Microfluidic production of snowman-shaped Janus hydrogel particles

Accepted Manuscript Title: Microfluidic production of snowman-shaped Janus hydrogel particles Authors: Somnath Ghosh, Peter Schurtenberger PII: DOI: Reference:

S0927-7757(19)30337-1 https://doi.org/10.1016/j.colsurfa.2019.04.034 COLSUA 23378

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

22 February 2019 12 April 2019 14 April 2019

Please cite this article as: Ghosh S, Schurtenberger P, Microfluidic production of snowman-shaped Janus hydrogel particles, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2019), https://doi.org/10.1016/j.colsurfa.2019.04.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Microfluidic production of snowman-shaped Janus hydrogel particles Somnath Ghosh*a and Peter Schurtenbergera a

Division of Physical Chemistry, Lund University, Lund, Sweden. * [email protected].

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Abstract: We present a simple microfluidic-based synthesis protocol to fabricate snowman like Janus hydrogel particles of poly(N-isopropylacrylamide) (PNIPAM) in micron length scale. Particles are synthesized by emulsifying a hydrogel solution (water (w)/oil (o)) followed by partial solvent evaporation. Solvent evaporation allows the hydrogels to concentrate within the droplet and phase separate into a monomer-abundant and a sparse phase. Due to the difference in interfacial tension and chemical properties of the two phases, emulsion droplets are deformed into snowman like Janus droplets. After cross-linking the monomer by UV irradiation, the particles are transferred into water. The resulting Janus colloids exhibit thermo-responsive behavior and form self-assembled structures.

Keywords: Janus particle, microfluidics, hydrogel particle 1. Introduction

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Particles with two distinct physical and/or chemical properties, known as Janus particles, have attracted enormous attention for their potential for various applications ranging from micro-actuators [1] to drug delivery [2]. Also, these particles are widely used as model colloid systems in many fundamental studies on self-assembly [3], active motion of colloids [4-6], colloidal aggregation [7], etc. Due to two different properties, Janus particles have emerged as a new class of colloids and offer the possibility to create smart materials and structures with new or enhanced properties through programmed self-assembly. As a result, the research interest in developing effective methods to synthesize those particles in large quantities is increasing. Over the years, many promising methods have been developed to produce hydrogel based Janus micro-spheres with varying surface chemistries [8-15]. Recently, microfluidic devices have appeared as a promising tool to fabricate functional materials with exciting morphologies and structures [13, 16-19]. They offer an excellent control on synthesizing anisotropic particles and a possibility to scale up with high precision [20]. However, most of the reported methods to synthesize Janus particles are very similar, and require laminar co-flowing streams of multiple polymeric solutions and systematic break-up into monodisperse Janus droplets by the hydrodynamic forces induced by a third continuous oil phase [21]. The polymer network within the droplet is cross-linked by rapid polymerization immediately after the drop generation within the microchannel to keep the anisotropic nature intact. In this approach, a stable interface between dissimilar solutions before cross-linking is a prerequisite to form Janus particles. It is also necessary to consider the diffusive length scale and transport of monomers across the interface, which significantly restrict the final size of the particles. Otherwise, any perturbation or high diffusivity of the polymer will lead to convective cross-mixing, and result in particles with undesirable blended internal architecture [1, 11]. Therefore, it is still challenging to synthesize microfluidic-based functional particles in the micron length scale. Also, very few methods have been developed [8, 9], so far, to produce Janus particles from the same base material with clearly distinguishable anisotropic features. poly(N-isopropylacrylamide) (PNIPAM) is one of the most commonly used polymers to synthesize bio-compatible soft-materials for drug delivery [22] and tissue engineering [23]. It is also sensitive to the changes in environmental conditions, such as, temperature, pH, ionic strength, electric fields, etc. At room temperature, the amide groups of PNIPAM are solvated and water is a good solvent for PNIPAM, which results in highly swollen networks. At an elevated temperature of about 32°C, known as the volume phase transition temperature (VPTT), hydrogen bonds with water molecules become weaker, water now becomes a poor solvent, and the networks collapse to hydrophobic spheres with a significantly higher density. Apart from the polymer chains, N-isopropylacrylamide (NIPAM) monomers also show an interesting phase behavior at room temperature. Recently, it has been reported that supersaturated NIPAM solutions undergo a liquid-liquid phase separation [8, 24]. Based on this phenomenon, binary droplets are formed by co-flowing those two immiscible phases in two separate channels to fabricate thermo-responsive Janus particles. However, PNIPAM-based Janus hydrogel particles developed so far are much larger (~ 100 µm) than the colloidal limit (10 nm to 10 µm), which potentially hinders many fundamental studies. In this article, we report a simple but effective method to prepare snowman shaped Janus hydrogel particles in micron length scale and study their assembly. In this protocol, instead of multiple phases, a single aqueous phase with a monomer solution flows through the microchannel and breaks into droplets, thus eliminating the problem of cross-mixing and overcoming the limitation of the final size of those Janus particles. We use emulsion droplets of a NIPAM monomer solution as a template. A partial evaporation of water in a nitrogen environment results in a concentration-induced phase separation of the NIPAM monomer solution in the initial droplets, and thus in the formation of Janus droplets. Janus particles are then created by photo cross-linking the monomers in the Janus droplets through UV irradiation. The resulting thermoresponsive PNIPAM particles have two independent anisotropic features: (a) geometric anisotropy due to the snowman-like shape and (b) chemical anisotropy as the two parts have a different polymer density, which results in a corresponding difference in charge density and temperature-dependent interaction parameters. Note that the phase separated Janus droplets are referred as particles once they are polymerized. 2. Experiment 2.1 Materials and method

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For this synthesis protocol, we used N-Isopropylacrylamide monomer (NIPAM, 97%, Aldrich) as base material, N, N’-methylenebis(acrylamide) (BIS, 99%, Sigma-Aldrich) cross-linker, 2,2′-Azobis(2methylpropionamidine) dihydrochloride (granular, 97%, Sigma-Aldrich) photo initiator, methacryloxyethyl thiocarbamoyl rhodamine B dye (MRB, Polysciences Inc.), and fluorinated oil (HFE-7500, 3M) with Pico-Surf 1 (Dolomite) surfactant. 1H,1H,2H,2H-Perfluoro-1-octanol (PFO, 97% Aldrich), hexane (95%, Sigma-Aldrich), span 80 (Sigma-Aldrich) and triton X-100 (laboratory grade, Sigma-Aldrich) were used to wash Janus particles. All chemicals were used as received. To prepare a homogeneous hydrogel solution, we added 50 mg NIPAM, 2.3 mg BIS, 10 mg UV initiator, 1 mg MRB in 10 ml Milli-Q water and vortexed the mixture for 5 min prior to emulsification. For the oil phase, 5 ml 5 wt% Pico-Surf 1 was diluted by pure oil to make a ~1.5 wt % surfactant solution. The surfactant concentration is sufficient enough to stabilize 20 µm emulsion drops for a long time, and we have verified that the emulsion remains stable even after four months of generation. Once the concentrated NIPAM monomer was phase separated and formed Janus emulsions, they were crosslinked through UV polymerization (365 nm, 27W/cm2). After UV cross-linking, the particles were washed and stored in Milli-Q water.

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2.2 Confocal microscopy A confocal laser scanning microscope (Leica SP5), operated in the inverted mode, was used to visualize the evolution of the Janus architecture in the w/o emulsion and investigate the self-assembly of those particles in water. Samples were prepared on glass cover slides with a spacer of 120 µm and illuminated with a He–Ne laser (543 nm wavelength) to visualize the emulsion droplets and particles that were dyed with rhodamine B. The confocal microscope is enclosed within a temperature controlled cabinet where temperature can be controlled with an accuracy of 0.2 ºC. To visualize the thermoresponsive behavior of the particles and monitor the formation of assembled structures, we increased the temperature of the chamber to 35 ºC and kept all the samples for 30 minutes to equilibrate before imaging.

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2.3 Microfluidics and drop generation In our experiments, microfluidic flow focusing devices (MFFD) have been used to generate emulsion drops at high frequency. Those devices were fabricated by replica molding [25] of Polydimethylsiloxane (PDMS) on negative (with respect to the channel) SU8 structures over a silicon wafer. First, the channel design (L-Edit software, Tanner Research) was imprinted on 4-inch chrome plated soda lime photomasks (Delta Mask B.V.) and then developed on spin coated negative SU8 photoresists of 10 µm thickness. Once the design was developed on SU8, the channel structure was hydrophobized by depositing a thin layer of 1H,1H,2H,2H-perfluorooctyltrichlorosilane (PFOTS, ABCR GmbH & Co. KG) to facilitate exfoliation of PDMS. PDMS monomer mixed with 10% curing agent (Sylgard 184, Dow Corning) was poured on SU8 and heated for 1 hour at 80 °C to cure it. The channel replica in PDMS was peeled off and bonded with a glass slide before connecting to the pressure pump. The method of drop production is shown schematically in Fig. 1a. All the edges and the top of the PDMS slab were glued with Norland Optical Adhesives (NOA) to make the channel air tight (Appendix A) so that any evaporation inside the channel after drop generation can be prevented. Fig. 1b is a typical example of a microfluidics device during drop production. The device consists of a rectangular cross-section of 10 x 100 µm2 at the inlet and 10 x 200 µm2 at the outlet. Droplets are generated at the square (10 x10 µm2) intersection area where two immiscible fluids meet.

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PDMS slabs with embedded channel design and the glass slides were treated with oxygen plasma (Plasma Preen II-862, Plasmatic Systems) for one minute prior to bonding with each other, and three holes were punched into them to Figure 1: (a) Schematic Illustration of drop generation (top view) in a microfluidic connect the channel with device. (b) Optical image of microfludic device, producing 20 µm drops. Scale bar reservoirs and the emulsion 50 µm. (c) Confocal image of the drops dispersed in HFE-7500 oil, after 2h of collector. The continuous generation. Scale bar 20 µm. oil phase, i.e. fluorinated oil (HFE-7500, 3M), and the aqueous mixture were injected via two inlets of the channel by a pressure pump (Fluigent). The aqueous solution flowed through the central inlet, whereas the oil phase was divided into two streams before it interacted with the aqueous phase at the cross-junction and split the aqueous stream into uniform droplets (Fig. 1b, c). Once NIPAM was phase separated after partial solvent evaporation, the monomer network was cross-linked by UV irradiation (Thorlabs, 365 nm, 27W/cm2), and the resulting hydrogel particles were washed and transferred to water. c)

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2.4 Washing protocol After UV photo polymerization, particles were washed based on the previous work by Klein et. al. [26]. In this protocol, first, the drops were demulsified by mixing 0.4 ml emulsion with 50 µl PFO and 1.5 ml hexane with 1% (v/v) span 80. The solvent mixture was vortexed for 30 s and centrifuged for 5 min at 3000 rpm. As the average density of the Janus particles (~1.0 g/cm3) is higher than the solvent mixture (~ 0.8 g/cm3), particles quickly settled as a sediment at the bottom. 1.5 ml of the top solvent was removed and replaced by the same amount of water with 0.1% (v/v) triton X-100 and centrifuged at 5000 rpm for 5 min. This step was repeated for a second time, and finally pure milli-Q water was added to suspend the Janus particles in a surfactant and oil free environment (see movie 1).

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3. Result and discussion 3.1 Evolution of Janus colloids Janus microspheres were produced in a two-step method as described schematically in Fig. 2a. First, the monomer solution was emulsified for 2h at a rate of 2000 drops per second and collected at the downstream emulsion collector. After collection, the water in the water-in-oil emulsion was evaporated slowly in a Nitrogen (N2) environment, resulting in a shrinkage of the emulsion droplets and an increasing NIPAM concentration. During evaporation, the sample was stirred at 300 rpm, and 150 µl of the emulsion was collected every 20 min to inspect the drying process and observe the evolution of the Janus droplets (Fig. 2b). In the first 60 minutes, the droplets shrunk without any obvious change of droplet shape. Further reduction of the drop size by evaporation triggered the phase separation, and each emulsion drop evolved into a Janus architecture with two different phases of the same material (Fig. 2a,b). The resulting Janus-like particles after cross-linking are shown in Fig. 3 before and after washing. It is interesting to note in Fig. 3a that when suspended in oil, all Janus particles seem to align with their dense part on top. As the oil is very volatile it evaporates quickly, leaving the Janus particles floating on a thin oil film above the glass substrate. As the concentrated NIPAM phase has a lower density (0.93 ± 0.01 g/cm3) than the dilute phase (0.99 ± 0.01 g/cm3)[8, 24], this results in a densitydriven alignment of the particles (movie 2). Phase separated Janus droplets were exposed to UV light for 30 s to polymerize and cross-link the monomers, and the resulting cross-linked PNIPAM Janus microgels were transferred to water (Fig. 3b) following the method described in section 2.4.

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Figure 2: (a) Schematic illustration showing the evolution of Janus particles with evaporation and subsequent photo polymerization. Blue arrows indicate evaporation of water from the droplet. (b) Change in drop size during evaporation as a function of time. Drop size is measured off-chip, and each data point is obtained by measuring the diameter of 40 to 50 droplets over 10 frames. t=0 indicates the time point when the droplets start evaporating in the presence of a rotating magnetic bar under N2. Solid line is a guide to the eye. Inset shows snowman Janus particles suspended in oil. Scale bar of the images below the line is 20 µm and above the line is 2 µm.

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Around ~ 20% of the surface area of each particle in the oil phase is occupied by the dense phase. During evaporation, the monomer concentration within each droplet increases and reaches a supersaturated state that undergoes liquid-liquid phase separation, similar to the bulk phase separation reported for NIPAM [8, 23]. Those two phases are immiscible and deform the droplets to create a snowman-like shape. In our experiments, we have observed that phase separation starts when the droplet size reduces to ~ 4 µm in diameter, which corresponds to an average NIPAM concentration of ~ 62.5 wt% inside the droplet. Driven by interfacial tension and the density difference, the Janus architecture evolves. Continued further evaporation of water results in the formation of single-phase deformed particles (Appendix B) which we could not recover after washing. a)

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Figure 3: (a) Janus particles suspended in HFE-7500 oil. As the oil is very volatile, it evaporates quickly, leaving the Janus particles floating on the thin film above the glass substrate. Due to low oil density, the particles align with their dense NIPAM part on top. (b) Janus particles suspended in water after washing. Inset: typical shape of individual particle. (c), (d) are the bright-field images of the particles in oil and water medium respectively. Scale bar 5µm.

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3.2 Self-assembly experiments After washing, the particles retain their shape and remain stable in water, as shown in Fig. 3b. Each particle has two distinct features; a dense head and a lower density body, and confocal images clearly reveal their anisotropic shape. We have further investigated the effect of temperature on their interactions. As the particles are weakly positively charged, where the charges originate from the photo initiator, the particles are soft repulsive at a temperature below and above the VPTT and do not form any aggregates (Fig. 4a). Once the surface charge is screened by adding potassium chloride (KCl) salt to the suspension, the particles assemble into clusters. We systematically varied the salt concentration and observed aggregation for T = 35 °C > VPTT. At 10mM salt solution, only a few particles form binary clusters and most particles remain individually suspended (Fig. 4b). Increasing the salt concentration increases the number of clusters and significantly less individual particles are observed. Once the salt concentration is increased to 100 mM, all the particles aggregate into larger clusters (movie 3). To our surprise, all the aggregates are formed based on the attraction between the less dense phases forming the body of the snowman particles, independent of salt concentration. As the less dense phase is larger even after shrinking at elevated temperatures, inter-particle attraction (van der Waals) through the body is larger than for the denser small top. Fig. 4b-d are typical examples of such aggregates in different salt solutions.

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Figure 4: Effects of salt concentration at a temperature (35 °C) above the VPTT (32°C). (a) no cluster formation in absence of KCl. (b) Few binary clusters in 10mM KCl solution. (c) Binary clusters in 20 mM KCl solution. (d) Random clusters in 100 MM KCl solution. Scale bar 2 µm.

3.3 Synthesis of Janus hydrogels in Mineral oil:

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Finally, we have verified the production of Janus particles in a different oil medium. The emulsification of the same aqueous solution in mineral oil produces similar snowman like Janus droplets. However, it takes longer time to form the Janus droplets, as the evaporation of water through the mineral oil is significantly slower than through the fluorinated oil. The formation of Janus droplets in mineral oil highlights the robustness of the synthesis protocol. To stabilize the w/o emulsion, ABIL EM 90 surfactant (5wt%) was used as emulsion stabilizer. As the mineral oil (density ~ 0.8 g/cm3) is lighter than water, unlike in HFE-7500 oil, emulsion droplets sediment at Figure 5: Janus particles synthesized and the bottom of the emulsion collector. Fig. 5 shows snowman suspended in mineral oil. Scale bar 5 µm. Janus particles synthesized in mineral oil. It is worthwhile to mention that the Janus droplets in Fig. 5 are formed in an air environment. However, a Nitrogen environment is essential during photopolymerization.

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4. Conclusion In summary, we have demonstrated a novel and facile synthesis method to produce micron-sized snowman-shaped Janus particles from the same monomer and investigated their temperature induced assembly. This synthesis protocol is highly versatile and can easily be scaled-up for mass production of thermoresponsive Janus particles. As the particles are formed by induced phase separation within the emulsion, it can easily be extended to any size range by adjusting the initial hydrogel monomer concentration and drop size. The ability to fabricate responsive Janus colloids, i.e. particles that are dominated by Brownian motion rather than sedimentation, is essential for future applications in controlled self-assembly. Here we have demonstrated that once the particle surface charge is screened through the addition of salt, particles self-assemble at a temperature above the VPTT. Due to their dual anisotropic nature, these particles can thus be used as a model colloid system for self-assembly and gel formation to understand the material efficient design of anisotropic colloids.

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Acknowledgements The authors acknowledge Feifei Peng for the chip design, Linda Månsson and Brijitta Joseph for fruitful discussions. The work was financially supported by the European Research Council (ERC339678-COMPASS and the Knut and Alice Wallenberg Foundation (project grant KAW 2014.0052). All the devices were fabricated in NanoLund at Lund University.

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Conflicts of interest There are no conflicts to declare.

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Appendix A Microfluidic device: In our experiments, microfluidic flow focusing device (MFFD) have been used to generate emulsion drops at high frequency. Fig. 6 is an example of a chip we used for our experiments. We glued the edge and top of PDMS slab to make the channel air tight so that any evaporation inside the channel after drop generation can be prevented.

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Figure 6: Camera image of a chip after sealing with NOA (Norland Optical Adhesives) glue.

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Appendix B Deformed particles We emulsify aqueous solution of NIPAM using MFFD at 2000 Hz for 2h and collect them in sealed glass vial. As the mass density of oil (~1.7 g/cm3) is much higher than the aqueous mixture, droplets stay at the top. We systematically observed the emulsion during evaporation under confocal microscope (Leica, 100X objective, 20 ºC, 543 nm He-Ne laser) to visualize the formation of snowman shaped Janus particle. While evaporation, emulsion droplets gradually shrink and evolve to anisotropic shapes. When a drop reaches to ~4µm in diameter, we first observe the phase separation; denser phase stays at the top (head) and emits stronger fluorescence signal under microscope compared to dilute phase which stays below(body). Further evaporation does not produce smaller Janus particles. Instead, all the monomer phase separates and form bowl shaped, deformed particles (Fig. 7).

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Figure 7: Typical image of deformed particles after 100 min of evaporation (in oil). Inset: close look of a deformed bean shaped particle. Scale bar is 2 µm.

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