Symposium Organizers
Anderson Shum, The University of Hong Kong
Takahiko Ban, Osaka University
Christine Keating, The Pennsylvania State University
Shuichi Takayama, University of Michigan
SM5.1: Hydrodynamics of Aqueous Two-Phase Systems (ATPS) Droplets
Session Chairs
Wednesday PM, April 19, 2017
PCC North, 100 Level, Room 122 B
9:15 AM - *SM5.1.01
Moving through Intracellular Phase Space
Clifford Brangwynne 1
1 Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States
Show AbstractIn this talk I will discuss our work showing that phase transitions play an important role in organizing the contents of living cells. We focus on a class of membrane-less RNA and protein rich organelles, known as RNP bodies, which help control the flow of genetic information within cells. The nucleolus is one such nuclear RNP body, which is important for cell growth and size homeostasis. We've shown that a phase transition model explains many features of nucleolar assembly. Recently, we've also shown that the internal subcompartments of the nucleolus arise from multi-phase coexistence, which may have important consequences for sequential RNA processing. I will also discuss our new "Optodroplet" approach, which enables spatiotemporal control of phase transitions within living cells, allowing us to begin quantitatively mapping intracellular phase diagrams. This approach has begun to yield rich insights into the link between intracellular liquids, gels, and the onset of pathological protein aggregation.
9:45 AM - *SM5.1.02
Membrane Formation by Interfacial Complexation in Aqueous Two-Phase Systems (ATPS)
Kathleen Stebe 1 , Sarah Hann 1 , Daeyeon Lee 1
1 , University of Pennsylvania, Philadelphia, Pennsylvania, United States
Show AbstractTo encapsulate fluid within a membrane, emulsion-based methods are often exploited; a drop of fluid is dispersed in an immiscible fluid, for example, an aqueous drop in an external oil phase. Complexing molecules or particles present in either phase adhere to the fluid interface, trapped there by interfacial tension, to form a membrane. Advantages to this technique include the broad ranges of materials/molecules that can be trapped at fluid interfaces and incorporated into membranes. There are important limitations, however, as oil-water interfaces can be hostile to delicate biological media as cargo or membrane components. We are developing strategies to exploit ATPS as droplet and external phases to encapsulate cargo based on complexation of polyelectrolytes dispersed in either phase. These systems pose interesting challenges, as the interfacial tension is extremely low, so interfaces may not be effective trapping sites. We show that the key to obtaining microcapsules is to tune the relative fluxes of the polyelectrolytes to meet and complex at the interface. New strategies to broaden this class of membranes to include other functional structures are discussed.
10:15 AM - SM5.1.03
Microfluidic Generation of Particle-Stabilized Water-in-Water Emulsions
Niki Abbasi 4 1 3 , Maryam Navi 2 1 3 , Scott Tsai 4 1 3
4 Department of Mechanical and Industrial Engineering, Ryerson University, Toronto, Ontario, Canada, 1 Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada, 3 Institute for Biomedical Engineering, Science and Technology (iBEST)-, Partnership between Ryerson University and St. Michael’s Hospital, Toronto, Ontario, Canada, 2 Department of Biomedical Engineering, Ryerson University, Toronto, Ontario, Canada
Show AbstractWe present a microfluidic system that generates water-in-water Pickering Emulsions, stabilized by Carboxylate microparticles, using an aqueous two-phase system (ATPS) of Polyethylene Glycol (PEG) and Dextran (DEX). DEX droplets are created at a flow-focusing junction in continuous PEG and carboxylate particle suspension phase, using weak hydrostatic pressure. Due to the low interfacial free energy of carboxylate particles on the ATPS interface, carboxylate particles partition to the interface of the ATPS and cover the DEX droplets; therefore, stabilizing the DEX droplets. Further downstream, stabilized droplets are collected in a reservoir. The effects of the carboxylate particle concentration and interfacial tension of the ATPS on the stability of the DEX droplets are studied. We anticipate that water-in-water Pickering Emulsions may have important biotechnological applications, due to their biocompatibility compared to traditional oil-in-water Pickering Emulsions.
10:30 AM - SM5.1.04
Magnetic Manipulation of Droplets in an Aqueous Two Phase Microfluidic System
Maryam Navi 1 2 3 , Niki Abbasi 4 2 3 , Scott Tsai 4 2 3
1 Biomedical Engineering, Ryerson University, Toronto, Ontario, Canada, 2 Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada, 3 Institute for Biomedical Engineering, Science and Technology (iBEST), A Partnership between Ryerson University and St. Michael’s Hospital, Toronto, Ontario, Canada, 4 Mechanical Engineering, Ryerson University, Toronto, Ontario, Canada
Show AbstractWe report on-chip magnetic droplet generation and manipulation using an aqueous two phase system of Polyethylene glycol (PEG) and Dextran (DEX). Droplets are hydrodynamically generated at a flow-focusing junction, with PEG being the sheath flow and the ferrofluid rich DEX being the dispersed phase. A permanent magnet is used downstream in a separation region to sort the droplets based on their size. Substitution of the continuous oil phase, usually used in micromagnetofluidics, with an all aqueous system eliminates the need for subsequent washing steps. This system of magnetic droplet manipulation may have potential in handling biomedical samples in lab-on- a-chip applications.
10:45 AM - SM5.1.05
Controlled Electrospray Generation of Non-Spherical Aqueous Microparticles
Morteza Jeyhani 1 3 4 , Sze Yi Mak 6 7 , Stephen Sammut 3 4 5 , Anderson Shum 6 7 , Dae Kun Hwang 2 3 4 , Scott Tsai 1 3 4
1 Department of Mechanical and Industrial Engineering, Ryerson University, Toronto, Ontario, Canada, 3 Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Ontario, Canada, 4 , Institute for Biomedical Engineering, Science, and Technology (iBEST)- a partnership between Ryerson University and St. Michael’s Hospital, Toronto, Ontario, Canada, 6 , HKU-Shenzhen Institute of Research and Innovation (HKU-SIRI), Shenzhen, Guangdong, China, 7 Department of Mechanical Engineering, University of Hong Kong, Hong Kong Hong Kong, 5 Department of Electrical and Computer Engineering, Ryerson University, Toronto, Ontario, Canada, 2 Department of Chemical Engineering, Ryerson University, Toronto, Ontario, Canada
Show AbstractProduction of aqueous polymeric microparticles has attracted increasing interest because they benefit a variety of new biomedical applications such as cell encapsulation. Here we present a technique based on the application of electrospray to generate aqueous non-spherical microparticles using sodium alginate as emulsion phase and calcium chloride for continues phase. This approach allows the formation of calcium alginate microbeads with tunable sizes and shapes. We use a high voltage power supply to form the electric field, we apply a charge to the alginate solution that flows through a glass capillary with a tapered tip, and we ground a metallic ring that is positioned beneath the capillary tip. We investigate the effects of changing various parameters such as voltage, flow rate, capillary tip size, reagent concentration, and the distance from the capillary tip to the free interface of the calcium chloride bath, and discover parameter spaces that yield a variety of different shape and size particles. This technique of aqueous microparticles may have applications in drug delivery, enzyme immobilization, cell encapsulation and drug screening and further investigation of this technique can lead to a simple but high throughput method for generation of three-dimensional culture system in cancer studies.
11:30 AM - *SM5.1.06
Microfluidic Water-in-Water Droplets—Passive Generation, Cargo Encapsulation, and Controlled Release
Scott Tsai 1 2
1 Department of Mechanical and Industrial Engineering, Ryerson University, Toronto, Ontario, Canada, 2 Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
Show AbstractAqueous two-phase systems (ATPS) are formed when two incompatible polymers are mixed in water and phase-separated above a critical polymer concentration. The all-biocompatible nature of ATPS makes ATPS-based water-in-water droplets promising for cellular encapsulation applications.
Despite the promise of all-compatibility, generating water-in-water droplets in microfluidics has remained challenging. Namely, applying classical water-in-oil droplet microfluidics approaches to generate water-in-water droplets has not been possible due to the very low interfacial tension of ATPS.
Here, we describe a simple and completely passive microfluidic technique that generates water-in-water droplets with a high degree of monodispersity. This is accomplished by applying hydrostatic pressure to create very slow and stable flows. We demonstrate this method's application to encapsulating particles and cells. Finally, we exploit the all-aqueous nature of ATPS to show that the generated water-in-water droplets can grow, shrink, and release its cargo, by simply tuning the phase-separation equilibrium of the ATPS in the microfluidic system.
12:00 PM - *SM5.1.07
Chemical Control of Hydrodynamics in Aqueous Systems
Yuichiro Nagatsu 1
1 Department of Chemical Engineering, Tokyo University of Agriculture and Technology, Tokyo Japan
Show AbstractWhen chemical reactions change property of liquid in flow regarding of flow motion such as density and viscosity, the hydrodynamics are changed by the reactions. In this talk, we introduce our studies on control of a hydrodynamics by chemical reactions in aqueous systems. We especially focus on viscous fingering as an instance of hydrodynamics. Viscous fingering is one of well-known instability which is formed when a more-viscous fluid is displaced by a less-viscous one in porous media. We have tried to control the hydrodynamic by chemical reactions which change viscosity and viscoelasticity of the aqueous solutions. Furthermore, we have tried to apply our fundamental study to environmental and energy fields. In this talk, we discuss applications of chemical control of hydrodynamics in aqueous system to medical and biological fields.
12:30 PM - SM5.1.08
Generation of Micron-Size All-Aqueous Emulsions by Interfacial Folding
Sze Yi Mak 1 2 , Youchuang Chao 1 2 , Anderson Shum 1 2
1 Department of Mechanical Engineering, The University of Hong Kong, Hong Kong Hong Kong, 2 , HKU-Shenzhen Institute of Research and Innovation (HKU-SIRI), Shenzhen China
Show AbstractAll-aqueous system has promising bio- and cyto-compatibility and hence is widely used in biotechnology and biomaterials.1,2 This has fostered efforts to generate all-aqueous emulsions with controlled size.3-6 However, current techniques are often limited by low droplet generation frequency, relatively large size of droplets and high size polydispersity. We present a microfluidic technique to generate all-aqueous emulsions from interfacial folding. We perturb a water-water jet in a microfluidic channel. Due to the low interfacial tension, the interface is folded and aqueous droplets of the outer phase are sheared off from the interface. We regulate the droplet size and generation rate by controlling the perturbation amplitude and frequency. The droplets are of micron-size (O(1) μm), generated at a high throughput (O(102-103) Hz) and with low size polydispersity (c.v. 5%). The all-aqueous droplets, added with suitable stabilizers, could serve as the building block of new bio- or cyto-mimetic materials.
1. S. Hardt and T. Hahn, Lab on a Chip, 2012, 12, 434-442.
2. Y. Song, A. Sauret and H. C. Shum, Biomicrofluidics, 2013, 7, 061301.
3. I. Ziemecka, V. van Steijn, G. J. M. Koper, M. T. Kreutzer and J. H. van Esch, Soft Matter, 2011, 7, 9878-9880.
4. A. Sauret and H. C. Shum, Applied Physics Letters, 2012, 100, 154106.
5. Y. Song, Y. K. Chan, Q. Ma, Z. Liu and H. C. Shum, ACS Applied Materials & Interfaces, 2015, 7, 13925-13933.
6. B.-U. Moon, N. Abbasi, S. G. Jones, D. K. Hwang and S. S. H. Tsai, Anal. Chem., 2016, 88, 3982-3989.
12:45 PM - SM5.1.09
Exploration of Emergent Collective Phenomena and Dynamic Behavior of Active Matter Subjected to Steep Spatiotemporal Thermal Gradients
Serim Ilday 1 , Ercag Pince 1 , Ghaith Makey 1 , Muhamet Ibrahimi 1 , Mutlu Erdogan 1 , Ozgun Yavuz 1 , Onur Tokel 1 , Oguz Gulseren 1 , Omer Ilday 1
1 , Bilkent University, Ankara Turkey
Show AbstractDespite numerous inspiring theoretical and experimental demonstrations to date, fundamental understanding on how dynamical behavior and collective motion emerge in living systems is still in its infancy. This is so because emergence of such complex phenomena occurs when conditions are far from equilibrium, where vastly diverse patterns and/or functionalities can only be captured by simultaneous exploration of spatial and temporal dynamics. Here, we show an experimental platform, whereby we demonstrate pattern formation along with a rich set of collective phenomena for both motile, rod-like bacteria colonies performing biased random walk due to chemotaxis and for immotile, spherical bacteria colonies displaying Brownian motion in a quasi-two dimensional setting. We show dissimilar and similar dynamical behaviors of these two distinct colonies, where the former emanates from their active swimming and from the individual interactions of the bacteria and the latter is the result of their collective behaviors shaped by steep spatiotemporal temperature gradients in the liquid environment. Moreover, we report much faster timescales (seconds) for pattern formation compared to earlier reports (minutes or hours), which has important implications for observation of motility-induced clustering and for dynamic behavior of active matter far from equilibrium.
SM5.2: Aqueous Phase Separation for Artificial Cells
Session Chairs
John Frampton
Anderson Shum
Wednesday PM, April 19, 2017
PCC North, 100 Level, Room 122 B
2:30 PM - *SM5.2.01
ATPS Deserves Plausible Real-World Modeling for the Structure and Function of Living Cells
Kanta Tsumoto 1
1 Graduate School of Engineering, Mie University, Tsu, Mie, Japan
Show AbstractAll living organisms on the Earth maintain their lives by utilizing micrometer-size aqueous compartment enclosed with phospholipid bilayers, and researchers have been fascinated toward the constitution of artificial cell systems with such vesicles (liposomes). Since lipid membranes assume a structure of water-oil-water layers, hydrophilic molecules cannot easily enter cells, and the membrane serves as a barrier to differentiate an intra- from an extra-cellular space. Encapsulating various biochemical reactions, the lumen can apparently enhance gene expression [1] and protect RNA products from RNase [2]. Pioneering studies have recently revealed essential functions of intracellular membraneless structures like RNA bodies caused by liquid-liquid phase separation (LLPS) [3]
It is, of course, understood that membraneous architectures are crucial in maintaining molecular traffic in order, when closely looking at the inside of cells, thus also important are such membrane-free regions separated from their local surroundings due to mutually exclusive effects by soluble crowding biopolymers. The aqueous two phase system (ATPS) is an established technique for moderate bioseparation [4], and we have adopted it as a simple model to investigate how aqueous medium crowding with multiple macromolecules creates exotic microstructures accompanied with the specific localization of biologically important molecules, such as DNA and actin. First, we here show that in a PEG/dextran (Dex) system, most of the DNA molecules were entrapped and located in the inner part of Dex-rich microdroplets, and each droplet containing DNA in a concentrated state were able to be transported using optical tweezers [5], which causes coalescence of the droplets. Trapping DNAs using the tweezers, however, we could not make them move across the interface of PEG/Dex droplets. Interestingly, we observed that the trapped DNAs apparently served as a core so that a Dex microdroplet could grow around it under laser radiation. When mixing the cytoskeletal protein actin with the PEG/Dex solution, the actin was partitioned inside/outside Dex droplets dependently on its polymerized-filamentous/depolymerized-monomeric form, respectively. Detailed observation indicated that the crowded environment altered localization of actin filament, polymerization condition, etc. Further mentioning other cases, we will discuss the physico-chemical mechanism together with the argument on possible biological significance.
References: [1] Nomura SM, Tsumoto K, Hamada T, Akiyoshi K, Nakatani Y, Yoshikawa K, ChemBioChem 4 (2003) 1172. [2] Tsumoto K, Nomura SM, Nakatani Y, Yoshikawa K, Langmuir 17 (2001) 7225. [3] Mitrea DM, Kriwacki RW, Cell Commun Signal 14 (2016)1; Feric M et al., Cell 165 (2016) 1686. [4] Albertsson P-A, Partition of Cell Particles and Macromolecules, 2nd ed., New York, Wiley-Interscience (1971) [5] Tsumoto K, Arai M, Nakatani N, Watanabe SN, Yoshikawa K, Life (Basel) 5 (2015) 459.
3:00 PM - *SM5.2.02
Assembly of Highly Stable and Self-Repairing Membrane-Mimetic 2D Materials from Lipid-Like Peptoids
Chun-Long Chen 1
1 , Pacific Northwest National Lab, Richland, Washington, United States
Show AbstractTwo-dimensional (2D) materials have attracted intense interest due to their novel properties and potential for applications in molecular separation, catalysis, optics, and biomedicine. An ability to develop sequence-defined synthetic molecules that mimic lipid amphiphilicity for assembly of membrane-mimetic 2D materials and exhibit protein-like, sequence-specific molecular recognition would significantly advance the development of functional 2D materials including artificial membranes.
Here I will report my group’s discovery in assembly of lipid-like peptoids into highly stable, crystalline, free-standing and self-repairable 2D membrane materials.1,2 They were formed through a solvent-induced crystallization process, in which inter-peptoid hydrophobic interactions drove the anisotropic packing of peptoids to form a bilayer-like membrane structure. These peptoid membranes exhibit a number of properties associated with cell membranes, including thicknesses in the 3.5 - 5.6 nm range, spontaneous assembly at interfaces and the ability to self-repair. We further demonstrated that these membranes are superior to lipid bilayers and other assembled 2D materials because: 1) they are free-standing, atomically ordered, and highly stable in pure organic solvents, biological environments, as well as high temperature; and 2) a broad range of active functional groups, including chromophores, can be incorporated and patterned within membranes through large side-chain diversity and/or co-crystallization approaches, leading to the development of biomimetic membranes tailored to specific applications (e.g. live cell imaging and drug delivery).
References
1. Jin et al., Nat. Commun. 2016, 7, 12252. DOI: 10.1038/ncomms12252.
2. Jiao et al., Adv. Funct. Mater., 2016, DOI: 10.1002/adfm.201602365.
4:30 PM - *SM5.2.03
The PURE System for Artificial Cells
Takuya Ueda 1
1 Department of Computational Biology and Medical Sciences, University of Tokyo, Kashiwa, Chiba-prefecture, Japan
Show AbstractTo address the construction of artificial cell, we first reconstituted cell-free translation system from translation factors individually purified from over-expressed E. coli cell. The reconstituted translation system, which we named PURE system, is capable of synthesizing prokaryotic and eukaryotic proteins.
On the basis of the PURE system, we are creating cell-like system in test tube by integrating lipid-bilayer to the system. We made an attempt to create energy-generating liposome by expressing the genes corresponding to ATPase subunits onto lipid membrane using the PURE system supplemented with liposome. We succeeded in construction of membrane insertion system using the PURE system and efficient integration of ATPase onto membrane was observed.
We are also addressing minimal genetic code and primitive ribosome. In order to realize the minimal genetic code, a minimal set of tRNA species was transcribed and their capabilities in the PURE system were examined. It turned out that 21 transcribed tRNAs are sufficient for translation reaction. It means that we can create simple and artificial genetic code system and would have realized reproduction of gene expression system in liposome. The reconstitution of ribosome small subunit in physiological condition will be also discussed.
5:00 PM - *SM5.2.04
Synthetic Biology in Aqueous Compartments at the Micro- and Nanoscale
Charles Collier 1 2
1 Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States, 2 Mechanical Aerospace and Biomedical Engineering, University of Tennessee, Knoxville, Tennessee, United States
Show AbstractAqueous two-phase systems and related emulsion-based structures defined within micro- and nanoscale environments enable a bottom-up synthetic biological approach to mimicking the dynamic compartmentation of biomaterial that naturally occurs within cells. Model systems we have developed to aid in understanding these phenomena include on-demand generation and triggering of reversible phase transitions in ATPS confined in microscale droplets, morphological changes in networks of femtoliter-volume aqueous droplet interface bilayers formulated in microfluidic channels, and temperature-driven phase transitions in interfacial lipid bilayer systems supported on nanostructured substrates. We have found that for each of these cases, the dynamics are intimately linked with changes in the chemical potential of water, which becomes increasingly susceptible to evaporation at nanoscopic and microscopic length scales, where interfacial and surface areas predominate over compartment volumes. These models have been used to study consequences of confinement and crowding in cell-sized microcompartments for increasingly complex scenarios, from single-molecule mobility measured with fluorescence correlation spectroscopy to spatiotemporal modulation of resource sharing in cell-free gene expression bursting.
5:30 PM - *SM5.2.05
Self-Propelled Vesicles Using Transient Interfacial Tension in ATPS
Takahiko Ban 1
1 , Osaka University, Osaka Japan
Show AbstractThe motion of vesicles has fundamental significance for the origin of force generation in cellular motility, coupling behavior between motility and shape deformation, and cell division, and is potentially useful in many applications, including targeted drug delivery systems and bioreactors. However, vesicles cannot utilize their interfacial energy for self-propulsion because they have extremely low interfacial energies. Thus, self-propulsion of vesicles requires the interaction of specially treated vesicle membranes with their environmental media. In this study, we have developed a simple method to propel vesicles using the transient interfacial energy generated by the mixing of two polymeric aqueous solutions without pretreatment of the vesicle membrane. The mixing of miscible liquids generates a concentration gradient in the boundary between the two liquids, giving rise to a transient interfacial tension in the mixing zone, and the resulting interfacial tension induces a convective flow. This phenomenon, which is called the Korteweg effect, occurs when two miscible liquids are in contact with each other far from equilibrium. We have recently reported the self−propulsion of soft matter at the macroscopic scale driven by the Korteweg effect using an aqueous two−phase system (ATPS). The advantages of the ATPS are that is less harmful to biological systems in comparison with organic solvent systems, and that is applicable to lipid bilayer membranes. The Korteweg effect using ATPS is exploited for the self-propulsion of vesicles.
Vesicles composed of Didodecyl dimethylammonium bromide (DDAB) were prepared to encapsulate the Dextran (Dextran 40, Mr 40 000) solution in a polyethylene glycol (PEG 4000, Mw = 3000) solution by a reverse emulsion method. Vesicles encapsulating 20 wt% DEX solution traveled spontaneously when the PEG concentration in the environmental media was > 15 wt%. Vesicles showed a variety of motions, such as uniform motion, reciprocatory motion, zigzag motion, and sudden cessation of motion. Its velocity is much faster than the Brownian motion.
We investigated the effect of PEG concentration on the permeability. The permeability increased with increasing PEG concentration. A discontinuous change in the slope was observed at PEG concentrations in the range of 15 – 20 wt%, shifting to a steeper slope at the higher concentrations. PEG promoted the transport of DEX and water molecules across the DDAB membrane. PEG produced two different effects required for self-propulsion: the leakage of macromolecules and the generation of the Korteweg effect. Therefore, the mixing of the two polymeric aqueous solutions served as an efficient driving force for the propulsion of vesicles, even in highly viscous solutions.
SM5.3: Poster Session
Session Chairs
Yuichiro Nagatsu
Hossein Tavana
Thursday AM, April 20, 2017
Sheraton, Third Level, Phoenix Ballroom
9:00 PM - SM5.3.01
High-Throughput 3D Neural Cell Culture Analysis Facilitated by Aqueous Two-Phase Systems
Kristin Robin Ko 1 , Rishima Agarwal 1 , John Frampton 1
1 School of Biomedical Engineering, Dalhousie University, Halifax, Nova Scotia, Canada
Show AbstractIntroduction: Neural cell culture models have contributed significantly to understanding neurodegenerative diseases and identifying potential treatments. Although two-dimensional (2D) cell culture techniques offer real-time analysis and high-throughput screening capabilities, unnatural 2D environments can negatively influence cell behaviour, morphology, and growth. Three-dimensional (3D) cell culture has been proposed as an alternative to provide more realistic mass transport, environmental cues and cell-cell interactions that simulate native tissue. 3D systems such as cell-seeded hydrogels can be combined with automated liquid-handling robotics for rapid sample dispensing into multi-well plates. However, air-liquid interfacial tension and evaporation of hydrogels when exposed to air can result in uneven, inconsistent, 3D cultures. Thick-layer hydrogels can be generated to counter these factors, but new problems arise including large diffusion distances, high cost, and incompatibility with standard imaging tools, plate readers and assays. To address these present limitations, we have developed a thin-layer, 3D Matrigel culture technique using aqueous two-phase systems (ATPSs).
Methods: Matrigel solutions containing the SH-SY5Y neuroblastoma cell line were dispensed into standard 96-well plates using a dextran T10 (D10) and hydroxypropyl methylcellulose 4000 cPs (MC4000) ATPS. D10 and MC4000 were selected from a range of polymer pairings as both were observed to be compatible with Matrigel. SH-SY5Y cell viability and morphology were observed by fluorescence microscopy. Cell viability was also analyzed using a standard plate reader. To determine the reproducibility of gel formation, construct thickness and volume were monitored by way of 3.0 µm microparticles distributed within Matrigel-D10 solutions without cells.
Results: D10 and MC4000 did not disrupt the gelation process and were capable of maintaining their phase separating properties following the addition of Matrigel to the bottom phase solution. Matrigel evaporation was effectively eliminated, and small volumes (20 µl and lower) were capable of forming thin gels that were evenly and consistently spread. In contrast, equivalent volumes dispensed into air formed irregular gels with non-uniform distribution of cells/microparticles. SH-SY5Y cells were observed to extend small neurite-like processes in three-dimensions within the Matrigel, and cell viability remained high, suggesting minimal negative impact of the experimental protocol on cell growth.
Conclusion: We demonstrate a low cost, simple, high-throughput, 3D neuronal cell culture system that is compatible with well-established equipment and hydrogel materials.
9:00 PM - SM5.3.02
Biopatterning of Keratinocytes in Aqueous Two-Phase Systems as a Potential Tool for Skin Tissue Engineering
Rishima Agarwal 1 , Kristin Robin Ko 1 , Paul Gratzer 1 , John Frampton 1
1 School of Biomedical Engineering, Dalhousie University, Halifax, Nova Scotia, Canada
Show AbstractIntroduction: Extrusion-based bioprinting (EBP) is a promising approach for printing fluids containing cells. EBP offers the ability to print viscous bioinks, the capability to deposit high-density cell suspensions, and the ability to precisely control patterning. However, this approach can be limited by loss of pattern fidelity when printing on wet substrates. This limitation can be overcome using aqueous two-phase systems (ATPSs) as novel ink formulations for EBP. ATPS-based inks are comprised of FDA-approved biocompatible polymeric solutions such as poly(ethylene) glycol (PEG) and dextran (DEX) that separate from each other at relatively low concentrations. Cells can be patterned by printing cell-rich DEX droplets onto a substrate covered with a PEG solution. The interfacial tension between the PEG and DEX solutions constrains the cells within the DEX droplets. In this study, ATPSs were used to pattern arrays of human epidermal keratinocyte (HEK001) cell colonies by EBP as a method to promote epithelial growth for future tissue engineering applications.
Methods: Concentrations of PEG and DEX were determined based on binodal phase diagrams. Four ATPS combinations (5.0% PEG 35 kDa/5.5% DEX 500 kDa, 5.0% PEG 35 kDa/5.0 % DEX 500 kDa, 5.0% PEG 35 kDa/4.5% DEX 500 kDa, and 5.0% PEG 35 kDa/4.0% DEX 500 kDa) were tested for cell viability, stable ATPS formation, and uniform cell patterning. A handheld pipette was used to accurately dispense 1 μl droplets of DEX (containing approximately 5000 cells each) onto standard tissue culture plates coated in PEG. This process was also tested on DermGENTM acellular dermal matrix by patterning HEK001 cells into discrete colonies with approximately 3 μm spacing between the colonies. Cell colony patterning and ATPS stability were observed by brightfield microscopy. A Calcein-AM/Propidium Iodide assay was performed to examine cell viability. Cell proliferation and formation of adherens junctions were analyzed by immunocytochemistry.
Results: Solutions of 5.0% PEG 35 kDa /4.5% DEX 500 kDa and 5.0% PEG 35 kDa /4.0% DEX 500 kDa did not form stable ATPSs, and therefore, were not useful for biopatterning. The Calcein-AM/Propidium Iodide assay suggested that 5.0% PEG 35 kDa /5.0% DEX 500 kDa had no adverse effects on cell viability. In addition, this formulation resulted in stable ATPS formation and high-fidelity cell patterning. Cells patterned in colonies displayed higher rates of cell viability, proliferation and E-cadherin junction formation compared to non-patterned cells. Moreover, when cells were patterned on DermGENTM (a decellularized tissue product that resembles the dermal component of skin), stable ATPS formation and discrete cell colonies were also observed.
Conclusion: These findings suggest that ATPS EBP is a very promising technique for biopatterning epidermal cells that may have future applications in skin tissue engineering.
9:00 PM - SM5.3.03
Microfluidic Platform for Examining the Phase Behavior of Condensed RNA/Protein Phases
Nicole Taylor 1 , Shana Elbaum-Garfinkle 1 , Howard Stone 1 2 , Clifford Brangwynne 1
1 Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States, 2 Mechanical and Aerospace Engineering, Princeton University, Princeton, New Jersey, United States
Show AbstractLiving cells contain numerous membrane-less RNA/protein (RNP) bodies that assemble by intracellular liquid-liquid phase separation. The properties of these condensed phase droplets are increasingly recognized as important in their physiological function within living cells, and also through the link to protein aggregation pathologies. However, techniques such as droplet coalescence analysis or standard microrheology do not always enable robust property measurements of model RNA/protein droplets in vitro. Here, we introduce a microfluidic platform that drives protein droplets into a single large phase, which facilitates viscosity measurements using passive microrheology and/or active two-phase flow analysis. We use this technique to study various phase separating proteins from structures including P granules and nucleoli. In both cases, droplets initially exhibit simple liquid behavior, with shear rate-independent viscosities. However, droplets show viscoelastic signatures after several hours, indicating that these condensed liquid phases are metastable. Complementary assays, including fluorescence recovery after photobleaching (FRAP) and transmission electron microscopy (TEM) elucidate the evolving dynamics of protein components and microstructure of protein droplets, respectively. Together, these results provide insight into the relationship between condensed protein liquid phases and gel-like protein aggregates.
9:00 PM - SM5.3.04
Dynamics of Non-Equilibrium w/w/o Double Emulsions towards Their Equilibrium State
Youchuang Chao 1 , Sze Yi Mak 1 , Anderson Shum 1
1 , The University of Hong Kong, Hong Kong Hong Kong
Show AbstractDouble emulsions have been widely used as templates to fabricate multifunctional micro-capsules and micro-particles, which are highly desirable for biomedical applications [1]. However, the double emulsions produced by microfluidics or industry are not always in their equilibrium morphology, and their transformation into the equilibrium state is very fast, preventing us utilizing the intermediate structures. Conventionally, the spreading coefficients are often used as criteria to determine the final morphology of the droplets, which is from the perspective of minimized interfacial energy rather than the dynamics [2, 3]. In this work, we use a microfluidic approach [4] to generate energetically unfavorable water-in-water-in-oil (w/w/o) double emulsions and observe how they transform into equilibrium configurations. Two of the phases are from the equilibrated aqueous two-phase systems (ATPSs), whose interfacial tension can be easily tuned by varying the concentrations, thus providing an excellent flexibility in tuning the interfacial tensions of the three-phase systems [5, 6]. We find that the transformation speed mainly depends on the interfacial tension of the two aqueous phases, and the viscosity of the core phase when the shell phase is not viscous. The understanding of the observed dynamics suggests a route to predict the lifetime of the non-equilibrium w/w/o double emulsions, and thus allow using the intermediate w/w double drops as templates to fabricate novel particles that will find applications in biomedical encapsulation and analyses.
1 C. H. Chen, A. R. Abate, D. Lee, E. M. Terentjev, and D. A. Weitz, Adv. Mater. 21, 3201 (2009).
2 L. D. Zarzar, V. Sresht, E. M. Sletten, J. A. Kalow, D. Blankschtein, and T. M. Swager, Nature 518, 520 (2015).
3 N. Pannacci, H. Bruus, D. Bartolo, I. Etchart, T. Lockhart, Y. Hennequin, H. Willaime, and P. Tabeling, Phys. Rev. Lett. 101, 164502 (2008).
4 A. S. Utada, E. Lorenceau, D. R. Link, P. D. Kaplan, H. A. Stone, and D. A. Weitz, Science 308, 537 (2005).
5 A. Sauret and H. C. Shum, Appl. Phys. Lett. 100, 154106 (2012).
6 Y. Song, Y. K. Chan, Q. Ma, Z. Liu and H. C. Shum, ACS Appl. Mater. Interfaces, 7, 13925 (2015).
9:00 PM - SM5.3.05
Effects of Acid Hydrolysis on the Fabrication of Cassava Starch Microspheres in Aqueous Two-Phase System
Huiping Xia 1 , Bing-zheng Li 2 , Qunyu Gao 1
1 Carbohydrate Laboratory, College of Light Industry and Food Sciences, South China University of Technology, Guang zhou, Guangdong, China, 2 State Key Laboratory of Non-Food Biomass and Enzyme Technology/National Engineering Research Center for Non-food Biorefinery/Guangxi Biomass Industrialization Engineering Institute/Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, Nanning, China, Nanning, Guangxi, China
Show AbstractStarch microspheres (SMs) were fabricated by an aqueous two-phase system (ATPS) emulsification technique, in which aqueous solutions of starch and polypropylene glycol (PEG) were employed as dispersed phase and continuous phase, respectively. Cassava starch were firstly subjected to acid hydrolysis pre-treatment and then heated in hot water to form dispersed phase. The molecular weight (Mw) of starch granules was analyzed using a gel permeation chromatography (GPC) system. The effect of degree of acid hydrolysis (AH) on the properties of SMs were investigated. During the process of AH, only the proper Mw of starch could recombine good shape of SMs. Scanning Electron Microscopy (SEM) pictures showed that “high quality” microspheres with non-damaged, round-shaped and well-defined particles with sharp contours and smooth surface. The particle size distribution was determined by a Mastersizer 2000 laser particle analyzer. X-ray diffraction (XRD) patterns revealed that crystalline structures of starch microspheres were changed compared to native starch. The gelatinization temperatures (To, Tp, and Tc), enthalpy of gelatinization (DH) decreased sharply in all starch microspheres compared with the controls. These starch microspheres could be preferentially used as drug delivery system for encapsulation of protein drugs.
9:00 PM - SM5.3.06
Controlling Convection in Rehydrating Aqueous Two-Phase Systems
Cameron Yamanishi 1 , C. Ryan Oliver 1 , Cedric Bathany 1 , Tasdiq Ahmed 1 , Shuichi Takayama 1 2
1 Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States, 2 Macromolecular Science and Engineering, University of Michigan, Ann Arbor, Michigan, United States
Show AbstractAs complex biological signals become better understood, measuring multiple proteins from the same solutions becomes critical. To meet this demand, multiplex immunoassays have been developed. Many of these techniques use spatial location of antibodies within a well to identify targets. However, this approach is susceptible to cross-reactions between reagents, particularly at higher number of targets. Aqueous two-phase systems (ATPS) have been used to confine assay reagents within microdroplets inside wells to reduce the cross-reaction problem. A pre-dried format enables a user-friendly multiplex assay. In this work, the convective mixing of rehydrating ATPS is quantified to optimize ATPS component parameters. Astigmatic microscopy, a technique to track 3D locations of particles, enables flow tracking in a rehydrating droplet.
9:00 PM - SM5.3.07
Biomimetic Membrane Platforms for Water Purification
Tae-Joon Jeon 1 , Hyunil Ryu 1 , Ahmed Fuwad 2 , Sun Min Kim 2
1 Biological Engineering, Inha University, Incheon Korea (the Republic of), 2 Mechanical Engineering, Inha University, Incheon Korea (the Republic of)
Show AbstractWater is the basic necessity for living organisms. Due to increase in population and industrial development, clean water resources are very rapidly being scarce and the world is demanding revolutionary technologies for water purification than ever before. Membrane based water purification technologies have played most important role in water purification to date. However, conventional technologies reached their performance limit and further enhancement in performance is needed by developing new materials for their membranes. Recently, biomimicry provides insights and technological basis to develop novel systems. Of particular interest, an aquaporin embedded biomimetic membranes are considered as an alternative to replace the conventional membranes. In this work, we show biomimetic membrane platforms for water purification and improved performance of the membranes by employing a novel substrate and chemical conjugation techniques.
Symposium Organizers
Anderson Shum, The University of Hong Kong
Takahiko Ban, Osaka University
Christine Keating, The Pennsylvania State University
Shuichi Takayama, University of Michigan
SM5.4: Molecular Properties of Aqueous Systems
Session Chairs
Christine Keating
Kanta Tsumoto
Thursday AM, April 20, 2017
PCC North, 100 Level, Room 122 B
9:15 AM - *SM5.4.01
Mesoscale Studies of Ionic Vesicles with Polyhedral Geometries
Mykola Tasinkevych 1 , Monica Olvera de la Cruz 1
1 Materials Science and Engineering, Northwestern University, Evanston, Illinois, United States
Show AbstractLarge crystalline molecular shells buckle spontaneously into icosahedra, while multicomponent shells buckle into various polyhedra. Continuum elastic theory explains the buckling of closed shells with one elastic component into icosahedra. A generalized elastic model, on the other hand, describes the spontaneous buckling of inhomogeneous shells into regular and irregular polyhedra. By co-assembling water-insoluble anionic (-1) amphiphiles with cationic (3+) amphiphiles we realized ionic vesicles. Results revealed that surface crystalline domains and the unusual shell shapes observed arise from the competition of ionic correlations with charge-regulation. We explain here the mechanism by which these ionic membranes generate a mechanically heterogeneous vesicle. We explore dynamic protocols for enlarging the shape space of both fluid and crystalline vesicles beyond the equilibrium zone. We performed coarse-grained molecular dynamics simulations of ionic vesicles. In continuously dehydrated vesicles, simulations show the appearance and merge of small flat areas over the surface. Analytical elasticity analysis has been performed to understand the emergent faceted polyhedra in crystalline vesicles. This work was supported by DOE-BES DE-FG02-08ER46539.
9:45 AM - *SM5.4.02
Properties of Aqueous Two-Phase Systems
Boris Zaslavsky 1
1 , Cleveland Diagnostics, Cleveland, Ohio, United States
Show AbstractAqueous two-phase systems formed by two polymers originate from polymer influence on the solvent properties of water. The phase forming polymers may include proteins and polysaccharides. The differences between solvent features of aqueous media in the two phases may be quantified and manipulated by polymers’ concentrations and additives of inorganic salts or small organic compounds, such as sucrose, sorbitol, etc. The differences between electrostatic properties of the phases may be measured by partitioning of a homologous series of ionic compounds, and those between solvent features may be quantified using solvatochromic dyes as molecular probes for the solvent dipolarity/polarizability, solvent H-bond donor acidity, and solvent H-bond acceptor basicity. The differences between solvent features and electrostatic properties of the phases govern distribution of proteins and other natural compounds in aqueous two phase systems. It will be shown that proteins, such as human heat shock protein B6 and plant dehydrin K2, may influence solvent features of water and their effects are similar or exceeding those displayed by common macromolecular crowding agents and organic osmolytes. It is suggested that the effects of proteins on the solvent features of aqueous media may regulate the phase separation in vivo.
10:15 AM - SM5.4.03
Supramolecular Hydrogels Compartmentalized Using Aqueous Multi-Phase Systems
Serhii Mytnyk 1 , Alexandre Olive 1 , Frank Versluis 1 , Eduardo Mendes 1 , Jan van Esch 1
1 , Delft University of Technology, Delft Netherlands
Show AbstractCompartmentalization plays an important role in numerous biological and industrial processes, such as, allowing many, otherwise incompatible, metabolic reactions to run in parallel in any living cell. So far, the level of complexity of cellular compartmentalization remains out of reach, though a number of useful approaches have been developed to mimic it. Many of these employ emulsions, microcapsules, liposomes and polymerosomes to isolate the compartments. Unfortunately, such methods generally require using organic phases to induce structuring, which may be undesirable due to the toxicity concerns or decreased permeability of membranes towards polar solutes. Aqueous multi-phase systems (AMPS) have been recently shown to be a highly useful tool for structuring of aqueous medium.1,2 In this contribution we describe an approach utilizing AMPS to create separate aqueous microcompartments in supramolecular hydrogel materials.3,4 We found that the self-assembly of supramolecular hydrogelators in 2- and 3-phase all-aqueous emulsions allowed us to prepare soft hydrogels with distinct microstructure. These soft hydrogels consist of different compartments which are not separated by hydrophobic boundaries, and can be addressed individually. Interestingly, the dynamic self-assembly of the supramolecular hydrogelators could be exploited to achieve dynamic compartmentalization. These features offer new opportunities for the design of cytoplasm-mimicking soft materials.
References
1. P. Torre, C. D. Keating and S. S. Mansy, Langmuir, 2014, 30, 5695–9.
2. Y. Song, Y. K. Chan, Q. Ma, Z. Liu and H. C. Shum, ACS Appl. Mater. Interfaces, 2015, 7, 13925–33.
3. J. Boekhoven, J. M. Poolman, C. Maity, F. Li, L. van der Mee, C. B. Minkenberg, E. Mendes, J. H. van Esch and R. Eelkema, Nat. Chem., 2013, 5, 433–7.
4. S. Mytnyk, A.G.L. Olive, F. Versluis, E. Mendes and J.H. van Esch (manuscript in preparation).
11:00 AM - *SM5.4.04
Nanostructured Protein Capsules
Tuomas Knowles 1 2
1 Department of Chemistry, University of Cambridge, Cambridge United Kingdom, 2 Department of Physics, University of Cambridge, Cambridge United Kingdom
Show AbstractThis talk outlines our efforts to explore the use of natural proteins as building blocks for the synthesis of microcapsules. We use the self-assembly of polypeptide chains into nanofibrils to define the structure of these materials on the nanoscale, and exploit microfluidics to determine their micron scale morphology. Such capsules can be used for the stabilisation, storage and release of sensitive materials, in particular aggregation prone antibodies. Moreover, we explore the use of peptide self-assembly within microdroplets as the basis of active materials and demonstrate chemo-mechanical actuation in such systems.
11:30 AM - *SM5.4.05
Molecular Engineering of Polyelectrolyte Complex Materials
Sarah Perry 1
1 , University of Massachusetts Amherst, Amherst, Massachusetts, United States
Show AbstractPolyelectrolyte complexation can be used in the self-assembly of a wide range of responsive, bioinspired soft materials ranging from dehydrated thin film and bulk solids to dense, polymer-rich liquid complex coacervates, and more complex hierarchical structures such as micelles and hydrogels. This responsivity can include swelling and dissolution, or liquid-to-solid transitions, which can be harnessed to facilitate encapsulation and the subsequent fabrication of functional materials. Drawing inspiration from proteins as sequence-controlled polymers, the patterning or presentation of charges and other chemical functionalities represents a powerful strategy for the design and manipulation of this type of responsiveness and the corresponding material properties. We utilize polypeptides and polypeptide derivatives as a model platform for the study of branching, chirality, sequence, and patterning effects on materials self-assembly. This experimental effort is supported by the parallel development of computational approaches for modeling and predicting the phase behavior of patterned polymeric materials. This molecular-level understanding of polyelectrolyte complexation is further enhanced by detailed rheological and thermodynamic examinations of the molecular nature of the various material transitions present in these systems. The goal of this systematic investigation is the elucidation of molecular engineering design rules to facilitate the tailored creation of materials based on polyelectrolyte complexation with defined properties for a wide range of applications. Looking beyond immediate translational efforts, these studies also have tremendous potential to elucidate parallel self assembly phenomena in nature.
12:00 PM - *SM5.4.06
Basic and Applied Aspects of "Microphase-Separation" on Biomimetic Membrane- Designed Bio-Inspired Membrane Can Achieve Chiral Recognition and Conversion of Target Molecules -
Hiroshi Umakoshi 1 , Keishi Suga 1 , Yukihiro Okamoto 1
1 , Osaka University, Toyonaka, Osaka Japan
Show AbstractA “Biomembrane” is a highly-organized self-assembly of biomolecules (i.e. lipid, protein etc.) and a key interface for the survival of biological cell. The “Membranome” can be defined as the properties of vesicle (or liposome), which arise from the bilayer molecular assembly of amphiphiles, focusing on “emergent properties” which are not present in the individual components, and is gradually recognized as an important research methodology to investigate the potential functions of vesicles (or liposome) and to apply them for the bioprocess design. “Self-Organizing System”, such as liposome or vesicle, possesses several benefits in the recognition of (bio)molecules, where it can recognize them with (i) electrostatic, (ii) hydrophobic interaction, and (iii) stabilization effect of hydrogen bonds at its surface. A key of next chemical engineering is the use of the “Self-Organizing System”, where “enthalpy-driven” nature of chemical process would be converted to “entropy-driven” one. We call this strategy as “Bio-Inspired Chemical Engineering”. In the presentation, the basic and applied aspects of the self-organizing system will be introduced: (1) Phase Equibrillium and Physicochemical Properties of Self-Organizing System, (2) Functions of Self-Organizing System (i.e. Chiral Recognition Function, Asymmetric Conversion etc.), and (3) Its Application to the Development of the Chemical Process Devices (i.e. Membrane Module for Optical Resolution etc.).
[Publications (Selected)] Langmuir, 24, 350-354 (2008) / Langmuir, 24, 4451-4455 (2008) / Langmuir, 24, 10537-10542 (2008) / Langmuir, 25, 4835–4840 (2009) / Colloid Surface B, 88, 221-230 (2011) / Nucleic Acid Res., 39, 8891-8900 (2011) / Biochem. Biophys. Res. Comm., 426, 165-171 (2012) / AIChE J., 57, 3625–3632 (2012) / Langmuir, 29, 1899–1907 (2013) / Langmuir, 29, 4830–4838 (2013) / Chem. Comm., 50, 10177-10197 (2014) / Anal. Chem., 87, 4772-4780 (2015) / Lab on a Chip, 15, 373-377(2015) / J. Phys. Chem. B, 119, 9772-9779 (2015) / ACS AMI, 7, 21065−21072 (2015) / Langmuir, 31, 12968–12974 (2015) / Langmuir, 32, 3630–3636 (2016) / J. Phys. Chem. B, 120, 2790–2795 (2016) / Langmuir, 32, 6011-6019 (2016) / Langmuir, 32, 6176–6184 (2016)/ J. Phys. Chem. B, 120, 5662–5669 (2016)
12:30 PM - SM5.4.07
Aqueous Emulsion Droplets Stabilized by Lipids Vesicles as Microcompartments for Biomimetic Mineralization
Andrew Rowland 1 , David Cacace 1 , Daniel Dewey 1 , Joshua Stapleton 1 , Christine Keating 1
1 Chemistry, The Pennsylvania State University, State College, Pennsylvania, United States
Show AbstractBiological systems are capable of producing biominerals with physical and mechanical properties that exceed non-biological minerals. For instance, calcium carbonate produced by certain organisms has increased sheer strength and structural hierarchy compared to the same mineral found in the earth’s crust. These unique properties are attributed to the organic components incorporated into the biomineral structure. Such complexity requires specialized mineralizing environments, with strict controls over the mineral precursors. These environments come in the form of mineralization vesicles, lipid-bound organelles capable of concentrating precursors and producing mineral. We draw inspiration from these mineralization systems, in the hopes of developing our own advanced materials.
We have used liposome-stabilized all-aqueous emulsion droplets to create simple artificial mineralization vesicles (AMVs). Droplets of a polymer-rich aqueous phase are suspended within a second continuous polymer-rich aqueous phase. These droplets are stabilized by liposomes that adsorb to the aqueous/aqueous interface. Mineralization is driven by an enzyme, which produces the carbonate needed for calcium carbonate formation. The calcium is held inside the droplets using a chelator, similar to how biology regulates mineral precursor. Both the chelated calcium and the enzyme partition favorably to the denser phase, localizing the mineralization reaction to the droplet interiors.
We can assert greater control over the mineral formation and location by introducing more phases to the AMVs. Anionic polymeric chelators bind the cation precursor to form a complex coacervate, which serves as a polymer-induced liquid precursor (PILP). The majority of mineral forms inside the PILP due to higher cation concentration. The shape of the mineral is affected by the original shape of the PILP. We can investigate the influence of the PILP shape using our AMVs. Furthermore, incorporating multiple PILPs further increases the potential for complex minerals. Each interior phase represents another distinct reaction site, with differing mineral structure and properties. This level of control is unprecedented in artificial mineralization systems, and our AMV’s present a new platform for developing complex materials.
12:45 PM - SM5.4.08
Formation of Biomimetic Materials through Short Peptide Self-Assembly Under Volume Confinement
Aviad Levin 1 2 , Thomas Michaels 2 , Lihi Adler-Abramovich 3 , Thomas Mason 2 , Thomas Mueller 2 4 , Bohan Zhang 2 , L. Mahadevan 5 6 , Ehud Gazit 1 7 , Tuomas Knowles 2 8
1 Department for Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv Israel, 2 Department of Chemistry, University of Cambridge, Cambridge United Kingdom, 3 Department of Oral Biology, The Goldschleger School of Dental Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv Israel, 4 , Fluidic Analytics Ltd, Cambridge United Kingdom, 5 School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, United States, 6 Department of Physics, Harvard University, Cambridge, Massachusetts, United States, 7 Department of Materials Science and Engineering, Tel Aviv University, Tel Aviv Israel, 8 Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge United Kingdom
Show AbstractIn nature, sophisticated materials and structures are formed through self-assembly, a process where chemically simple building blocks form complex arrays of biomolecules functioning cooperatively in living systems. This phenomenon has inspired a sustained research effort to elucidate the basic physical principles which govern self-assembly and the nature of the structures that emerge from this process, in contexts ranging from artificial materials to understanding human disease. Current research in the context of molecular design of materials assembly units is mainly focused on exploiting bottom-up approaches using existing experimental methodologies through which the synthesis of specific building blocks is used to modulate the final product attributes. In this work, we explored a fundamentally orthogonal approach by utilizing microfluidic techniques for restricting the reaction volume, leading to the triggering of the self-assembly and the resulting formation of products under confinement on a length scale that is exploited by natural systems. The high surface to volume ratio achieved by encapsulation of the reaction solution into microdroplets has allowed for the decoupling of the system’s volume and surface properties, providing insights into the driving forces responsible for early selfassembly events. The results of this research provide the basis on which a number of phenomena can be investigated in a quantitative manner by exploiting the spatial confinement of the self-assembly reaction. Moreover, the phenomena revealed in small volumes illustrates the ability to mimic complex natural processes through simple, self-assembling modules, in a fundamental new approach than could not be achieved under bulk conditions (Levin et al. Nature Physics, 2016). The results of this study establish elastic-instability actuation by supramolecular polymers as a high power-density mode of chemo-mechanical transduction and open up the possibility of using elastic instabilities for future applications in other short peptide, protein and synthetic polymer systems.
SM5.5: Biotechnology Based on Aqueous Systems
Session Chairs
Chun-Long Chen
Shuichi Takayama
Thursday PM, April 20, 2017
PCC North, 100 Level, Room 122 B
2:45 PM - *SM5.5.01
Incorporating Aqueous Two-Phase Systems and the Lateral-Flow Immunoassay into a Single Point-of-Care Diagnostic
Daniel Kamei 1
1 , University of California, Los Angeles, Los Angeles, California, United States
Show AbstractThe lateral-flow immunoassay (LFA) is an inexpensive point-of-care paper-based diagnostic device with the potential to rapidly detect disease biomarkers in resource-poor settings. LFA is inexpensive, light in weight, easy to use, rapid, and requires no laboratory equipment or trained personnel. Although LFA has seen much success as a pregnancy test, it exhibits low sensitivity when detecting infectious disease biomarkers at low concentrations, and remains inferior to laboratory-based assays for these applications. This presentation summarizes the evolution of our efforts in integrating aqueous two-phase systems (ATPSs) with LFA in order to lower biomarker detection limits and improve sensitivity. Our initial investigations focused on first allowing the ATPS to phase separate in a test tube before manually extracting the phase containing the concentrated biomarker and applying it to LFA. Although this improved the detection limit of LFA, it required waiting for the phase separation to occur in a test tube and an additional extraction step. Subsequently, our laboratory integrated ATPSs and LFA within a three-dimensional (3D), paper-based device to simultaneously and seamlessly concentrate and detect biomarkers. This device revolves around a new phenomenon we discovered where the ATPS solution very rapidly phase separates and concentrates the target biomarker as it flows through paper. Therefore, rather than using the test tube for phase separation, the ATPS was added directly to the paper to reduce the time to result and eliminate an extraction step to improve ease-of-use. This 3D paper diagnostic was the first to demonstrate ATPS phase separation in paper, allowing for concentration of the target analyte as the sample flowed through the paper to the downstream detection zone. Most recently, our paper-based diagnostic was further engineered to incorporate dehydrated ATPS components so that a user only simply needs to add sample, removing the mixing step with the ATPS components. Our investigations over the past several years have led to a diagnostic that improves the sensitivity of conventional LFA devices while maintaining ease-of-use and time-to-result. This portable device requires no electricity or sophisticated laboratory equipment and is ideal for point-of-care applications in resource-poor settings. This research has the potential to vastly improve disease diagnosis in underserved populations by being able to lower the detection limit, transforming the current state of healthcare through the development of next generation LFA tests.
3:15 PM - *SM5.5.02
Aqueous Two-Phase System Solution Micropatterning—Applications in Biomaterial Development and Clinical Chemistry
John Frampton 1
1 , Dalhousie University, Halifax, Nova Scotia, Canada
Show AbstractAqueous two-phase systems (ATPSs) can be formed when two incompatible polymers are dissolved in water above certain critical concentrations. These systems have been used extensively for separation and purification of biological materials by way of a phenomenon known as partitioning, in which molecules and particles preferentially distribute to one phase or the other, or to the interface between the phases. Recently, this phenomenon has been exploited to enable a range of novel solution micropatterning technologies compatible with various liquid handling tools ranging from simple micropipettes to precision liquid handling robots. This presentation will highlight emerging solution micropatterning applications for several existing ATPS formulations, including the commonly used polyethylene glycol (PEG)-dextran system. Preliminary work utilizing rapid and cost-effective assays for characterization, optimization and application-testing of the PEG-dextran system, other less commonly used ATPSs and novel ATPSs will be described in the first part of the talk. The second part of the talk will focus on recent work utilizing ATPSs for microtissue fabrication. This includes the development of approaches for self-assembly of multilayer tissue constructs at the ATPS interface, as well as the fabrication of high throughput thin-layer hydrogel arrays for 3D cell culture. Work on epithelial cell micropatterning on a variety of substrates including acellular dermal matrices will also be discussed. The final part of the talk will cover advances in adapting solution micropatterning for multiplex biomarker analysis of complex immunological diseases. Work to expand a previously developed 4-plex antibody solution microarray to enable detection of 16 biomarkers from a single sample, including both circulating serum IgM and IgG antibodies and cytokines linked to early infection and inflammation, will be described.
3:45 PM - SM5.5.03
Lipid Multilayer Grating Arrays as Label-Free Cytomimetic Aqueous Sensors
Troy Lowry 1 , Plengchart Prommapan 1 , Steven Lenhert 1
1 , Florida State University, Tallahassee, Florida, United States
Show AbstractBiological cells are uniquely capable of detecting and responding to a wide variety of different analytes in complex environments. Typically, this sensing is carried out at a cellular level by detection of a binding event to a cell membrane by means of a receptor, which subsequently triggers a signal amplification cascade within the cell leading to a cellular response. Lipid multilayer gratings are a novel and promising approach to the synthetic recreation of biological sensing.[1] In this case, diffraction gratings are formed from biological lipids, and the binding of analytes to these gratings results in a shape change in the fluid lipid multilayer that can be read out optically by monitoring the intensity of light diffracted from the grating. Arrays of different lipid gratings can be generated by a nanointaglio process with potential scalability to thousands of sensors per square centimeter or more.[2] Exposure of lipid multilayer nanostructures and microstructures to membrane binding and remodeling proteins provides a new label-free assay for protein activity, providing new insights into how cells organize their molecules in three dimensions in terms of lipid mesophases.[3] Recent progress in the functionalization of these nanostructured two-phase sensor array systems using DNA aptamers will be presented.
References:
[1] S. Lenhert, F. Brinkmann, T. Laue, S. Walheim, C. Vannahme, S. Klinkhammer, M. Xu, S. Sekula, T. Mappes, T. Schimmel, H. Fuchs, Nat Nanotechnol 2010, 5, 275.
[2] T. W. Lowry, P. Prommapan, Q. Rainer, D. Van Winkle, S. Lenhert, Sensors 2015, 15, 20863.
[3] T. W. Lowry, H. Hariri, P. Prommapan, A. Kusi-Appiah, N. Vafai, E. A. Bienkiewicz, D. V. Winkle, S. M. Stagg, S. Lenhert, Small 2016, 12, 506.
4:30 PM - *SM5.5.04
Membrane Wetting, Budding and Tubulation in Vesicles Exposed to Aqueous Two-Phase Systems
Rumiana Dimova 1
1 , Max Planck Institute of Colloids and Interfaces, Potsdam Germany
Show AbstractGiant unilamellar vesicles, an artificial cell-like system with sizes in the 10-micron range, can be loaded with aqueous solutions of macromolecules. The phase separation of these solutions generates spatial compartments within the vesicles. We employed GUVs to study various phenomena related to molecular crowding and microcompartmentation in cells. We mimicked the crowded environment in cells by aqueous polymer solutions of poly(ethylene glycol) (PEG) and dextran. These solutions exhibit phase separation at polymer concentrations above a few weight percent. Similarly to the wetting behavior of liquid droplets in contact with surfaces, the different aqueous phases in contact with the vesicle membrane as a substrate can undergo complete to partial wetting transition [J. Am. Chem. Soc. 130:12252, 2008]. The degree of wetting is characterized by a hidden material parameter - the intrinsic contact angle, which can be determined from effective contact angles observed via optical microscopy [Phys. Rev. Lett. 103:238103, 2009]. Osmotic deflation of the vesicles induces a variety of vesicle shape transformations [Soft Matter 8:6409, 2012; Adv. Mater. Interfaces 1600451, 2016]. One such transformation is droplet-induced budding of the vesicles [J. Phys. Chem. B 116:1819, 2012]. Another, particularly striking transformation is the spontaneous tube formation [Proc. Natl. Acad. Sci. USA. 108:4731, 2011], which reveals a substantial asymmetry and spontaneous curvature of the membranes [ACS Nano 10, 463], arising from the different polymer compositions across the membrane. Phase separation in the interior of vesicles can lead to stable and retractable membrane nanotubes, which is relevant for membrane area storing and regulation in cells.
This talk will also discuss some of our more recent observations on membrane-less organelles enriched in intrinsically disordered proteins such as FUS. We find that depending on the environmental conditions and/or the molecular composition of the membranes, FUS-enriched droplets in contact with vesicles can attain three different wetting morphologies corresponding to dewetting, partial wetting, and complete wetting of the membranes.
5:00 PM - *SM5.5.05
Experimental and Modeling Investigation of Cell Partition in Aqueous Two-Phase Systems
Ehsan Atefi 1 , Jay Mann 2 , Hossein Tavana 1
1 Biomedical Engineering, The University of Akron, Akron, Ohio, United States, 2 Chemical Engineering, Case Western Reserve University, Cleveland, Ohio, United States
Show AbstractSelective partition of living cells in aqueous two-phase systems (ATPS) is used in various areas of biomedical research including stem cells, tissue engineering, and cancer. ATPS consist of a pair of immiscible, aqueous phases and provide a mild environment for cell partition. This unique property of ATPS to partition cells non-invasively and without exerting any thermal, mechanical, or chemical stresses is critical to maintain viability and functionality of cells. To enable cell partition in ATPS, small amounts of two biocompatible polymers, such as polyethylene glycol (PEG) and dextran (DEX), are dissolved in a cell culture medium of predefined volume. Resulting solutions are thoroughly mixed with a known number of cells and allowed to equilibrate. This generates an ATPS consisting of PEG-rich and a DEX-rich aqueous phases. During the phase separation process, cells partition between the two phases and their interface. The ability to control the partition of cells between the two phases has recently been utilized to generate a variety of cell-based assays.
We model cell partition in ATPS using a thermodynamic approach to determine free energy of displacement of cells between the two aqueous phases and their interface. This theoretical model, coupled with systematic measurements of physical properties of ATPS, shows that the interfacial tension (IFT) between the two aqueous phases plays a key role on partition of cells in ATPS. We use a drop shape technique and implement mathematical criteria to consistently measure ultralow IFT of several two-phase solutions with varying concentrations of PEG (Mw: 35,000) and DEX (Mw: 500,000). These two-phase solutions are also used for cell partition experiments. After counting the number of cells partitioned to each phase, the ratio of cells in the bottom DEX phase to the total number of cells is calculated as the partition coefficient. The system with an IFT of 0.030 mJ/m2 results in a partition coefficient of 88±5% for a cancer cell line. An increase in the IFT distributes the cells toward the interface. An independent spheroid formation assay confirms these observations: a sub-microliter volume of the DEX phase containing cancer cells is dispensed into the immersion PEG phase to form a cell-containing drop. Only at very small IFTs do cells remain within the drop to aggregate into a spheroid. Our model shows that increasing the IFT shifts the minimum energy and partition of cells toward the interface of the two phases. Examining differences in the partition behavior and minimum free energy modeling of cancer cells and mouse embryonic stem cells shows that the surface properties of cells further modulate partition in ATPS. This combined approach provides a fundamental understanding of cell partition in ATPS and a framework for future studies.
5:30 PM - SM5.5.06
Water Dehydration for Successful Underwater Adhesion
Dong Soo Hwang 1 , Sangsik Kim 1
1 , POSTECH, Pohang Korea (the Republic of)
Show AbstractWater is a common medium and looks simple, yet its interaction mechanisms with biological molecules and biomaterials are very complicated. Therefore, the structure and localization of the water molecules could determine intermolecular interactions of biomaterials in aqueous conditions . In this presentation, the recent understanding in metal-ligand interactions, cation-pi interactions, lock and key interactions, and complex coacervations of mussel adhesive proteins and carbohydrates will be presented. We directly have measured these interactions using a surface forces apparatus (SFA) in aqueous solutions and some insights with regard to water dehydration have been discovered. Some case studies which apply the insights from the measured interactions to translate to the design of underwater adhesive will be also introduced.
5:45 PM - SM5.5.07
Phase Separated Proteins at Work—A Biomechanical Study of Endocytic Coat Proteins in Yeast
Louis-Philippe Bergeron-Sandoval 1 , Hossein Khavidi Heris 2 , Adam Hendricks 2 , Allen Ehrlicher 2 , Paul Francois 2 , Stephen Michnick 1
1 , Université de Montréal, Montreal, Quebec, Canada, 2 , McGill University, Montreal, Quebec, Canada
Show AbstractAt the cellular level, evolution of biomechanical strategies to shape cells and interact with the environment has generated the wide variety of life we observe today. As an example, mammalian cells can rely on a complex cytoskeleton to adapt specific shapes whereas bacteria, yeast and plants use a combination of turgor pressure and cell walls to have their characteristic bloated form.
We are exploring basic physical phenomenon, in particular protein aqueous-aqueous phase separation and adhesion from interface free energy, as simple and efficient ways for cells to organize internal matter and accomplish work to shape internal structures and surfaces.
We observed, in the context of clathrin-mediated endocytosis (CME) in yeast, that a group of disordered proteins that simultaneously condense on cortical sites are assembled through phase separation into nanometer sized viscoelastic bodies. The dynamic nature and material properties of these cortical bodies were determined by imaging and micro rheological techniques. When these cortical droplets are nucleated between the membrane and cytoplasm, new interfaces are created and we propose that free energy available on the droplet surface can produce work to deform the surrounding materials. Based on our results, we developed a mechanical model that account for deformation of the membrane under action of the cortical bodies alone. We further hypothesize that this mechanism enables CME to proceed in absence of turgor and F-actin polymerization, which constitutes an elusive phenomenon in yeast.