Efficient Separation of Methanol Single-Micron Droplets by Tailing Phenomenon Using a PDMS Microfluidic Device.

Microdroplet-based fluidic systems have the advantages of small size, short diffusion time, and no cross-contamination; consequently, droplets often provide a fast and precise reaction environment as well as an analytical environment for individual molecules. In order to handle diverse reactions, we developed a method to create organic single-micron droplets (S-MDs) smaller than 5 µm in diameter dispersed in silicone oil without surfactant. The S-MD generation microflow device consists of a mother droplet (MoD) generator and a tapered separation channel featuring multiple side channels. The tapered channel enhanced the shear forces to form tails from the MoDs, causing them to break up. Surface treatment with the fluoropolymer CYTOP protected PDMS fluid devices from organic fluids. The tailing separation of methanol droplets was accomplished without the use of surfactants. The generation of tiny organic droplets may offer new insights into chemical separation and help study the scaling effects of various chemical reactions.

Deformability-Based Microfluidic Microdroplet Screening to Obtain Agarolytic Bacterial Cells.

Environmental microorganisms possess enzymes that can digest macromolecules such as agarose into smaller molecules that can be utilized for growth. These enzymes could be valuable for the effective utilization of global resources. However, since most of the microorganisms on Earth remain uncultured, there is significant untapped enzymatic potential in nature. Therefore, it is necessary to develop innovative tools and strategies for exploring these enzymatic resources. To address this, we developed a method for screening microbial cells that secrete hydrogel-degrading enzymes using deformability-based microfluidic microdroplet sorting. In this method, microbial cells are encapsulated as single cells in water-in-oil (W/O) microdroplets with a hydrogel whose shape becomes deformable as the hydrogel is progressively degraded into smaller molecules. Screening is achieved using a microfluidic device that passively sorts the deformed W/O microdroplets. Using this method, we successfully sorted agarose-containing microdroplets, encapsulating single bacterial cells that hydrolyzed agarose. This method can be used to screen various hydrogel-degrading microbial cells.

Efficient Synthesis of a Schiff Base Copper(II) Complex Using a Microfluidic Device.

The efficient synthesis of amino acid Schiff base copper(II) complexes using a microfluidic device was successfully achieved. Schiff bases and their complexes are remarkable compounds due to their high biological activity and catalytic function. Conventionally, products are synthesized under reaction conditions of 40 °C for 4 h using a beaker-based method. However, in this paper, we propose using a microfluidic channel to enable quasi-instantaneous synthesis at room temperature (23 °C). The products were characterized using UV-Vis, FT-IR, and MS spectroscopy. The efficient generation of compounds using microfluidic channels has the potential to significantly contribute to the efficiency of drug discovery and material development due to high reactivity.

High-Efficiency Single-Cell Containment Microdevices Based on Fluid Control.

In this study, we developed a comb-shaped microfluidic device that can efficiently trap and culture a single cell (bacterium). Conventional culture devices have difficulty in trapping a single bacterium and often use a centrifuge to push the bacterium into the channel. The device developed in this study can store bacteria in almost all growth channels using the flowing fluid. In addition, chemical replacement can be performed in a few seconds, making this device suitable for culture experiments with resistant bacteria. The storage efficiency of microbeads that mimic bacteria was significantly improved from 0.2% to 84%. We used simulations to investigate the pressure loss in the growth channel. The pressure in the growth channel of the conventional device was more than 1400 PaG, whereas that of the new device was less than 400 PaG. Our microfluidic device was easily fabricated by a soft microelectromechanical systems method. The device was highly versatile and can be applied to various bacteria, such as Salmonella enterica serovar Typhimurium and Staphylococcus aureus.

Dielectrophoresis-Based Selective Droplet Extraction Microfluidic Device for Single-Cell Analysis.

We developed a microfluidic device that enables selective droplet extraction from multiple droplet-trapping pockets based on dielectrophoresis. The device consists of a main microchannel, five droplet-trapping pockets with side channels, and drive electrode pairs appropriately located around the trapping pockets. Agarose droplets capable of encapsulating biological samples were successfully trapped in the trapping pockets due to the difference in flow resistance between the main and side channels. Target droplets were selectively extracted from the pockets by the dielectrophoretic force generated between the electrodes under an applied voltage of 500 V. During their extraction from the trapping pockets, the droplets and their contents were exposed to an electric field for 400-800 ms. To evaluate whether the applied voltage could potentially damage the biological samples, the growth rates of Escherichia coli cells in the droplets, with and without a voltage applied, were compared. No significant difference in the growth rate was observed. The developed device enables the screening of encapsulated single cells and the selective extraction of target droplets.

Efficient Generation of Microdroplets Using Tail Breakup Induced with Multi-Branch Channels.

In recent years, research on the application of microdroplets in the fields of biotechnology and chemistry has made remarkable progress, but the technology for the stable generation of single-micrometer-scale microdroplets has not yet been established. In this paper, we developed an efficient and stable single-micrometer-scale droplet generation device based on the fragmentation of droplet tails, called "tail thread mode", that appears under moderate flow conditions. This method can efficiently encapsulate microbeads that mimic cells and chemical products in passively generated single-micrometer-scale microdroplets. The device has a simple 2D structure; a T-junction is used for droplet generation; and in the downstream, multi-branch channels are designed for droplet deformation into the tail. Several 1-2 µm droplets were successfully produced by the tail's fragmentation; this continuous splitting was induced by the branch channels. We examined a wide range of experimental conditions and found the optimal flow rate condition can be reduced to one-tenth compared to the conventional tip-streaming method. A mold was fabricated by simple soft lithography, and a polydimethylsiloxane (PDMS) device was fabricated using the mold. Based on the 15 patterns of experimental conditions and the results, the key factors for the generation of microdroplets in this device were examined. In the most efficient condition, 61.1% of the total droplets generated were smaller than 2 µm.

Controlling Microdroplet Inner Rotation by Parallel Carrier Flow of Sesame and Silicone Oils.

We developed a method for passively controlling microdroplet rotation, including interior rotation, using a parallel flow comprising silicone and sesame oils. This device has a simple 2D structure with a straight channel and T-junctions fabricated from polydimethylsiloxane. A microdroplet that forms upstream moves into the sesame oil. Then, the largest flow velocity at the interface of the two oil layers applies a rotational force to the microdroplet. A microdroplet in the lower oil rotates clockwise while that in the upper oil rotates anti-clockwise. The rotational direction was controlled by a simple combination of sesame and silicone oils. Droplet interior flow was visualized by tracking microbeads inside the microdroplets. This study will contribute to the efficient creation of chiral molecules for pharmaceutical and materials development by controlling rotational direction and speed.

Development of Microdroplet Generation Method for Organic Solvents Used in Chemical Synthesis.

Recently, chemical operations with microfluidic devices, especially droplet-based operations, have attracted considerable attention because they can provide an isolated small-volume reaction field. However, analysis of these operations has been limited mostly to aqueous-phase reactions in water droplets due to device material restrictions. In this study, we have successfully demonstrated droplet formation of five common organic solvents frequently used in chemical synthesis by using a simple silicon/glass-based microfluidic device. When an immiscible liquid with surfactant was used as the continuous phase, the organic solvent formed droplets similar to water-in-oil droplets in the device. In contrast to conventional microfluidic devices composed of resins, which are susceptible to swelling in organic solvents, the developed microfluidic device did not undergo swelling owing to the high chemical resistance of the constituent materials. Therefore, the device has potential applications for various chemical reactions involving organic solvents. Furthermore, this droplet generation device enabled control of droplet size by adjusting the liquid flow rate. The droplet generation method proposed in this work will contribute to the study of organic reactions in microdroplets and will be useful for evaluating scaling effects in various chemical reactions.

Microdroplet synthesis of azo compounds with simple microfluidics-based pH control.

Conventional solution-phase synthesis of azo compounds is complicated by the need for precise pH and temperature control, high concentrations of pH control reagents, and by-product removal. In this work, we exploited the advantages of microdroplet chemistry to realize the simple and highly efficient synthesis of an azo compound using microfluidics-based pH control. Owing to the small size of microdroplets, heat exchange between a microdroplet and its environment is extremely fast. Furthermore, chemical reactions in microdroplets occur rapidly due to the short diffusion distance and vortex flow. Formation of the azo compound reached completion in less than 3 s at room temperature, compared with 1 h at 0 °C under conventional conditions. pH control was simple, and the pH control reagent concentration could be reduced to less than one-tenth of that used in the conventional method. No by-products were generated, and thus this procedure did not require a recrystallization step. The time course of the chemical reaction was elucidated by observing the growth of azo compound microcrystals in droplets at various locations along the channel corresponding to different mixing times.

Integration of Horizontal and Vertical Microfluidic Modules for Core-Shell Droplet Generation and Chemical Application.

This paper presents a method for utilizing three-dimensional microfluidic channels fully to realize multiple functions in a single device. The final device structure was achieved by combining three independent modules that consisted of horizontal and vertical channels. The device allowed for the one-step generation of water-in-oil-in-water droplets without the need for partial treatment of the polydimethylsiloxane channel surface using separate modules for generating water-in-oil droplets on the horizontal plane and oil-in-water droplets on the vertical plane. The second vertically structured module provided an efficient flow for the generation of highly wettable liquid droplets, and tuning of the first horizontally structured module enabled different modes of inner-core encapsulation within the oil shell. The successful integration of the vertical and horizontal channels for core-shell droplet generation and the chemical synthesis of a metal complex within the droplets were evaluated. The proposed approach of integrating independent modules will expand and enhance the functions of microfluidic platforms.

Structural Formation of Oil-in-Water (O/W) and Water-in-Oil-in-Water (W/O/W) Droplets in PDMS Device Using Protrusion Channel without Hydrophilic Surface Treatment.

This paper presents a simple method of droplet formation using liquids that easily wet polydimethylsiloxane (PDMS) surfaces without any surface treatment. Using only structural features and uniform flow focusing, Oil-in-Water (O/W) and Water-in-Oil-in-Water (W/O/W) droplets were formed in the full PDMS structure. Extrusion channel and three-dimensional flow focusing resulted in effective fluidic conditions for droplet formation and the droplet size could be precisely controlled by controlling the flow rate of each phase. The proposed structure can be utilized as an important element for droplet based research, as well as a droplet generator.

Size-Dependent and Property-Independent Passive Microdroplet Sorting by Droplet Transfer on Dot Rails.

A fully passive microdroplet sorting method is presented in this paper. On the rails with dot patterns, the droplets were sorted in different ways depending on their size. However, the effect of droplet properties on the threshold size of the sorting was eliminated. The droplet positions on two railways and the Laplace pressure of the droplets on the dot patterns allowed selective droplet transfer according to size. Different gaps between the rails altered the threshold size of the transfer. However, the threshold size was independent of the droplet's surface tension and viscosity because the droplet transfer utilized only the droplet position and Laplace pressure without lateral flow to sort targets. This feature has a high potential for bio/chemical applications requiring categorization of droplet targets consisting of various mixtures as pre- or post-elements.

Construction of integrated gene logic-chip.

In synthetic biology, the control of gene expression requires a multistep processing of biological signals. The key steps are sensing the environment, computing information and outputting products1. To achieve such functions, the laborious, combinational networking of enzymes and substrate-genes is required, and to resolve problems, sophisticated design automation tools have been introduced2. However, the complexity of genetic circuits remains low because it is difficult to completely avoid crosstalk between the circuits. Here, we have made an orthogonal self-contained device by integrating an actuator and sensors onto a DNA origami-based nanochip that contains an enzyme, T7 RNA polymerase (RNAP) and multiple target-gene substrates. This gene nanochip orthogonally transcribes its own genes, and the nano-layout ability of DNA origami allows us to rationally design gene expression levels by controlling the intermolecular distances between the enzyme and the target genes. We further integrated reprogrammable logic gates so that the nanochip responds to water-in-oil droplets and computes their small RNA (miRNA) profiles, which demonstrates that the nanochip can function as a gene logic-chip. Our approach to component integration on a nanochip may provide a basis for large-scale, integrated genetic circuits.

Stochastic expression of lactate dehydrogenase A induces Escherichia coli persister formation.

Bacterial persisters are phenotypic variants that survive the treatment of lethal doses of growth-targeting antibiotics without mutations. Although the mechanism underlying persister formation has been studied for decades, how the persister phenotype is switched on and protects itself from antibiotics has been elusive. In this study, we focused on the lactate dehydrogenase gene (ldhA) that was upregulated in an Escherichia coli persister-enriched population. A survival rate assay using an ldhA-overexpressing strain showed that ldhA expression induced persister formation. To identify ldhA-mediated persister formation at the single-cell level, time-lapse microscopy with a microfluidic device was used. Stochastic ldhA expression was found to induce dormancy and tolerance against high-dose ampicillin treatment (500 µg/ml). To better understand the underlying mechanism, we investigated the relationship between ldhA-mediated persister formation and previously reported persister formation through aerobic metabolism repression. As a result, ldhA expression enhanced the proton motive force (PMF) and ATP synthesis. These findings suggest that ldhA-mediated persister formation pathway is different from previously reported persister formation via repression of aerobic metabolism.

Liquid Chromatography Chip with Low-Dispersion and Low-Pressure-Drop Turn Structure Utilizing a Distribution-Controlled Pillar Array.

To realize efficient, fast separations on pillar array columns with turns, a novel turn with low-dispersion and low-pressure-drop properties was developed. This "pillar-distribution-controlled" (PDC) turn was designed as a constant-radius turn filled with octagonal pillars that were arranged to control the linear velocity of the mobile phase in the radial direction. After the pillar positions were adjusted by computational fluid dynamics analysis, 27 mm long pillar array columns with two turns were fabricated on a 20 × 20 mm(2) silicon glass plate. The PDC turns suppressed the sample dispersion to a similar extent as the previously developed tapered turn, and the pressure drop of the newly designed turn was reduced to ∼1/6 that of the tapered turn. Moreover, the C18-modified pillar array column with the PDC turns showed good bioanalytical applicability; five fluorescently labeled amino acids were separated in only 24 s at a linear velocity of 7.5 mm/s. The developed turn structure offers the advantages of longer pillar array columns with more turns for the fast analysis of complex samples.

Rapid quantitative method for the detection of phenylalanine and tyrosine in human plasma using pillar array columns and gradient elution.

This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140 s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field.

Microfluidic Stamping on Sheath Flow.

A microfluidic stamping method to form functional shapes on a cross section in fiber-shaped flow is proposed. Microfluidic stamping and overstamping allow various cross sectional shapes on the 3D flow. The shapes can be controlled by a change in combination of structures and fluidic conditions which correspond to stamp type and stamping force.

Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform.

Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel ß-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes.

Droplet-based microfluidics for high-throughput screening of a metagenomic library for isolation of microbial enzymes.

This paper proposes a high-throughput, function-based screening approach of a metagenomic library for isolating novel microbial enzymes by droplet-based microfluidics. We used gel microdroplets (GMDs) dispersed in oil as picoliter-volume reaction vessels for lipolytic enzyme by encapsulating cells in individual GMDs. Using this approach, we monitored the growth of individual cells encapsulated in GMDs and assessed the enzyme reaction activities at the level of an individual GMD. We then applied this method to screen lipolytic enzyme genes from the metagenomic library constructed from soil collected from a quercus serrate forest of Mount Tsukuba, Ibaraki, Japan. In the workflow presented in this study, metagenomic library clones were encapsulated in 100-pL GMDs with a fluorogenic reporter substrate. A total of 67,000 metagenomic library clones can be screened in only 24 h with reduced consumption of reagents (i.e., <10 µL). As a result, we identified a novel lipolytic enzyme, EstT1, belonging to the EstD2 family of esterases and containing a putative signal peptide, which facilitates enzyme export and catalyzation of substrates in the periplasm. Our study demonstrates the potential of microfluidic GMDs as an efficient tool for metagenomic library screening of industrially relevant enzymes with the potential of significantly reducing the cost and time factors involved in successful practical application of microbial enzymes.

Active microdroplet merging by hydrodynamic flow control using a pneumatic actuator-assisted pillar structure.

This paper describes a microdroplet merging device that can actively control the merging of various droplets under a wide range of flow conditions, using a simple structure. The microdroplets were trapped and merged in a wide chamber divided by pillars, and their behavior was controlled by two horizontal pneumatic microactuators. Hydrodynamic flow control by the actuation was evaluated numerically, and the trapping and merging of droplets were achieved experimentally and controlled via pressure applied to the microactuators. Furthermore, two independently generated droplets were merged under four different modes, ranging from no merging to four-droplet merging, with different ratios and volumes. The pneumatic actuators allowed not only the control of the number of merged droplets, but also a wide range of applied droplet volumes. The device was fabricated simply using a single-layer PDMS (polydimethylsiloxane) structure, and the continuous merging performance operated using only hydrodynamic flow control without any surfactant. Finally, chemical synthesis of a metal complex was performed by the droplet merging method. Crystallization of the complex was visualized in real time, and the synthesis was verified by ultraviolet-visible spectroscopy.