Development of a Microfluidic Droplet-Based Microbioreactor for Microbial Cultivation.

Droplet microfluidics creates new opportunities for microbial engineering. Most microbial cultivations are carried out in bioreactors, which are usually bulky and consume a large amount of reagents and media. In this paper, we propose a microfluidic droplet-based microbioreactor for microbial cultivation. A microfluidic device was designed and fabricated to produce many droplet-based microbioreactors integrated with an AC electric field for the manipulation of these microbioreactors. Droplets encapsulating fluorescent Escherichia coli cells were generated, sorted, and trapped individually in small chambers. Fluorescence intensity was monitored to determine cell growth. An electric field with varying voltages and frequencies manipulates the droplets, simulating an oscillation effect. Initial results showed that electric field does not affect cell growth. A comparison with shake flask showed that a similar standard growth curve is obtained when cultivating at room temperature. This device has the potential for making droplet-based microbioreactors an alternative for microbial engineering research.

On-Demand Droplet Merging with an AC Electric Field for Multiple-Volume Droplet Generation.

We introduce a unique system to achieve on-demand droplet merging and splitting using a perpendicular AC electric field. The working mechanism involves a micropillar to split droplets, followed by electrocoalescence using an AC electric field. Adjusting the parameters of the AC signal and conductivity of the fluid result in different merging regimes. We observed a minimum threshold voltage and a strong influence of the surfactant. We hypothesize that the merging process is caused by dipole-dipole coalescence between the daughter droplets. At the same time, adjustment of the conductivity reveals a shift in the merging regimes and can be explained with an electric circuit diagram. Size-based sorting using this merging phenomenon is subsequently demonstrated, where alternate, single, double, and triple droplets sorting were achieved. The concept presented in this paper is potentially useful for drug dispensing or multivolume digital polymerase chain reaction, as droplets of multiple sizes can be generated simultaneously.

Opto-acousto-fluidic microscopy for three-dimensional label-free detection of droplets and cells in microchannels.

This paper reports a novel method, opto-acousto-fluidic microscopy, for label-free detection of droplets and cells in microfluidic networks. Leveraging the optoacoustic effect, the microscopic system possesses capabilities of visualizing flowing droplets, analyzing droplet contents, and detecting cell populations encapsulated in droplets via the sensing of acoustic waves induced by the intrinsic light-absorbance of matter.

A stretchable conductive Polypyrrole Polydimethylsiloxane device fabricated by simple soft lithography and oxygen plasma treatment.

This paper reports a simple method used to fabricate a stretchable conductive polypyrrole (PPy) rough pore-shape polydimethylsiloxane (p-PDMS) device. An abrasive paper is first used to imprint rough micro-structures on the SU-8 micromold. The p-PDMS microchannel is then fabricated using a standard soft-lithography process. An oxygen plasma treatment is then applied to form an irreversible sealing between the microchannel and a blank cover PDMS. The conductive layer is formed by injecting the PPy mixture into the microchannel which polymerizes in the rough pore-shape micro-structures; The PPy/p-PDMS hybrid device shows good electrical property and stretchability. The electrical properties of different geometrical designs of the PPy/p-PDMS microchannel under stretching were investigated, including straight, curved, and serpentine. Mouse embryonic fibroblasts (NIH/3 T3) were also cultured inside the PPy/p-PDMS device to demonstrate good biocompatibility and feasibility using the conductive and stretchable microchannel in cell culture microfluidics applications. Finally, cyclic stretching and bending tests were performed to evaluate the reliability of PPy/p-PDMS microchannel.

Microfluidic Formation of Coculture Tumor Spheroids with Stromal Cells As a Novel 3D Tumor Model for Drug Testing.

Three-dimensional (3D) tumor spheroids offer unprecedented capability for drug screening because of their unique features such as spatial 3D structure, relevant physiological responses, more complex intercellular network, and stroma-cancer cell interactions. Microfluidic technology provides a facile strategy to make uniform tumor spheroids with potential of high-throughput production. In this article, we develop a microfluidic approach to produce core-shell alginate particles, which allows the separate confinement of different cells in the core and shell structure. To reconstitute the complex tumor structure, we encapsulated tumor cells in the core and stromal fibroblast cells in the shell. These coculture tumor spheroids were applied for drug evaluation showing similar drug resistance as those prepared using conventional methods in well plates. These results demonstrated that our microfluidic approach are facile and versatile for making various tumor spheroids with uniform size but different components to better mimic tumor microenvironment. Moreover, the production rate of around 200 spheroids/min indicates the great potential of this approach for high-throughput drug screening.

High-Throughput Separation of White Blood Cells From Whole Blood Using Inertial Microfluidics.

White blood cells (WBCs) constitute only about 0.1% of human blood cells, yet contain rich information about the immune status of the body; thus, separation of WBCs from the whole blood is an indispensable and critical sample preparation step in many scientific, clinical, and diagnostic applications. In this paper, we developed a continuous and high-throughput microfluidic WBC separation platform utilizing the differential inertial focusing of particles in serpentine microchannels. First, separation performance of the proposed method is characterized and evaluated using polystyrene beads in the serpentine channel. The purity of 10-µm polystyrene beads is increased from 0.1% to 80.3% after two cascaded processes, with an average enrichment ratio of 28 times. Next, we investigated focusing and separation properties of Jurkat cells spiked in the blood to mimic the presence of WBCs in whole blood. Finally, separation of WBCs from human whole blood was conducted and separation purity of WBCs was measured by the flow cytometry. The results show that the purity of WBCs can be increased to 48% after two consecutive processes, with an average enrichment ratio of ten times. Meanwhile, a parallelized inertial microfluidic device was designed to provide a high processing flow rate of 288 ml/h for the diluted (×1/20) whole blood. The proposed microfluidic device can potentially work as an upstream component for blood sample preparation and analysis in the integrated microfluidic systems.

On-Chip Microparticle and Cell Washing Using Coflow of Viscoelastic Fluid and Newtonian Fluid.

This work investigates the on-chip washing process of microparticles and cells using coflow configuration of viscoelastic fluid and Newtonian fluid in a straight microchannel. By adding a small amount of biocompatible polymers into the particle medium or cell culture medium, the induced viscoelasticity can push particles and cells laterally from their original medium to the coflow Newtonian medium. This behavior can be used for particle or cell washing. First, we demonstrated on-chip particle washing by the size-dependent migration speed using coflow of viscoelastic fluid and Newtonian fluid. The critical particle size for efficient particle washing was determined. Second, we demonstrated continuous on-chip washing of Jurkat cells using coflow of viscoelastic fluid and Newtonian fluid. The lateral migration process of Jurkat cells along the channel length was investigated. In addition, the cell washing quality was verified by hemocytometry and flow cytometry with a recovery rate as high as 92.8%. Scanning spectrophotometric measurements of the media from the two inlets and the two outlets demonstrated that diffusion of the coflow was negligible, indicating efficient cell washing from culture medium to phosphate-buffered saline medium. This technique may be a safer, simpler, cheaper, and more efficient alternative to the tedious conventional centrifugation methods and may open up a wide range of biomedical applications.

Negative Pressure Induced Droplet Generation in a Microfluidic Flow-Focusing Device.

We introduce an effective method to actively induce droplet generation using negative pressure. Droplets can be generated on demand using a series of periodic negative pressure pulses. Fluidic network models were developed using the analogy to electric networks to relate the pressure conditions for different flow regimes. Experimental results show that the droplet volume is correlated to the pressure ratio with a power law of 1.3. Using a pulsed negative pressure at the outlet, we are able to produce droplets in demand and with a volume proportional to the pulse width.

Active droplet sorting in microfluidics: a review.

The ability to manipulate and sort droplets is a fundamental issue in droplet-based microfluidics. Various lab-on-a-chip applications can only be realized if droplets are systematically categorized and sorted. These micron-sized droplets act as ideal reactors which compartmentalize different biological and chemical reagents. Array processing of these droplets hinges on the competence of the sorting and integration into the fluidic system. Recent technological advances only allow droplets to be actively sorted at the rate of kilohertz or less. In this review, we present state-of-the-art technologies which are implemented to efficiently sort droplets. We classify the concepts according to the type of energy implemented into the system. We also discuss various key issues and provide insights into various systems.

Editorial for the Special Issue on the Insights and Advancements in Microfluidics.

We present a total of 19 articles in this special issue of Micromachines entitled, "Insights and Advancements in Microfluidics."[...].

A novel viscoelastic-based ferrofluid for continuous sheathless microfluidic separation of nonmagnetic microparticles.

Separation of microparticles has found broad applications in biomedicine, industry and clinical diagnosis. In a conventional aqueous ferrofluid, separation of microparticles usually employs a sheath flow or two offset magnets to confine particle streams for downstream particle sorting. This complicates the fluid control, device fabrication, and dilutes the particle sample. In this work, we propose and develop a novel viscoelastic ferrofluid by replacing the Newtonian base medium of the conventional ferrofluid with non-Newtonian poly(ethylene oxide) (PEO) aqueous solution. The properties of both viscoelastic 3D focusing and negative magnetophoresis of the viscoelastic ferrofluid were verified and investigated. By employing the both properties in a serial manner, continuous and sheathless separation of nonmagnetic particles based on particle size has been demonstrated. This novel viscoelastic ferrofluid is expected to bring more flexibility and versatility to the design and functionality in microfluidic devices.

A Microfluidic Method for Investigating Ion-Specific Bubble Coalescence in Salt Solutions.

This paper reports the direct and precise measurement of bubble coalescence in salt solutions using microfluidics. We directly visualized the bubble coalescence process in a microchannel using high-speed imaging and evaluated the shortest coalescence time to determine the transition concentration of sodium halide solutions. We found the transition concentration is ion-specific, and the capacity of sodium halide salts to inhibit bubble coalescence follows the order of NaF > NaCl > NaBr > NaI. The microfluidic method overcomes the inherent uncertainties in conventional large-scale devices and methods.

AC electrified jets in a flow-focusing device: Jet length scaling.

We use a microfluidic flow-focusing device with integrated electrodes for controlling the production of water-in-oil drops. In a previous work, we reported that very long jets can be formed upon application of AC fields. We now study in detail the appearance of the long jets as a function of the electrical parameters, i.e., water conductivity, signal frequency, and voltage amplitude. For intermediate frequencies, we find a threshold voltage above which the jet length rapidly increases. Interestingly, this abrupt transition vanishes for high frequencies of the signal and the jet length grows smoothly with voltage. For frequencies below a threshold value, we previously reported a transition from a well-behaved uniform jet to highly unstable liquid structures in which axisymmetry is lost rather abruptly. These liquid filaments eventually break into droplets of different sizes. In this work, we characterize this transition with a diagram as a function of voltage and liquid conductivity. The electrical response of the long jets was studied via a distributed element circuit model. The model allows us to estimate the electric potential at the tip of the jet revealing that, for any combination of the electrical parameters, the breakup of the jet occurs at a critical value of this potential. We show that this voltage is around 550 V for our device geometry and choice of flow rates.

Nanoscale silicon surface-assisted laser desorption/ionization mass spectrometry: environment stability and activation by simple vacuum oven desiccation.

Nanoscale silicon surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is an emerging matrix-free, highly sensitive MS analysis method. An important challenge in using nanoscale silicon SALDI-MS analysis is the aging and stability of silicon after storage in various environments. No proper nanoscale silicon SALDI-MS activation procedure has been reported to solve this issue. This study investigated the sensitivity, wettability, and surface oxidation behavior of nanoscale silicon surface SALDI-MS in a room, an inert gas atmosphere, and a vacuum environment. A simple vacuum oven desiccation was proposed to activate the SALDI-MS surface, and the limit of detection was further enhanced 1000 times to a 500 attomole level using this approach. The long-term stability and desorption/ionization mechanism were also investigated.

AC electric field induced droplet deformation in a microfluidic T-junction.

We present for the first time an experimental study on the droplet deformation induced by an AC electric field in droplet-based microfluidics. It is found that the deformation of the droplets becomes stronger with increasing electric field intensity and frequency. The measured electric field intensity dependence of the droplet deformation is consistent with an early theoretical prediction for stationary droplets. We also proposed a simple equivalent circuit model to account for the frequency dependence of the droplet deformation. The model well explains our experimental observations. In addition, we found that the droplets can be deformed repeatedly by applying an amplitude modulation (AM) signal.

Active droplet generation in microfluidics.

The reliable generation of micron-sized droplets is an important process for various applications in droplet-based microfluidics. The generated droplets work as a self-contained reaction platform in droplet-based lab-on-a-chip systems. With the maturity of this platform technology, sophisticated and delicate control of the droplet generation process is needed to address increasingly complex applications. This review presents the state of the art of active droplet generation concepts, which are categorized according to the nature of the induced energy. At the liquid/liquid interface, an energy imbalance leads to instability and droplet breakup.

Self-Aligned Interdigitated Transducers for Acoustofluidics.

The surface acoustic wave (SAW) is effective for the manipulation of fluids and particles at microscale. The current approach of integrating interdigitated transducers (IDTs) for SAW generation into microfluidic channels involves complex and laborious microfabrication steps. These steps often require full access to clean room facilities and hours to align the transducers to the precise location. This work presents an affordable and innovative method for fabricating SAW-based microfluidic devices without the need for clean room facilities and alignment. The IDTs and microfluidic channels are fabricated using the same process and thus are precisely self-aligned in accordance with the device design. With the use of the developed fabrication approach, a few types of different SAW-based microfluidic devices have been fabricated and demonstrated for particle separation and active droplet generation.

Silicon etching using only Oxygen at high temperature: An alternative approach to Si micro-machining on 150 mm Si wafers.

Using a combination of low-pressure oxygen and high temperatures, isotropic and anisotropic silicon (Si) etch rates can be controlled up to ten micron per minute. By varying the process conditions, we show that the vertical-to-lateral etch rate ratio can be controlled from 1:1 isotropic etch to 1.8:1 anisotropic. This simple Si etching technique combines the main respective advantages of both wet and dry Si etching techniques such as fast Si etch rate, stiction-free, and high etch rate uniformity across a wafer. In addition, this alternative O2-based Si etching technique has additional advantages not commonly associated with dry etchants such as avoiding the use of halogens and has no toxic by-products, which improves safety and simplifies waste disposal. Furthermore, this process also exhibits very high selectivity (>1000:1) with conventional hard masks such as silicon carbide, silicon dioxide and silicon nitride, enabling deep Si etching. In these initial studies, etch rates as high as 9.2 µm/min could be achieved at 1150 °C. Empirical estimation for the calculation of the etch rate as a function of the feature size and oxygen flow rate are presented and used as proof of concepts.

Acoustofluidic control of bubble size in microfluidic flow-focusing configuration.

This paper reports a method to control the bubble size generated in a microfluidic flow-focusing configuration. With an ultrasonic transducer, we induce acoustic streaming using a forward moving, oscillating gas-liquid interface. The induced streaming substantially affects the formation process of gas bubbles. The oscillating interface acts as a pump that increases the gas flow rate significantly and forms a larger bubble. This method is applicable to a wide range of gas pressure from 30 to 90 kPa and flow rate from 380 to 2700 µL h(-1). The bubble size can be tuned repeatedly with the response time on the order of seconds. We believe that this method will enhance the capability of a microfluidic bubble generator to produce a tunable bubble size.

The microfluidic jukebox.

Music is a form of art interweaving people of all walks of life. Through subtle changes in frequencies, a succession of musical notes forms a melody which is capable of mesmerizing the minds of people. With the advances in technology, we are now able to generate music electronically without relying solely on physical instruments. Here, we demonstrate a musical interpretation of droplet-based microfluidics as a form of novel electronic musical instruments. Using the interplay of electric field and hydrodynamics in microfluidic devices, well controlled frequency patterns corresponding to musical tracks are generated in real time. This high-speed modulation of droplet frequency (and therefore of droplet sizes) may also provide solutions that reconciles high-throughput droplet production and the control of individual droplet at production which is needed for many biochemical or material synthesis applications.