Sunwoong Bae

Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure

A perforated hemispherical microwell structure is shown to efficiently capture single Chlamydomonas reinhardtii (C. reinhardtii) cells, culture them to form colonies, and retrieve these colonies to serve as seeds for large-scale cultivation. This solution-phase formation and recovery of colonies could overcome the tedious and time-consuming process of selecting colonies from a solid-phase agar plate. The fabricated microdevice was composed of three layers: a top layer consisting of a cell solution for injection and recovery of a microalgal solution, a hemispherical perforated microwell array in the middle, and a bottom layer in which the solution is manipulated by controlling the hydrodynamic force. The microalgal (wild type and hygromycin B-resistant mutant) cells loaded in the top layer rapidly diffused into the microwell holes, and individual such cells were captured with high efficiency (>90%) and within 1 min by applying a withdraw mode in the bottom layer. Single-cell-based cultivation in a medium containing hygromycin B was then performed to generate colonies in the hemispherical microwell. While the wild type cells died, mutant cells resistant to hygromycin B survived well and grew into a colony within 2 days. The produced colonies in the microwells were recovered by applying a release mode in the bottom layer, so that a hydrodynamic force was exerted vertically to push out the colonies through the outlet in 10 s. The recovered cells were cultured on a large scale in medium by using a flask. The recovered C. reinhardtii was confirmed as a hygromycin B-resistant mutant by identifying the hygromycin gene in the polymerase chain reaction (PCR). The microdevice described here could in solution perform single-cell capture, colony formation, and retrieval of colonies for further large-scale cultivation, which could replace tedious and time-consuming solid-phase agar plate processes with a 7-fold reduction in the duration of the process.

Exogenous Gene Integration for Microalgal Cell Transformation Using a Nanowire-Incorporated Microdevice.

Superior green algal cells showing high lipid production and rapid growth rate are considered as an alternative for the next generation green energy resources. To achieve the biomass based energy generation, transformed microalgae with superlative properties should be developed through genetic engineering. Contrary to the normal cells, microalgae have rigid cell walls, so that target gene delivery into cells is challengeable. In this study, we report a ZnO nanowire-incorporated microdevice for a high throughput microalgal transformation. The proposed microdevice was equipped with not only a ZnO nanowire in the microchannel for gene delivery into cells but also a pneumatic polydimethylsiloxane (PDMS) microvalve to modulate the cellular attachment and detachment from the nanowire. As a model, hygromycin B resistance gene cassette (Hyg3) was functionalized on the hydrothermally grown ZnO nanowires through a disulfide bond and released into green algal cells, Chlamydomonas reinhardtii, by reductive cleavage. During Hyg3 gene delivery, a monolithic PDMS membrane was bent down, so that algal cells were pushed down toward ZnO nanowires. The supply of vacuum in the pneumatic line made the PDMS membrane bend up, enabling the gene delivered algal cells to be recovered from the outlet of the microchannel. We successfully confirmed Hyg3 gene integrated in microalgae by amplifying the inserted gene through polymerase chain reaction (PCR) and DNA sequencing. The efficiency of the gene delivery to algal cells using the ZnO nanowire-incorporated microdevice was 6.52 × 10(4)- and 9.66 × 10(4)-fold higher than that of a traditional glass bead beating and electroporation.

Rapid, High-Throughput, and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire-Incorporated and Pneumatic Pressure-Driven Microdevice

Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real-time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire-incorporated and pneumatic pressure-driven microdevice for rapid, high-throughput, and direct MB delivery to human breast cancer MCF-7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump-associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire-patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB-coated nanowire array can be manipulated. The combination of the nanowire-mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment.

A large-area hemispherical perforated bead microarray for monitoring bead based aptamer and target protein interaction

Herein, we present a large-area 3D hemispherical perforated microwell structure for a bead based bioassay. Such a unique microstructure enables us to perform the rapid and stable localization of the beads at the single bead level and the facile manipulation of the bead capture and retrieval with high speed and efficiency. The fabrication process mainly consisted of three steps: the convex micropatterned nickel (Ni) mold production from the concave micropatterned silicon (Si) wafer, hot embossing on the polymer matrix to generate the concave micropattened acrylate sheet, and reactive ion etching to make the bottom holes. The large-area hemispherical perforated micropatterned acrylate sheet was sandwiched between two polydimethylsiloxane (PDMS) microchannel layers. The bead solution was injected and recovered in the top PDMS microchannel, while the bottom PDMS microchannel was connected with control lines to exert the hydrodynamic force in order to alter the flow direction of the bead solution for the bead capture and release operation. The streptavidin-coated microbead capture was achieved with almost 100% yield within 1 min, and all the beads were retrieved in 10 s. Lysozyme or thrombin binding aptamer labelled microbeads were trapped on the proposed bead microarray, and the in situ fluorescence signal of the bead array was monitored after aptamer-target protein interaction. The protein-aptamer conjugated microbeads were recovered, and the aptamer was isolated for matrix assisted laser desorption/ionization time-of-flight mass spectrometry analysis to confirm the identity of the aptamer.

Investigation on the Nucleation Stage of Palladium Nanoparticles Using a Microfluidic Droplet Generator Integrated with In Situ Sol-Gel Quencher

The nanoparticle (NP) synthesis undergoes stepwise processes starting from the input metal ions: nucleation, coalescence, ripening, and growth. Considering the whole process is completed in a very short time, the conventional flask-scale method, which requires at least minutes, is not adequate to trace the mechanism of NP nucleation. In this study, a microfluidic droplet generator is developed, which is capable of in situ sol-gel polymerization for synthetic reaction quenching. As a model, palladium (Pd) NPs are synthesized within microdroplets, and the reaction time is controlled by tuning the length of the microchannel. In the microfluidic design, the outmost microchannel is incorporated, in which tetraethyl orthosilicate (TEOS) dissolved in ethanol is injected. The generated droplets are merged to the outmost flow under the variety of time interval (50 to 5,000 ms), so that the tens of milliseconds observation on NP nucleation is conducted via flash-like sol-gel quenching. Based on the result analysis, the seeds of Pd NPs have undergone slight size fluctuation and then a thermodynamically stable aggregation/coalescence step within 5 s before moving into the growth stage. This microfluidic platform permits the study of the fundamental and initial stage of the NP synthesis, which cannot be approached by the conventional methodology.

Molecular Delivery: Rapid, High-Throughput, and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire-Incorporated and Pneumatic Pressure-Driven Microdevice (Small 46/2015)

A nanowire-incorporated and pneumatic pressure-driven microdevice for rapid, high-throughput and direct molecular beacon (MB) delivery is described by I. Park, T. S. Seo, and co-workers. On page 6215, they show how the microdevice, composed of a manifold layer, a monolithic PDMS membrane, and a nanowire-patterned microchannel layer, could manipulate cellular movement, so the attachment and detachment from the MB-functionalized nanowire array is performed in a controllable way to increase MB delivery efficiency.