Kwanseop Lim

Poly(dimethylsiloxane) microchip for precolumn reaction and micellar electrokinetic chromatography of biogenic amines

We have demonstrated that precolumn derivatization and capillary electrophoresis separation on a poly(dimethylsiloxane) (PDMS) microchip can be realized as efficient as those on glass microchips. In an optimized condition of micellar electrokinetic chromatography (MEKC), using 25 mM sodium borate buffer (pH 10.0) with 25 mM sodium dodecyl sulfate (SDS) and 5% v/v methanol, the electroosmotic flow in an oxidized PDMS microchip is stabilized within 3% for days. By employing a fluorometric derivatization with o‐phthaldialdehyde (OPA) in an optimally designed reaction chamber, four most important biogenic amines occurring in foods, histamine, tyramine, putrescine, and tryptamine, are quantitatively determined in less than 1 min at the levels applicable to real samples. The migration behaviors of anionic OPA‐derivatized biogenic amines under the MEKC conditions are analyzed, and it has been found that under our separation conditions, the electrophoretic mobility of the SDS micelles is significantly greater than those of the anions in the aqueous phase. The channel manifold in a PDMS substrate is fabricated using replica molding against a thick photoresist, SU‐8, pattern generated by photolithography. The plate with the microchannel pattern is strongly, irreversibly bonded to another PDMS plate by using a new bonding technique, which employs surface oxidation by corona discharge generated from a cheap, handy source, Tesla coil.

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase.

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two‐state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two‐state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X‐ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7 Å in 0 M urea, 17.3 Å in 5.2 M urea, and 25.1 Å in 7 M urea by SAXS. The size of KSI in 5.2 M urea was significantly decreased compared with those in 0 M and 7 M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2 M urea is distinctly different from native and fully‐unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using 1H NMR spectroscopy and Kratky plot supported the existence of a partially‐folded form of KSI which is distinct from those of native and fully‐unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.

Adhesive and Dead Volume Free Interfacing between PDMS Microfluidic Channels

We have developed an adhesive and dead volume free poly(dimethylsiloxane) (PDMS) microfluidic channel interfacing technique through capillaries. Capillary zone electrophoresis (CZE) with a bare capillary and capillary electrochromatography (CEC) with a beads-packed separation column successfully separated fluorescein, FITC and coumarin440 and 450 respectively using the adhesive free feature.

PDMS Microchip for Precolumn Reaction and Micellar Electrokinetic Chromatography of Biogenic Amines

We have developed a microchip to perform fast and efficient precolumn reactions and micellar electrokinetic chromatography (MEKC) of biogenic amines sequentially in poly(dimethylsiloxane) (PDMS). The relative mixing and reaction efficiencies of three different structures of precolumn reactors are investigated. The separation efficiency in a PDMS chip is comparable to that in a glass chip.

Squeezing micropumps for elastomer microchips; picoliter-handling capability

We have developed a squeezing micropump for elastomeric micropumps, which is featured by capabilities of bi-directional pumping, highly reproducible and ultra-small-volume handling down to picoliters.