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.

Sizing biological cells using a microfluidic acoustic flow cytometer.

We describe a new technique that combines ultrasound and microfluidics to rapidly size and count cells in a high-throughput and label-free fashion. Using 3D hydrodynamic flow focusing, cells are streamed single file through an ultrasound beam where ultrasound scattering events from each individual cell are acquired. The ultrasound operates at a center frequency of 375 MHz with a wavelength of 4 µm; when the ultrasound wavelength is similar to the size of a scatterer, the power spectra of the backscattered ultrasound waves have distinct features at specific frequencies that are directly related to the cell size. Our approach determines cell sizes through a comparison of these distinct spectral features with established theoretical models. We perform an analysis of two types of cells: acute myeloid leukemia cells, where 2,390 measurements resulted in a mean size of 10.0 ± 1.7 µm, and HT29 colorectal cancer cells, where 1,955 measurements resulted in a mean size of 15.0 ± 2.3 µm. These results and histogram distributions agree very well with those measured from a Coulter Counter Multisizer 4. Our technique is the first to combine ultrasound and microfluidics to determine the cell size with the potential for multi-parameter cellular characterization using fluorescence, light scattering and quantitative photoacoustic techniques.

Simultaneous acoustic and photoacoustic microfluidic flow cytometry for label-free analysis.

We developed a label-free microfluidic acoustic flow cytometer (AFC) based on interleaved detection of ultrasound backscatter and photoacoustic waves from individual cells and particles flowing through a microfluidic channel. The AFC uses ultra-high frequency ultrasound, which has a center frequency of 375 MHz, corresponding to a wavelength of 4 µm, and a nanosecondpulsed laser, to detect individual cells. We validate the AFC by using it to count different color polystyrene microparticles and comparing the results to data from fluorescence-activated cell sorting (FACS). We also identify and count red and white blood cells in a blood sample using the AFC, and observe an excellent agreement with results obtained from FACS. This new label-free, non-destructive technique enables rapid and multi-parametric studies of individual cells of a large heterogeneous population using parameters such as ultrasound backscatter, optical absorption, and physical properties, for cell counting and sizing in biomedical and diagnostics applications.

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.

Stable microfluidic flow focusing using hydrostatics.

We present a simple technique to generate stable hydrodynamically focused flows by driving the flow with hydrostatic pressure from liquid columns connected to the inlets of a microfluidic device. Importantly, we compare the focused flows generated by hydrostatic pressure and classical syringe pump driven flows and find that the stability of the hydrostatic pressure driven technique is significantly better than the stability achieved via syringe pumps, providing fluctuation-free focused flows that are suitable for sensitive microfluidic flow cytometry applications. We show that the degree of flow focusing with the hydrostatic method can be accurately controlled by the simple tuning of the liquid column heights. We anticipate that this approach to stable flow focusing will find many applications in microfluidic cytometry technologies.

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.