 sensors,11-13 cell encapsulation14 and the detection

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of metal ions.15 Anisotropic particles with rough surfaces can be used for Raman

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signal enhancement7 and hydrophobic coatings,16-17 due to their enlarged surface area 1

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compared to that of smooth particles. Droplet-based microfluidic techniques have

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been widely used to fabricate anisotropic microparticles.4,

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polymer microgels with different components has been achieved using water in oil

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(w/o) emulsion droplets combined with UV-induced polymerization or ionic

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crosslinking to solidify droplets. The solidification process mostly occurs inside the

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microfluidic device, known as on-chip solidification method.31 Seiffert et al. have

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developed a double T-junction PDMS-based microfluidic device to fabricate Janus

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pNIPAAm microgels by polymerizing Janus precursor droplets inside the microfluidic

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device.32 The Janus pNIPAAm precursor droplets were formed from two independent

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streams of linear pNIPAAm solutions with photo-chemical reaction groups and

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polymerized using strong UV light32. UV-polymerized anisotropic microgels can also

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be fabricated using stop-flow lithography microfluidic techniques.19,

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coworkers synthesized Janus microgels by injecting NIPAAm-rich and NIPAAm-poor

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phase into a hydrodynamic focusing microfluidic device (HFMD), followed by the

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on-chip polymerization of Janus droplets via UV light.34 Ma et al. prepared concave

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microgels by selectively polymerizing double emulsion droplets template.35 Besides

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UV-induced solidification of polymers, on-chip ionic crosslinking methods can be

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used to fabricate anisotropic microgels. Marquis et al. prepared both homo-Janus and

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hetero-Janus biopolymer microbeads by using a flow-focusing device for

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diffusion-controlled ionic crosslinking system.

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Janus alginate microgels by injecting sodium alginate solutions into a double

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T-junction microfluidic device and used on-chip cross-linking of the sodium alginate

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droplets.37 Lu et al. fabricated Janus-like chitosan-based dimer capsules using a

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T-junction microfluidic chip with four inlets.38 However, all the above-mentioned

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methods are limited to requiring the use of a complicated microfluidic device with

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multiple inlets to inject three or more precursor solutions. To overcome this limitation,

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Choi et al. employed dewetting to induce phase separation, followed by UV

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polymerization, obtaining complex structured microgels through single emulsion

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precursor droplets.39 This approach provides a simple method to fabricate anisotropic

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gel particles, but it is still limited to UV polymerized precursor droplets. To the best of

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The fabrication of

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Kim and

Zhao et al. fabricated magnetic

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our knowledge, there are no reports on the fabrication of anisotropic complex

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microgels by single emulsion droplets through an off-chip ionic crosslinking method.

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In this study, we performed a simple and versatile approach to fabricate

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shape-controllable poly (N-isopropylacrylamide) (pNIPAAm)/sodium alginate (SA)

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microgels using single emulsion droplets. We use a glass capillary microfluidic device

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to generate single water-in-oil emulsions; the monodisperse pNIPAAm/sodium

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alginate emulsions were collected in hot collecting solutions, composed of barium

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acetate (BA) mixed with different concentrations of glycerol aqueous solutions. We

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exploit a non-equilibrium phase separation, crosslinking properties of SA and

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coil-to-globule phase transition of pNIPAAm within the emulsions to fabricate

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microgels of different optical properties, geometries and surface microstructures. We

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control particle morphology, simply by tuning experimental parameters, such as the

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initial droplet diameter and the extent of phase separation, concentration of linear

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pNIPAAm and concentration of glycerol in the collecting solution. These complex

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microgels can potentially be used in the design of stimulus-responsive drug delivery

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vehicles, cell attachment materials, and for building porous three-dimensional tissue

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constructs.

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Materials. Poly (N-isopropyl acrylamide) (carboxylic acid terminated pNIPAAm,

MATERIALS AND METHODS

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average Mn 5000, Sigma-Aldrich),

sodium

alginate

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hexadecane (Anhydrous, 95%), barium acetate (purity > 99%), FeCl3 (Purity >97%),

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FeSO4·7H2O (Purity ≥ 99%), ammonia water (28.0-30.0% NH3 basis) have been

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obtained from Sigma-Aldrich and used without further purification. Glycerol and

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Cetyl PEG/PPG-10/1 Dimethicone Abil EM90 have been supplied by Macron Chem.

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and Evonik Co., respectively.

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Microfluidic setup

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The microfluidic device is composed of coaxially aligned tapered cylindrical glass

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capillaries, inserted into a square capillary and fixed on a glass slide. The schematic of

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the flow-focusing capillary-based microfluidic device is shown in Figure 1. Prior to

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inserting the cylindrical capillaries into the square capillary, we taper them using a 3

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micropipette puller to create a desired orifice diameter. The square capillary serves as

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a flow channel for the outer fluid stream and also facilitates the alignment of

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cylindrical capillaries. The left and right cylindrical capillaries serve as injection and

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collection tubes, respectively.

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Figure 1 Schematic of a co-flow glass capillary-based microfluidic device. Off-chip

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crosslinking was achieved by keeping the tip of the collecting tube in contact with the

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interface of a hot collecting solution, composed of barium acetate mixed with glycerol

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aqueous solution. Mushroom-like and Janus microgels are fabricated from droplets of

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300 µm and 150 µm diameter, respectively. The droplets were produced within the

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glass capillary microfluidic device at room temperature (~25

) and collected into

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glass beakers containing 4 mL of collecting solutions. The temperature of the

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collecting solution was maintained at 45

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The glass beakers were covered with aluminum foil during this process to minimize

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the evaporation of water.

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Formation of droplets

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Aqueous solutions of pNIPAAm/sodium alginate are emulsified in continuous

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Hexadecane oil phase containing 5 wt% Abil EM 90 that serves as surfactant (Figure

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S1). The collecting aqueous solutions are composed of 10 wt% barium acetate and

using a hot plate for at least 12 hours.

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0-40 wt% glycerol. The flow rates of the dispersed and continuous phases are fixed at

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150 µL/h and 3000 µL/h, respectively.

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Mushroom-like microgels are fabricated from initial droplets of about 300 µm

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diameter. The orifice diameter of the injecting tube is about 80 µm, and the orifice

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diameter of the collecting tube is 310 µm. Experiments are performed for 1 wt%

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pNIPAAm/1wt% SA and 2 wt% pNIPAAm/1wt% SA. Larger concentrations of

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pNIPAAm and sodium alginate lead to higher drop viscosity and formation of

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spherical particles. Droplets are collected in glass beakers containing aqueous solution

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of 10 wt% barium acetate and different glycerol concentrations. The collecting

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beakers are placed on a hot plate, whose temperature is kept at 45 °C. After about 12

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hours, the resultant fully crosslinked microgels are brought to the room temperature

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for further characterization.

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Janus microgels are formed out of initial droplets of 150 µm or smaller. In this case,

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the orifice diameter of the injecting tube is 60 µm and the orifice diameter of the

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collecting tube is 200 µm. The aqueous phase has the same composition as in the case

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of mushroom-like microgels.

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To observe droplet deformation at the air-water interface, we collect droplets in a

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Polystyrene Cuvette (VWR-5ml) containing 2 mL of collecting solution. To perform

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the temperature control experiment, we place the cuvette inside a thermostatic water

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bath with a constant temperature of 45 °C. The high speed camera (SA4 Fastcam,

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Photron) was focused at the interface of air and the collecting solution to monitor the

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droplets evolution process. To investigate the effect of droplet size, we utilize

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microfluidic devices with different injection and collection orifice sizes and we

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operate them under the same flow rates.

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Scanning Electron Microscope (SEM) measurements

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To prepare samples for SEM characterization, we wash and freeze-dried Janus

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microgels. In a typical experiment, we transferred crosslinked 150 µm microgels into

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a 10 mL centrifuge tube and centrifuged them at 2000 rpm for 2 min. The supernatant

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is removed and de-ionized water is added into the centrifuge tube followed by

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redispersion of microgels; the procedure is repeated 3 times. A drop of water 5

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containing purified microgels is put on a cleaned glass wafer (~8 mm×8 mm) and

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freeze-dried for 2 days. The freeze-dried samples are analyzed by a Field

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Emission-Scanning Electron Microscope (FE-SEM, Magellan 400) at a speed voltage

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of 3 KV.

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RESULTS AND DISCUSSIONS

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The monodispersed droplets created inside the microfluidic device and collected in

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collecting solutions are shown in the optical images in Fig. S1. The droplets exiting

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the collecting tube are slowed down at the interface of the collecting solution because

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their density is less than the density of the collecting solution (Table S1). The droplets

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deform and crosslink at the interface, and finally settle to the bottom of the beaker,

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which is in agreement with previous reports.40-42

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Mushroom-like microgels

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The mushroom-like microgels are formed out of 300 µm droplets. The observed

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mushroom shape has been reported for pure alginate hydrogel particles in a previous

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report.40 The shape is generated by the slow transition of the initially spherical

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droplets at the oil/collecting solution interface and by the accompanying crosslinking

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of the alginate via the inwards diffusion of Ba2+.41-42

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to the sodium alginate, droplets are composed of linear pNIPAAm polymer, which is

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one of the most widely used temperature responsive polymers, possessing a low

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critical solution temperature (LCST) of approximately 32°C. When the temperature of

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droplets increases above the LCST, the linear polymer undergo a coil-to-globule

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transition and shrinks, while below the LCST the polymer chain shows a swelling

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state. 43 Use of alcohol can results in pNIPAAm aggregation and limits its diffusivity44.

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Consequently, we employ glycerol within the collection bath to further control the

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morphology of microgels. This approach also enables us to tune the transparency of

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the resultant microgels selectively due to aggregation and formation of pNIPAAm

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microparticles within the main matrix (Figure S2). This indicates that both the

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temperature and glycerol cause the pNIPAAm chains to shrink, and lead to a change

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in the refractive index of the pNIPAAm/SA droplets. When the pNIPAAm/SA

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droplets flow out of the collecting tube and contact the hot collecting solutions, the

In the present study, in addition

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pNIPAAm chain instantly shrinks into a globule state, accompanied by a change in

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the refractive index from 1.32 to 1.39. 45 Thus, the collapsed pNIPAAm shows darker

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contrast due to light scattering of the aggregates and the refractive index mismatch

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with water (nwater = 1.33). In addition, the coiled pNIPAAm is gradually entrapped into

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the alginate network undergoing an ionic crosslinking process. Figure 2a shows

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microgels formed using a glycerol-free collecting solution. In this case, the resultant

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microgels exhibit an almost transparent mushroom shape. The transparent microgels

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indicate that little to no coiled pNIPAAm is entrapped in this case. When collected in

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40 wt% glycerol-collecting solutions, the microgels exhibit a mushroom shape with

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large amounts of interior black dots. These black dots in the mushroom microgels are

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pNIPAAm globules that are aggregated due to the addition of glycerol (Figure 2e).

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This phenomenon may be attributed to the diffusion of glycerol into the

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pNIPAAm/SA droplets and increase in the viscosity of the droplet. The inward

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diffusion of glycerol into the droplets limits the mobility of pNIPAAm globules and

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force them to form larger aggregates, resulting in a homogeneous entrapment of

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pNIPAAm particles (3~5 µm) in the resultant microgels. Different concentration of

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glycerol gives rise to different inward diffusion velocities and consequently different

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amount of aggregates. As a result, different features are observed for individual

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microgels collected in different glycerol concentrations. The mushroom microgel

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droplets collected in 10 wt% glycerol solutions display two different optical contrasts

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for each microgel, with one half nearly transparent, while the other half being opaque

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(Figure 2b). The different contrast is attributed to the inhomogeneous distribution of

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pNIPAAm aggregates. With increasing glycerol concentration, the mushroom-like

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microgels gradually show a homogeneous optical contrast (Figure 2c-e). This

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phenomenon suggests that higher glycerol concentration in the collecting solution

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inhibit the migration of pNIPAAm globules in the droplets, and as a result, the

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pNIPAAm particles will be uniformly distributed in the mushroom microgels.

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Not only is the microgels’ optical properties influenced by the collecting solutions,

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but their geometry also changes as a function of glycerol concentration in the

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collecting solutions. The geometric factor is defined here, as the length ratio of the 7

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distance between the poles (defined as L) to the diameter of the equator (defined as B),

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shown in the inset of Figure 2k. Clearly, the geometric factor decreases with the

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increase of glycerol concentration in the collecting solutions. The geometric factor

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falls within the interval 0.70~0.90 (Figure 2k), and continues to decrease with

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increasing glycerol concentration. By increasing the velocity of a continuous phase, a

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Figure 2. (a-e) Optical microscope images of microgels formed out of 300 µm

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diameter droplets, composed of 1 wt% pNIPAAm and 1 wt% sodium alginate and

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collected in collecting solutions with different glycerol concentrations: 0, 10 wt%, 20

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wt%, 30 wt% to 40 wt%. (f-j) Optical microscope images of microgels formed out of

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300 µm diameter droplets, composed of 2 wt% pNIPAAm and 1 wt% sodium alginate,

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collected in solutions with different glycerol concentrations: 0, 10 wt%, 20 wt%, 30

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wt% to 40 wt%. (k) The plot shows the geometric factor for different glycerol

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concentrations. All the samples were heated for 12 hours and then cooled down. All

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the images were taken at the room temperature. 8

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droplet falling into the interface shows greater degree of deformability, which

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provides another level of control to microgel geometry12.

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Similarly, when the concentration of pNIPAAm inside of the droplets was increased to

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2 wt%, more globule pNIPAAm were entrapped inside of the resultant microgels,

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compared to that of 1 wt% pNIPAAm. Figure 2f-j show the optical microscope

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images of microgels obtained from 2 wt% pNIPAAm/1 wt% SA droplets. As shown in

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Figure 2f-j, the microgels collected in the same glycerol collecting solution possesses

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higher contrast value compared to that of Figure 2a-e. The higher contrast was

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attributed to the higher amount of globule pNIPAAm entrapped in the microgels.

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Janus microgels

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By controlling the droplet size, we can also control the microgel morphology and

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obtain Janus shape particles. In the present experiment, when the initial droplet

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diameter is decreased to 175 µm or below, the resultant microgels show Janus

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morphology rather than the mushroom-like morphology formed out of 300 µm initial

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droplets. When the initial droplet diameter is below this critical value, Janus particles

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form, whereas above this value, the resultant microgels show mushroom morphology

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(Figure S4). We attribute this change in the microgel morphology to the combined

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effects of two factors: a) smaller droplets experience a greater extent of phase

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separation between pNIPAAm and SA and possess pNIPAAm-rich and SA-rich

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regions, prior to touching (impacting) the collecting solution interface46-47, and b) the

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viscosity, surface tension and osmotic pressure difference between drops and the

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collecting solution. The collecting solutions with higher glycerol concentrations

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possess higher viscosity and lower interfacial tension, these properties result in a

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greater degree of deformability in drops upon impacting the interface. Smaller

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droplets tend to form Janus particles because they are more difficult to deform due to

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their larger Laplace Pressure difference. The Laplace Pressure difference can be

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calculated by the following equation ∆ =

 

, where  is the interfacial tension

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between the droplet and outer oil phase, and is the droplet radius. We used the

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pendant drop method to measure the surface tension between aqueous solution of 9

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sodium alginate (2 wt%) and pNIPAAm (1 wt%) and hexadecane containing 5 wt. %

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Abil EM90. The measured  is 2.35 mN / m , and the ∆ values of the 150 µm and

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300 µm droplets are 31.33 Pa and 15.66 Pa, respectively. Larger droplets are easily

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deformed to a mushroom shape under the driving force of Osmotic pressure

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difference (the minimum osmotic pressure of the collecting solutions is ~ 390 mOsm,

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which is much higher than that of droplet’s osmotic pressure (~ 10 mOsm)) between

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inside and outside of droplets, as shown in Figure 2. Janus microgels collected in

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different glycerol concentrations demonstrate different geometry as shown in Figure 3.

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Figure 3a-e and Figure 3f-j represent the microgels fabricated out of droplets of 1wt%

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pNIPAAm/1wt% SA and 2 wt% pNIPAAm/1 wt% SA, respectively. Both images

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show the resultant microgels with two distinguishable parts: a relatively smaller part,

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which exhibits a darker contrast, richer in pNIPAAm, and a relatively larger

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alginate-rich part. The formation of two different phases is caused by the gradual

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phase separation of pNIPAAm inside the droplets when in contact with the hot

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collecting solutions (Figure S5). Compared to the larger droplets, i.e., 300 µm

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diameter droplets, the pNIPAAm chain in smaller droplets more easily migrate to the

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other side of the droplets and result in Janus morphology. The glycerol and the effect

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of heat could both induce the pNIPAAm chains to shrink into a globule state (Figure

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S2-4). Interestingly, the resultant microgels in glycerol-free collecting solutions only

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show the convex alginate dominant part (Figure 3a and 3f). This is due to the phase

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separation of pNIPAAm and SA within the droplets. However, the size of

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pNIPAAm-rich part gradually decreases with increasing the glycerol concentration of

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the collecting solution due to the entrapment of some of the coiled pNIPAAm in the

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alginate-rich side, as shown in Figure 3b-e and 3 g-j. To quantify the relative volume

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change of the two parts, we define the volume ratio as V1/V2 (V1 and V2 denote the

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volumes of pNIPAAm-rich and alginate-rich parts, respectively). Similar to the

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geometric factor for the mushroom-shaped microgels, the volume ratio of Janus

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particles decreases with increasing the glycerol concentration of the collecting

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solutions (See Figure 3k). The volume ratio decreased from the initial values of 0.20

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and 0.23 for 10 wt% glycerol collecting solutions to the final values of 0.11 and 10

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nearly 0.02 for 40 wt% glycerol collecting solutions. The decrease is attributed to the

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inward diffusion of glycerol from the collecting solutions into the droplets, which

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hinders the pNIPAAm separation from the main droplets and leads to the uniform

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distribution of pNIPAAm globules inside the microgels at high concentrations of

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glycerol collecting solutions. Furthermore, the phase separation process is inhibited

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by higher sodium alginate concentrations in the droplets (Figure S6, S7).

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Figure 3. (a-e). Optical microscope images of microgels formed out of droplets of

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150 µm diameter composed of 1 wt% pNIPAAm and 1 wt% sodium alginate and

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collected in collecting solutions of different glycerol concentrations: 0, 10wt%, 20

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wt%, 30 wt% to 40wt%. (f-j) Optical microscope images of microgels formed out of

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droplets of 150 µm diameter composed of 2 wt% pNIPAAm and 1 wt% sodium

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alginate and collected in collecting solutions of different glycerol concentrations: 0,

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10 wt%, 20 wt%, 30 wt% to 40 wt%. (k) The plot shows the volume ratio for Janus

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particles for different glycerol concentration of the collecting solutions.

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Figure 4. Scanning Electron Microscope (SEM) images of microgels collected in

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different concentrations of glycerol solutions: (a) 0; (b) 10 wt%; (c) 20 wt%; (d) 30

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wt%; (e) 40 wt%.

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Figure 4 represents SEM images of alginate-rich part of microgels formed by using

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collecting solutions of different glycerol concentrations. It can be clearly observed

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that the surface morphology of the microgel changes with glycerol concentration of

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the collecting solutions. As shown in the SEM images, the number of dents on the

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surface increases but their size decreases as the glycerol concentration in the

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collecting solution increases.

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Ultrasonication of Janus microgels

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The present study demonstrates that not only we can controllably fabricate microgels

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of different geometries by carefully adjusting the composition of collecting solution,

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but also we can selectively isolate different part of microgel using ultrasonication.

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Janus microgels formed in collecting solutions containing 10wt% glycerol are purified

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by centrifugation at 2000 rpm to remove the supernatants. This procedure is repeated

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2~3 times. The purified microgels are transferred into a 5 mL centrifuge tube and

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sonicated in an ultrasonicator (VWR, 97043-968) for 1 min. Figure 5 shows the

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breakup process of the initial Janus microgel formed from 10 wt% glycerol collecting

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solution. The Janus microgels breakup when they are placed in an ultrasonicator for 1

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min. The pNIPAAm-rich part is separated from the alginate-rich part due to the weak

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tangling interaction between alginate molecule chains and pNIPAAm molecule

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chains.48 Once further sonicated, the separated pNIPAAm-rich part gradually

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dissolves (as illustrated by red arrows in Figure 5d). The pNIPAAm is a water soluble

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polymer and gradually swells in the aqueous solution and the collapsed pNIPAAm

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chains recover their coil structure. The same behavior is observed when Janus 12

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particles are fabricated using higher glycerol concentrations (such as 50wt% and

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60wt%, corresponding to Figure S5 c and d, respectively) in the collecting solutions.

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Figure 5. (a) and (c) shows the Janus particles (b) and (d) shows that Janus microgels

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are broken to two parts by using external ultrasonication. Red arrows in (d) illustrate

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disintegrated pNIPAAm. The experiment was conducted at the room temperature.

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CONCLUSIONS

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In summary, we report a simple method to fabricate pNIPAAm/sodium alginate

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microgels through single emulsion generated in microfluidics combined with an

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external ionic crosslinking process. Monodispersed droplets composed of linear

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pNIPAAm and sodium alginate are collected in hot collecting solutions of barium

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acetate and glycerol at different concentrations. Starting out of initial droplets of 300

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µm and 150 µm in diameter, mushroom-like and Janus microgels can be attained. The

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geometric factor and optical properties of the mushroom-like microgels can be simply

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tuned by changing the glycerol concentration of the collecting solutions. Similarly, by

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varying the glycerol concentration of the collecting solutions, the relative volume

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ratio and the surface roughness of the Janus microgels can be varied. The Janus

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microgels can be broken to two parts by using ultrasound and can be potentially

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extended to other stimulus responsive materials. The resultant microgels may be

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applied in novel optical materials, superstructure microgels, target drug delivery

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materials, lock-key particles and highly efficient cell attachment (or regenerative 13

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medicine).

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ASSOCIATED CONTENT

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Supporting Information

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Supporting Information including the microfluidic fabrication of droplets and

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other supplementary data are available free of charge at http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author

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*E-mail: [email protected]

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Notes

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The authors declare no conflict of interest.

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ACKNOWLEDGEMENTS

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This work was supported by the NSF Grant No. CBET-1150348. We thank Notre

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Dame Integrated Imaging Facility Centre for the SEM measurements.

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