Kyeong-Ohn Kim

Facile single step fabrication of microchannels with varying size

In this report, we present a non-lithographic embedded template method for rapid and cost-effective fabrication of a monolithic microfluidic device with channels of various sizes. The procedure presented here enables the preparation of microchannels with varying dimensions in a single device without using any sophisticated micromachining instrumentation. In addition, this non-lithographic technique has also been used to fabricate a multilayer-multilevel biopolymer microdevice in a single step. To demonstrate the versatility of the presented method, we have fabricated microfluidic devices with four different materials under different curing/cross linking conditions. We have also demonstrated the application of the fabricated device to generate structured copper alginate microbeads, in vitro protein synthesis in three phase flow, and alternate plugs with liquid spacers.

Silica-coated alginate beads for in vitro protein synthesis via transcription/translation machinery encapsulation

Silica-coated alginate beads successfully encapsulate transcriptional and translational machinery for the prolonged cell-free synthesis of functional protein. The silica coating, identified by optical microscope and energy-dispersive spectroscopy, made the beads more resistant-than bare or chitosan-coated beads-to an alginate-dissolving environment. The permeation of small molecules through the silica coat was confirmed by the efficient intake of small fluorescent molecules. Macromolecules such as enhanced green fluorescent protein (EGFP) and fluorescence-labeled antibody were retained much longer in the silica-coated beads than in other beads. These characteristics of silica-coated beads have found utility in cell-free protein synthesis, which was verified by the successful synthesis of EGFP in the beads including DNAs and protein synthesis machinery. The productivity of the target protein increased by two-fold in the silica-coated beads over the bare alginate beads, and the activity of the protein synthesis machinery was elongated by five-fold and three-fold over bare and chitosan-coated beads, respectively. The individual protein synthesis units of silica-coated alginate beads can be utilized for the parallel synthesis of diverse functional proteins in designed combinations, which may lead to the development of artificial cells in the future.

Rapid and cost-effective fabrication of selectively permeable calcium-alginate microfluidic device using "modified" embedded template method.

In this paper, we have presented a non-lithographic embedded template method for rapid and cost-effective fabrication of a selectively permeable calcium-alginate (Ca-alginate) based microfluidic device with long serpentine delay channel. To demonstrate the versatility of the presented method, we have demonstrated two different strategies to fabricate serpentine long delay channels without using any sophisticated microfabrication techniques, in formal lab atmosphere. The procedure presented here, also, enables the preparation of a multilayered microfluidic device with channels of varying dimensions, in a single device without using any sophisticated micromachining instrumentation. In addition, we have also qualitatively studied the diffusion of small and large molecules from a Ca-alginate based microfluidic device and proposed a method to effectively control the out-flow of macro biomolecules from the crosslinked Ca-alginate matrix to create a selectively permeable matrix required for various biological and biomimetic applications, as mentioned in the Introduction section of this work.

Cell-free synthesis of functional proteins using transcription/translation machinery entrapped in silica sol–gel matrix

Herewith we report the encapsulation of functional protein synthesis machinery in a silica sol-gel matrix. When the sol-gel reaction using alkoxysilane monomers was carried out in the presence of Escherichia coli cell extract, macromolecular protein synthesis machinery in the cell extract was successfully immobilized within a silica gel matrix, catalyzing the translation of co-immobilized DNA when supplied with small-molecular-weight substrates for protein synthesis. The efficiency of protein synthesis was affected by the pore size of the gel structure, which was controlled through the use of appropriate additives during the sol-gel reactions. To the best of our knowledge, this is the first report describing the reproduction of the entire set of complicated biological process within an inorganic gel matrix, and we expect that the developed technology will find many applications in numerous fields such as high-throughput gene expression and the development of multifunctional biosensors.

Preparation of template plasmids for cell-free protein synthesis using a regenerated anion-exchange column

In this report, we describe how reactions of cell-free protein synthesis can be successfully conducted using plasmids prepared with regenerated anion-exchange columns. When washed, stripped, and equilibrated with appropriated buffers, regenerated columns were able to be used repeatedly to prepare plasmids with consistent yield and purity. The regenerated columns exhibited comparable performance to a fresh column with respect to the efficiency of protein synthesis using the plasmids prepared from them. Overall, we expect that the presented results will contribute significantly to economizing the technology of cell-free protein synthesis as a practical method for protein production in preparative scales.