Controlled Tempering of Lipid Concentration and Microbubble Shrinkage as a Possible Mechanism for Fine-Tuning Microbubble Size and Shell Properties.

The acoustic response of microbubbles (MBs) depends on their resonance frequency, which is dependent on the MB size and shell properties. Monodisperse MBs with tunable shell properties are thus desirable for optimizing and controlling the MB behavior in acoustics applications. By utilizing a novel microfluidic method that uses lipid concentration to control MB shrinkage, we generated monodisperse MBs of four different initial diameters at three lipid concentrations (5.6, 10.0, and 16.0 mg/mL) in the aqueous phase. Following shrinkage, we measured the MB resonance frequency and determined its shell stiffness and viscosity. The study demonstrates that we can generate monodisperse MBs of specific sizes and tunable shell properties by controlling the MB initial diameter and aqueous phase lipid concentration. Our results indicate that the resonance frequency increases by 180-210% with increasing lipid concentration (from 5.6 to 16.0 mg/mL), while the bubble diameter is kept constant. Additionally, we find that the resonance frequency decreases by 260-300% with an increasing MB final diameter (from 5 to 12 µm), while the lipid concentration is held constant. For example, our results depict that the resonance frequency increases by ∼195% with increasing lipid concentration from 5.6 to 16.0 mg/mL, for ∼11 µm final diameter MBs. Additionally, we find that the resonance frequency decreases by ∼275% with increasing MB final diameter from 5 to 12 µm when we use a lipid concentration of 5.6 mg/mL. We also determine that MB shell viscosity and stiffness increase with increasing lipid concentration and MB final diameter, and the level of change depends on the degree of shrinkage experienced by the MB. Specifically, we find that by increasing the concentration of lipids from 5.6 to 16.0 mg/mL, the shell stiffness and viscosity of ∼11 µm final diameter MBs increase by ∼400 and ∼200%, respectively. This study demonstrates the feasibility of fine-tuning the MB acoustic response to ultrasound by tailoring the MB initial diameter and lipid concentration.

Microfluidic nanobubbles: observations of a sudden contraction of microbubbles into nanobubbles.

Microfluidic devices are often utilized to generate uniform-size microbubbles. In most microfluidic bubble generation experiments, once the bubbles are formed the gas inside the bubbles begin to dissolve into the surrounding aqueous environment. The bubbles shrink until they attain an equilibrium size dictated by the concentration and type of amphiphilic molecules stabilizing the gas-liquid interface. Here, we exploit this shrinkage mechanism, and control the solution lipid concentration and microfluidic geometry, to make monodisperse bulk nanobubbles. Interestingly, we make the surprising observation of a critical microbubble diameter above and below which the scale of bubble shrinkage dramatically changes. Namely, microbubbles generated with an initial diameter larger than the critical diameter shrinks to a stable diameter that is consistent with previous literature. However, microbubbles that are initially smaller than the critical diameter experience a sudden contraction into nanobubbles whose size is at least an order-of-magnitude below expectations. We apply electron microscopy and resonance mass measurement methods to quantify the size and uniformity of the nanobubbles, and probe the dependence of the critical bubble diameter on the lipid concentration. We anticipate that further analysis of this unexpected microbubble sudden contraction regime can lead to more robust technologies for making monodisperse nanobubbles.

Controlled Shrinkage of Microfluidically Generated Microbubbles by Tuning Lipid Concentration.

Monodisperse microbubbles with diameters less than 10 µm are desirable in several ultrasound imaging and therapeutic delivery applications. However, conventional approaches to synthesize microbubbles, which are usually agitation-based, produce polydisperse bubbles that are less desirable because of their heterogeneous response when exposed to an ultrasound field. Microfluidics technology has the unique advantage of generating size-controlled monodisperse microbubbles, and it is now well established that the diameter of microfluidically made microbubbles can be tuned by varying the liquid flow rate, gas pressure, and dimensions of the microfluidic channel. It is also observed that once the microbubbles form, the bubbles shrink and eventually stabilize to a quasi-equilibrium diameter, and that the rate of stabilization is related to the lipid solution. However, how the lipid solution concentration affects the degree of bubble shrinkage, and the stable size of microbubbles, has not been thoroughly examined. Here, we investigate whether and how the lipid concentration affects the degree of microbubble shrinkage. Namely, we utilize a flow-focusing microfluidic geometry to generate monodisperse bubbles, and observe the effect of gas composition (2.5, 1.42, and 0.17 wt % octafluoropropane in nitrogen) and lipid concentration (1-16 mg/mL) on the degree of microbubble shrinkage. For the lipid system and gas utilized in these experiments, we observe a monotonic increase in the degree of microbubble shrinkage with decreasing lipid concentration, and no dependency on the gas composition. We hypothesize that the degree of shrinkage is related to lipid concentration by the self-assembly of lipids on the gas-liquid interface during bubble generation and subsequent lipid packing on the interface during shrinkage, which is arrested when a maximum packing density is achieved. We anticipate that this approach for creating and tuning the size of monodisperse microbubbles will find utility in biomedical applications, such as contrast-enhanced ultrasound imaging and ultrasound-triggered gene delivery.

Microfluidic Generation of Monodisperse Nanobubbles by Selective Gas Dissolution.

Nanotechnology currently enables the fabrication of uniform solid nanoparticles and liquid nano-emulsions, but not uniform gaseous nanobubbles (NBs). In this article, for the first time, a method based on microfluidics that directly produces monodisperse NBs is reported. Specifically, a two-component gas mixture of water-soluble nitrogen and water-insoluble octafluoropropane as the gas phase are used in a microfluidic bubble generator. First, monodisperse microbubbles (MBs) with a classical microfluidic flow-focusing junction is generated, then the MBs shrink down to ≈100 nm diameter, due to the dissolution of the water-soluble components in the gas mixture. The degree of shrinkage is controlled by tuning the ratio of water-soluble to water-insoluble gas components. This technique maintains the monodispersity of the NBs, and enables precise control of the final NB size. It is found that the monodisperse NBs show better homogeneity than polydisperse NBs in in vitro ultrasound imaging experiments. Proof-of-concept in vivo kidney imaging is performed in live mice, demonstrating enhanced contrast using the monodisperse NBs. The NB monodispersity and imaging results make microfluidically generated NBs promising candidates as ultrasound contrast and molecular imaging agents.

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.

Dancing with the Cells: Acoustic Microflows Generated by Oscillating Cells.

The interaction of a sound or ultrasound wave with an elastic object, such as a microbubble, can give rise to a steady-state microstreaming flow in its surrounding liquid. Many microfluidic strategies for cell and particle manipulation, and analyte mixing, are based on this type of flow. In addition, there are reports that acoustic streaming can be generated in biological systems, for instance, in a mammalian inner ear. Here, new observations are reported that individual cells are able to induce microstreaming flow, when they are excited by controlled acoustic waves in vitro. Single adherent cells are exposed to an acoustic field inside a microfluidic device. The cell-induced microstreaming is then investigated by monitoring flow tracers around the cell, while the structure and extracellular environment of the cell are altered using different chemicals. The observations suggest that the maximum streaming flow induced by an MDA-MB-231 breast cancer cell can reach velocities on the order of mm s-1 , and this maximum velocity is primarily governed by the overall cell stiffness. Therefore, such cell-induced microstreaming measurements, including flow pattern and velocity magnitude, may be used as label-free proxies of cellular mechanical properties, such as stiffness.

Evaporation-Driven Water-in-Water Droplet Formation.

We present new observations of aqueous two-phase system (ATPS) thermodynamic and interfacial phenomena that occur inside sessile droplets due to water evaporation. Sessile droplets that contain polymeric solutions, which are initially in equilibrium in a single phase, are observed at their three-phase liquid-solid-air contact line. As evaporation of a sessile droplet proceeds, we find that submicron secondary water-in-water (W/W) droplets emerge spontaneously at the edges of the mother sessile droplet due to the resulting phase separation from water evaporation. To understand this phenomenon, we first study the secondary W/W droplet formation process on different substrate materials, namely, glass, polycarbonate (PC), thermoplastic elastomer (TPE), poly(dimethylsiloxane)-coated glass slide (PDMS substrate), and Teflon-coated glass slide (Teflon substrate), and show that secondary W/W droplet formation arises only in lower-contact-angle substrates near the three-phase contact line. Next, we characterize the size of the emergent secondary W/W droplets as a function of time. We observe that W/W drops are formed, coalesced, aligned, and trapped along the contact line of the mother droplet. We demonstrate that this W/W multiple emulsion system can be used to encapsulate magnetic particles and blood cells, and achieve size-based separation. Finally, we show the applicability of this system for protein sensing. This is the first experimental observation of evaporation-induced secondary W/W droplet generation in a sessile droplet. We anticipate that the phenomena described here may be applicable to some biological assay applications, for example, biomarker detection, protein sensing, and point-of-care diagnostic testing.

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.

Controlled Electrospray Generation of Nonspherical Alginate Microparticles.

Electrospraying is a technique used to generate microparticles in a high throughput manner. For biomedical applications, a biocompatible electrosprayed material is often desirable. Using polymers, such as alginate hydrogels, makes it possible to create biocompatible and biodegradable microparticles that can be used for cell encapsulation, to be employed as drug carriers, and for use in 3D cell culturing. Evidence in the literature suggests that the morphology of the biocompatible microparticles is relevant in controlling the dynamics of the microparticles in drug delivery and 3D cell culturing applications. Yet, most electrospray-based techniques only form spherical microparticles, and there is currently no widely adopted technique for producing nonspherical microparticles at a high throughput. Here, we demonstrate the generation of nonspherical biocompatible alginate microparticles by electrospraying, and control the shape of the microparticles by varying experimental parameters such as chemical concentration and the distance between the electrospray tip and the particle-solidification bath. Importantly, we show that these changes to the experimental setup enable the synthesis of different shaped particles, and the systematic change in parameters, such as chemical concentration, result in monotonic changes to the particle aspect ratio. We expect that these results will find utility in many biomedical applications that require biocompatible microparticles of specific shapes.

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.

Honey, I shrunk the bubbles: microfluidic vacuum shrinkage of lipid-stabilized microbubbles.

We present a microfluidic technique that shrinks lipid-stabilized microbubbles from O(100) to O(1) µm in diameter - the size that is desirable in applications as ultrasound contrast agents. We achieve microbubble shrinkage by utilizing vacuum channels that are adjacent to the microfluidic flow channels to extract air from the microbubbles. We tune a single parameter, the vacuum pressure, to accurately control the final microbubble size. Finally, we demonstrate that the resulting O(1) µm diameter microbubbles have similar stability to microfluidically generated microbubbles that are not exposed to vacuum shrinkage. We anticipate that, with additional scale-up, this simple approach to shrink microbubbles generated microfluidically will be desirable in ultrasound imaging and therapeutic applications.

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.

ATPSpin: A Single Microfluidic Platform that Produces Diversified ATPS-Alginate Microfibers.

Microfluidic spinning is emerging as a useful technique in the fabrication of alginate fibers, enabling applications in drug screening, disease modeling, and disease diagnostics. In this paper, by capitalizing on the benefits of aqueous two-phase systems (ATPS) to produce diverse alginate fiber forms, we introduce an ATPS-Spinning platform (ATPSpin). This ATPS-enabled method efficiently circumvents the rapid clogging challenges inherent to traditional fiber production techniques by regulating the interaction between alginate and cross-linking agents like Ba2+ ions. By varying system parameters under the guidance of a regime map, our system produces several fiber forms─solid, hollow, and droplet-filled─consistently and reproducibly from a single device. We demonstrate that the resulting alginate fibers possess distinct features, including biocompatibility. We also encapsulate HEK293 cells in the microfibers as a proof-of-concept that this versatile microfluidic fiber generation platform may have utility in tissue engineering and regenerative medicine applications.

Microfluidically-generated Encapsulated Spheroids (µ-GELS): An All-Aqueous Droplet Microfluidics Platform for Multicellular Spheroids Generation.

Spheroids are three-dimensional clusters of cells that serve as in vitro tumor models to recapitulate in vivo morphology. A limitation of many existing on-chip platforms for spheroid formation is the use of cytotoxic organic solvents as the continuous phase in droplet generation processes. All-aqueous methods do not contain cytotoxic organic solvents but have so far been unable to achieve complete hydrogel gelation on chip. Here, we describe an enhanced droplet microfluidic platform that achieves on-chip gelation of all-aqueous hydrogel multicellular spheroids (MCSs). Specifically, we generate dextran-alginate droplets containing MCF-7 breast cancer cells, surrounded by polyethylene glycol, at a flow-focusing junction. Droplets then travel to a second flow-focusing junction where they interact with calcium chloride and gel on chip to form hydrogel MCSs. On-chip gelation of the MCSs is possible here because of an embedded capillary at the second junction that delays the droplet gelation, which prevents channel clogging problems that would otherwise exist. In drug-free experiments, we demonstrate that MCSs remain viable for 6 days. We also confirm the applicability of this system for cancer drug testing by observing that dose-dependent cell death is achievable using doxorubicin.

Materials and methods for droplet microfluidic device fabrication.

Since the first reports two decades ago, droplet-based systems have emerged as a compelling tool for microbiological and (bio)chemical science, with droplet flow providing multiple advantages over standard single-phase microfluidics such as removal of Taylor dispersion, enhanced mixing, isolation of droplet contents from surfaces, and the ability to contain and address individual cells or biomolecules. Typically, a droplet microfluidic device is designed to produce droplets with well-defined sizes and compositions that flow through the device without interacting with channel walls. Successful droplet flow is fundamentally dependent on the microfluidic device - not only its geometry but moreover how the channel surfaces interact with the fluids. Here we summarise the materials and fabrication techniques required to make microfluidic devices that deliver controlled uniform droplet flow, looking not just at physical fabrication methods, but moreover how to select and modify surfaces to yield the required surface/fluid interactions. We describe the various materials, surface modification techniques, and channel geometry approaches that can be used, and give examples of the decision process when determining which material or method to use by describing the design process for five different devices with applications ranging from field-deployable chemical analysers to water-in-water droplet creation. Finally we consider how droplet microfluidic device fabrication is changing and will change in the future, and what challenges remain to be addressed in the field.

Plug and Pop: A 3D-Printed, Modular Platform for Drug Delivery Using Clinical Ultrasound and Microbubbles.

Targeted drug and gene delivery using ultrasound and microbubbles (USMB) has the potential to treat several diseases. In vitro investigation of USMB-mediated delivery is of prime importance prior to in vivo studies because it is cost-efficient and allows for the rapid optimization of experimental parameters. Most in vitro USMB studies are carried out with non-clinical, research-grade ultrasound systems, which are not approved for clinical use and are difficult to replicate by other labs. A standardized, low-cost, and easy-to-use in vitro experimental setup using a clinical ultrasound system would facilitate the eventual translation of the technology to the bedside. In this paper, we report a modular 3D-printed experimental setup using a clinical ultrasound transducer that can be used to study USMB-mediated drug delivery. We demonstrate its utility for optimizing various cargo delivery parameters in the HEK293 cell line, as well as for the CMT167 lung carcinoma cell line, using dextran as a model drug. We found that the proportion of dextran-positive cells increases with increasing mechanical index and ultrasound treatment time and decreases with increasing pulse interval (PI). We also observed that dextran delivery is most efficient for a narrow range of microbubble concentrations.

Water-in-water droplet microfluidics: A design manual.

Droplet microfluidics is utilized in a wide range of applications in biomedicine and biology. Applications include rapid biochemical analysis, materials generation, biochemical assays, and point-of-care medicine. The integration of aqueous two-phase systems (ATPSs) into droplet microfluidic platforms has potential utility in oil-free biological and biomedical applications, namely, reducing cytotoxicity and preserving the native form and function of costly biomolecular reagents. In this review, we present a design manual for the chemist, biologist, and engineer to design experiments in the context of their biological applications using all-in-water droplet microfluidic systems. We describe the studies achievable using these systems and the corresponding fabrication and stabilization methods. With this information, readers may apply the fundamental principles and recent advancements in ATPS droplet microfluidics to their research. Finally, we propose a development roadmap of opportunities to utilize ATPS droplet microfluidics in applications that remain underexplored.

Phase transition modulation and biophysical characterization of biomolecular condensates using microfluidics.

Membraneless organelles (MLOs) formed through liquid-liquid phase separation (LLPS) are becoming increasingly relevant to understanding viral-host interactions, neurodegenerative disease, and cancer. The modulation of LLPS involves many parameters and components. To describe these modulators, typical in vitro studies require laborious, manual sample preparation of different concentrations and costly biological reagents. Here, we introduce a minimal reagent, microfluidic platform to systematically generate samples of different concentrations and trigger phase separation. We demonstrate the platform's utility by constructing phase diagrams describing the modulation of LLPS using an aqueous two-phase system (ATPS) and an MLO-based phase separating system. We also show on-chip biophysical characterization typical of in vitro studies. We expect that this platform will be utilized by scientists to study the growing number of MLOs and inform clinical treatments for pathology related to LLPS.