Evidence for biosurfactant-induced flow in corners and bacterial spreading in unsaturated porous media.

The spread of pathogenic bacteria in unsaturated porous media, where air and liquid coexist in pore spaces, is the major cause of soil contamination by pathogens, soft rot in plants, food spoilage, and many pulmonary diseases. However, visualization and fundamental understanding of bacterial transport in unsaturated porous media are currently lacking, limiting the ability to address the above contamination- and disease-related issues. Here, we demonstrate a previously unreported mechanism by which bacterial cells are transported in unsaturated porous media. We discover that surfactant-producing bacteria can generate flows along corners through surfactant production that changes the wettability of the solid surface. The corner flow velocity is on the order of several millimeters per hour, which is the same order of magnitude as bacterial swarming, one of the fastest known modes of bacterial surface translocation. We successfully predict the critical corner angle for bacterial corner flow to occur based on the biosurfactant-induced change in the contact angle of the bacterial solution on the solid surface. Furthermore, we demonstrate that bacteria can indeed spread by producing biosurfactants in a model soil, which consists of packed angular grains. In addition, we demonstrate that bacterial corner flow is controlled by quorum sensing, the cell-cell communication process that regulates biosurfactant production. Understanding this previously unappreciated bacterial transport mechanism will enable more accurate predictions of bacterial spreading in soil and other unsaturated porous media.

Flows of a nonequilibrated aqueous two-phase system in a microchannel.

Liquid-liquid phase separation is a rich and dynamic process, which recently has gained new interest, especially in biology and for material synthesis. In this work, we experimentally show that co-flow of a nonequilibrated aqueous two-phase system within a planar flow-focusing microfluidic device results in a three-dimensional flow, as the two nonequilibrated solutions move downstream along the length of the microchannel. After the system reaches steady-state, invasion fronts from the outer stream are formed along the top and bottom walls of the microfluidic device. The invasion fronts advance towards the center of the channel, until they merge. We first show by tuning the concentration of polymer species within the system that the formation of these fronts is due to liquid-liquid phase separation. Moreover, the rate of invasion from the outer stream increases with increasing polymer concentrations in the streams. We hypothesize the invasion front formation and growth is driven by Marangoni flow induced by the polymer concentration gradient along the width of the channel, as the system is undergoing phase separation. In addition, we show how at various downstream positions the system reaches its steady-state configuration once the two fluid streams flow side-by-side in the channel.

Microfluidic Generation of All-Aqueous Double and Triple Emulsions.

Higher order emulsions are used in a variety of different applications in biomedicine, biological studies, cosmetics, and the food industry. Conventional droplet generation platforms for making higher order emulsions use organic solvents as the continuous phase, which is not biocompatible and as a result, further washing steps are required to remove the toxic continuous phase. Recently, droplet generation based on aqueous two-phase systems (ATPS) has emerged in the field of droplet microfluidics due to their intrinsic biocompatibility. Here, a platform to generate all-aqueous double and triple emulsions by introducing pressure-driven flows inside a microfluidic hybrid device is presented. This system uses a conventional microfluidic flow-focusing geometry coupled with a coaxial microneedle and a glass capillary embedded in flow-focusing junctions. The configuration of the hybrid device enables the focusing of two coaxial two-phase streams, which helps to avoid commonly observed channel-wetting problems. It is shown that this approach achieves the fabrication of higher-order emulsions in a poly(dimethylsiloxane)-based microfluidic device, and controls the structure of the all-aqueous emulsions. This hybrid microfluidic approach allows for facile higher-order biocompatible emulsion formation, and it is anticipated that this platform will find utility for generating biocompatible materials for various biotechnological applications.

Controlled generation of spiky microparticles by ionic cross-linking within an aqueous two-phase system.

Microparticles are used in a variety of different fields, such as drug delivery. Recently, non-spherical microparticle generation has become desirable. The high surface-to-volume ratio of non-spherical microparticles allows for enhanced targeting, and attachment to cells and tissue. Current non-spherical microparticle generation techniques require complicated setup, and utilizing natural micrograins, such as pollen grains, as non-spherical delivery vehicles, requires extensive post-processing. Here, we describe a unique and facile chemical synthesis approach, for controlled generation of pollen-like microparticles, based on ionic cross-linking of alginate and calcium chloride (CaCl2), within an all-biocompatible aqueous two-phase system (ATPS) of dextran (DEX) and polyethylene glycol (PEG). Our technique controls the length of spikes that emerge on the surface of these microparticles. We anticipate that these pollen-like spiky microparticles may be used as drug delivery vehicles, and this new chemical synthesis approach may be used for generating other biomaterials.

Microfluidic Generation of Particle-Stabilized Water-in-Water Emulsions.

Herein, we present a microfluidic platform that generates particle-stabilized water-in-water emulsions. The water-in-water system that we use is based on an aqueous two-phase system of polyethylene glycol (PEG) and dextran (DEX). DEX droplets are formed passively, in the continuous phase of PEG and carboxylated particle suspension at a flow-focusing junction inside a microfluidic device. As DEX droplets travel downstream inside the microchannel, carboxylated particles that are in the continuous phase partition to the interface of the DEX droplets due to their affinity to the interface of PEG and DEX. As the DEX droplets become covered with carboxylated particles, they become stabilized against coalescence. We study the coverage and stability of the emulsions, while tuning the concentration and the size of the carboxylated particles, downstream inside the reservoir of the microfluidic device. These particle-stabilized water-in-water emulsions showcase good particle adsorption under shear, while being flowed through narrow microchannels. The intrinsic biocompatibility advantages of particle-stabilized water-in-water emulsions make them a good alternative to traditional particle-stabilized water-in-oil emulsions. To illustrate a biotechnological application of this platform, we show a proof-of-principle of cell encapsulation using this system, which with further development may be used for immunoisolation of cells for transplantation purposes.

Water-in-Water Droplets by Passive Microfluidic Flow Focusing.

We present a simple microfluidic system that generates water-in-water, aqueous two phase system (ATPS) droplets, by passive flow focusing. ATPS droplet formation is achieved by applying weak hydrostatic pressures, with liquid-filled pipette tips as fluid columns at the inlets, to introduce low speed flows to the flow focusing junction. To control the size of the droplets, we systematically vary the interfacial tension and viscosity of the ATPS fluids and adjust the fluid column height at the fluid inlets. The size of the droplets scales with a power law of the ratio of viscous stresses in the two ATPS phases. Overall, we find a drop size coefficient of variation (CV; i.e., polydispersity) of about 10%. We also find that when drops form very close to the flow focusing junction, the drops have a CV of less than 1%. Our droplet generation method is easily scalable: we demonstrate a parallel system that generates droplets simultaneously and improves the droplet production rate by up to one order of magnitude. Finally, we show the potential application of our system for encapsulating cells in water-in-water emulsions by encapsulating microparticles and cells. To the best of our knowledge, our microfluidic technique is the first that forms low interfacial tension ATPS droplets without applying external perturbations. We anticipate that this simple approach will find utility in drug and cell delivery applications because of the all-biocompatible nature of the water-in-water ATPS environment.

Microfluidic magnetic self-assembly at liquid-liquid interfaces.

We present a microfluidic method that controllably self-assembles microparticles into clusters at an aqueous two-phase liquid-liquid interface. The liquid-liquid interface is formed between converging flows of aqueous dextran and polyethylene glycol, in a microfluidic cross-slot device. We control the size of the self-assembled particle clusters as they pass through the liquid-liquid interface, by systematically varying the applied magnetic field gradient, and the interfacial tension of the liquid-liquid interface. We find that upon penetration through the interface, the number of particles within a cluster increases with increasing interfacial tension, and decreasing magnetic field gradient. We also develop a scaling model of the number of particles within a cluster, and observe an inverse scaling of the number of particles within a cluster with the dimensionless magnetic Bond number. Upon cluster penetration across the liquid-liquid interface, we find magnetic Bond number regimes where the fluid coating drains away from the surface of the cluster, and where the clusters are encapsulated inside a thin film coating layer. This self-assembly technique may find application in controlling the size of microscale self-assemblies, and coating such assemblies; for example, in clustering and coating of cells for immunoisolated cell transplants.

Magnetic water-in-water droplet microfluidics: Systematic experiments and scaling mathematical analysis.

A major barrier to the clinical utilization of microfluidically generated water-in-oil droplets is the cumbersome washing steps required to remove the non-biocompatible organic oil phase from the droplets. In this paper, we report an on-chip magnetic water-in-water droplet generation and manipulation platform using a biocompatible aqueous two-phase system of a polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (PEG-PPG-PEG) and dextran (DEX), eliminating the need for subsequent washing steps. By careful selection of a ferrofluid that shows an affinity toward the DEX phase (the dispersed phase in our microfluidic device), we generate magnetic DEX droplets in a non-magnetic continuous phase of PEG-PPG-PEG. We apply an external magnetic field to manipulate the droplets and sort them into different outlets. We also perform scaling analysis to model the droplet deflection and find that the experimental data show good agreement with the model. We expect that this type of all-biocompatible magnetic droplet microfluidic system will find utility in biomedical applications, such as long-term single cell analysis. In addition, the model can be used for designing experimental parameters to achieve a desired droplet trajectory.

Microneedle-assisted microfluidic flow focusing for versatile and high throughput water-in-water droplet generation.

Microdroplets have been utilized for a wide range of applications in biomedicine and biological studies. Despite the importance of such droplets, their fabrication is associated with difficulties in practice that emerge from the incompatible nature of chemicals, such as surfactants and organic solvents, with biological environments. Therefore, microfluidic methods have recently emerged that create biocompatible water-in-water droplets based on aqueous two-phase systems (ATPS), most commonly composed of water and incompatible polymers, dextran (DEX) and polyethylene glycol (PEG). However, so far, DEX- and PEG-based water-in-water droplet generation schemes have been plagued with low throughput, and most systems can only generate DEX-in-PEG droplets; PEG-in-DEX droplets have been elusive due to chemical interactions between the polymers and channel walls. Here, we describe a simple approach to generate water-in-water microdroplets passively at a high throughput of up to 850 Hz, and obtain both DEX-in-PEG and PEG-in-DEX droplets. Specifically, our method involves a simple modification to the conventional microfluidic flow focusing geometry, by the insertion of a microneedle to the flow focusing junction, which causes three-dimensional (3D) flow focusing of the dispersed phase fluid. We observe that the 3D flow focusing of the dispersed phase enables excellent control of droplet diameters, ranging from 5 to 65 µm, and achieves a high throughput. Moreover, we report the passive microfluidic generation of PEG-in-DEX droplets for the first time, because in our system the 3D flow focusing of the disperse phase separates the disperse PEG phase from the channel walls, negating the commonly observed wall wetting issues of the PEG phase. We expect this microfluidic approach to be useful in increasing the versatility and throughput of water-in-water droplet microfluidics, and help enable future biotechnological applications, such as microparticle-based drug delivery, cell encapsulation for single cell analysis, and immunoisolation for cell transplantation.

Microfluidic diamagnetic water-in-water droplets: a biocompatible cell encapsulation and manipulation platform.

Droplet microfluidics enables cellular encapsulation for biomedical applications such as single-cell analysis, which is an important tool used by biologists to study cells on a single-cell level, and understand cellular heterogeneity in cell populations. However, most cell encapsulation strategies in microfluidics rely on random encapsulation processes, resulting in large numbers of empty droplets. Therefore, post-sorting of droplets is necessary to obtain samples of purely cell-encapsulating droplets. With the recent advent of aqueous two-phase systems (ATPS) as a biocompatible alternative of the conventional water-in-oil droplet systems for cellular encapsulation, there has also been a focus on integrating ATPS with droplet microfluidics. In this paper, we describe a new technique that combines ATPS-based water-in-water droplets with diamagnetic manipulation to isolate single-cell encapsulating water-in-water droplets, and achieve a purity of 100% in a single pass. We exploit the selective partitioning of ferrofluid in an ATPS of polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (PEG-PPG-PEG) and dextran (DEX), to achieve diamagnetic manipulation of water-in-water droplets. A cell-triggered Rayleigh-Plateau instability in the dispersed phase thread results in a size distinction between the cell-encapsulating and empty droplets, enabling diamagnetic separation and sorting of the cell-encapsulating droplets from empty droplets. This is a simple and biocompatible all-aqueous platform for single-cell encapsulation and droplet manipulation, with applications in single-cell analysis.