Dong Hyun Yoon

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 and Precise Control of Microdroplet Division Using Horizontal Pneumatic Valves in Bifurcating Microchannel

This paper presents a microfluidic system for the active and precise control of microdroplet division in a micro device. Using two horizontal pneumatic valves formed at downstream of bifurcating microchannel, flow resistances of downstream channels were variably controlled. With the resistance control, volumetric ratio of downstream flows was changed and water-in-oil microdroplets were divided into two daughter droplets of different volume corresponding to the ratio. The microfluidic channels and pneumatic valves were fabricated by single-step soft lithography process of PDMS (polydimethylsiloxane) using SU-8 mold. A wide range control of the daughter droplets’ volume ratio was achieved by the simple channel structure. Volumetric ratio between large and small daughter droplets are ranged from 1 to 70, and the smallest droplet volume of 14 pL was obtained. The proposed microfluidic device is applicable for precise and high throughput droplet based digital synthesis.

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 β-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes.

Hydrodynamic on-rail droplet pass filter for fully passive sorting of droplet-phase samples

A hydrodynamic droplet pass filter for droplet-phase sample sorting was developed in this study. Using only groove rails, without additional components or external controls, droplets were sorted based on their physical properties. This is the first report of a droplet pass filter used for effective sorting, and the sorting structure provides a novel fluidic component for fluidic circuits for many applications. Depending on the number of rails, we obtained high-pass, low-pass, band-pass, and multi band-pass filters for sorting droplet samples, and their filtration performance was controlled by varying the dimensions of the rail structures. We evaluated in detail the effect of the rail width on sorting, threshold size of droplets sorted into each rail, and capillary number-dependent sample sorting. Furthermore, band-pass droplet sorting, useful for sample quantification, was provided, and multi-step rail ways allowed multi band-pass droplet sorting that was independent of flow conditions. The proposed sorting method does not require any external systems or skilled operation, and thus, it is expected that the device can contribute to on-site sample treatment and analysis in various fields such as medical care or the military.

Selective droplet sampling using a minimum number of horizontal pneumatic actuators in a high aspect ratio and highly flexible PDMS device

This paper presents a droplet sampling device driven by horizontal pneumatic actuators. A high aspect ratio and highly flexible PDMS (polydimethylsiloxane) structure was proposed for carrying out larger number of sampling than the number of actuators. Large deformation of the actuators caused domino-deformation of parallel walls, and the deformations allowed for selective collection of target droplets. The dimensions of the PDMS structure and the ratio of resin to curing agent were optimized for efficient sampling under the low pressure applied to the actuators. Five sampling modes were achieved in the simple one-layer structure consisting of one inlet, four walls, one drain channel, and two pneumatic actuators, formed by single-step soft lithography process.

Formation of polymeric hollow microcapsules and microlenses using gas-in-organic-in-water droplets

This paper presents methods for the formation of hollow microcapsules and microlenses using multiphase microdroplets. Microdroplets, which consist of a gas core and an organic phase shell, were generated at a single junction on a silicon device without surface treatment of the fluidic channels. Droplet, core and shell dimensions were controlled by varying the flow rates of each phase. When the organic solvent was released from the organic phase shell, the environmental conditions changed the shape of the solidified polymer shell to either a hollow capsule or a microlens. A uniform solvent release process produced polymeric capsules with nanoliter gas core volumes and a membrane thickness of approximately 3 μm. Alternatively physical rearrangement of the core and shell allowed for the formation of polymeric microlenses. On-demand formation of the polymer lenses in wells and through-holes polydimethylsiloxane (PDMS) structures was achieved. Optical properties of the lenses were controlled by changing the dimension of these structures.

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.

Controllable Active Micro Droplets Merging Device Using Horizontal Pneumatic Micro Valves

We present an active droplet merging device, which can merge various sizes of micro droplets in different numbers by using pneumatically controlled horizontal PDMS microvalves. The merging part consists of a main and side channels separated by a pillar array. The pillar array structure is contained within a microfuidic channel. The function of the pillar array provides a bypass path to the continuous flow (oil) inside the merging chamber. Droplets are successfully generated within the channel and achieve merging by controlling the selective different numbers and diameters of droplets through varying the flow resistance of main and side channel. In the merging chamber, a droplet will enter and slow down its movement. It will wait and then merge with the sequential droplets. These experiments demonstrate that such a merging device can controllably select and adjust the distance between the different adjacent micro droplets without any generation of sister droplets in the side channel. The device has no desynchronization problems. Thus, it can be applied for efficiently mixing the droplets in various diameters and numbers without changing the structure of the merging chamber. Hence, this device can be a more effective choice when applying microfluidics to chemical and biological applications.

Improvement of Filtration Performance Using Self-Tuning of Flow Resistance

In this paper, we present the filtration of a liquid sample from polystyrene microparticles analogous to the separation of a biological liquid from mixed particles such as whole blood. The proposed self-tuning of flow resistance can prevent the excessive clogging of microparticles in the microfilter by allowing the automatic change of the flow direction when the microfilter is clogged. Numerically, at about 80% of the clogging of microparticles in the pillar channel, the sample flow is regulated suddenly to the bypass channel. Experimentally, the clogging behavior at the five successive pillar channels and the self-tuning of flow are compared by measuring the clogging area and volume with time. Also, the microfilter array connected in a series can provide an increase in the sample volume proportionally without excessive pressure build-up. This implies the potential to reduce cell fracture in the filtration of biological cells.

Separation of Different Sized Nanoparticles with Time Using a Rotational Flow

In this paper, we describe the development of a microfluidic centrifuge with two inlets and two outlets potentially capable of rapidly separating nanoparticles and nanovesicles. Compared with the microfluidic centrifuge with a single inlet and outlet, the 2 ×2 microfluidic centrifuge gives improved centrifugation performance by increasing momentum flux transfer, angular velocity, and centrifugal acceleration. The center of flow rotation and the symmetry of the horizontal velocity in the microchamber were examined numerically. On the basis of the determined maximum velocity, the angular velocity and centrifugal acceleration were also evaluated. The centrifugation time of three different nanoparticles was examined by calculating the time when the nanoparticles left the microchamber for the first time. For visual observation and quantitative measurement of nanoparticle centrifugation, a 2 ×2 microfluidic centrifuge was fabricated and the experimental results demonstrate similar physical behavior to those of a mechanical centrifuge. On the basis of a comparison of the centrifugation time of two different nanoparticle populations of 300 and 700 nm in diameter, we propose that nanoparticles of different sizes can be physically separated by time under a range of inlet volume flow rates.