Biomedical subjects

Junhoe Cha

Publications and source records attributed to Junhoe Cha.

3 recordsLinked to original sources

PubMed2003

DNA hybridization electrochemical sensor using conducting polymer.

We report the use of poly(thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydro-isoindol-2-yl ester (PTAE) for application to electrochemical hybridization sensor. A synthetic route for the thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydro-isoindol-2-yl ester (TAE) is described, which is used as a monomer of conducting polymer sensor. A direct chemical substitution of probe oligonucleotide to good leaving group site in the PTAE is carried out on the conducting polymer film. A biological recognition can be monitored by comparison with the electrochemical signal (cyclic voltammogram) of single and double strand state oligonucleotide. The sensitivity of the electrochemical sensor is 0.62 microA/nmole and the detection limit is 1 nmole. The oxidation current of double strand state oligonucleotide is a half of that of single strand, that is corresponding to the decrease of electrochemical activity of conducting polymer with increase of stiffness of side group of the polymer. The oxidation current decreasing ratios of perfect matched and single nucleotide mismatched samples are 52 and 25-30%, respectively. The more decreasing ratio is attributable to the more steric hindrance of single nucleotide mismatched sample.

Biosensing Techniques↗

PubMedDate not supplied

Simultaneous in vitro protein synthesis using solid-phase DNA template.

In vitro protein synthesis is rapidly becoming an accepted tool in functional genomic analysis. We have demonstrated the in vitro synthesis of firefly luciferase on solid-phase template DNA, bound to wells in 96-well plates, using simultaneous transcription and translation in a wheat-germ extract system. The bound DNA template was stable and did not release during transcription. Coupled translation resulted in ca. 1.2 ng/microL luciferase synthesized, which is ca. one-fifth of that synthesized using conventional solution-phase coupled transcription and translation. Reuse of the DNA template was influenced by the complexity of the wheat-germ extract, which resulted in fouling of the transcription surface and reduction of protein synthesis after extended use. The approach developed in this study may enable the development of high-throughput, microscale protein synthesis platforms for use in functional genomic analysis.

Cell-Free System↗

PubMedDate not supplied

Chip-based polyketide biosynthesis and functionalization.

We demonstrate construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microfluidic platform. THNS immobilized to Ni-NTA agarose beads is prepacked into a microfluidic channel, while SBP is covalently attached to the walls of a second microfluidic channel precoated with a reactive poly(maleic anhydride) derivative. The result is a tandem, two-step biochip that enables the synthesis of novel polyketide derivatives. The first microchannel, consisting of THNS, results in the conversion of malonyl-CoA to flaviolin in yields up to 40% with a residence time of 6 min. This conversion is similar to that obtained in several-milliliter batch reactions after 2 h. Linking this microchannel to the SBP microchannel results in biflaviolin synthesis. During the course of this work, we discovered that the substrate specificity of THNS could be manipulated by simply changing the reaction pH. As a result, the starter acyl-CoA specificity can be broadened to yield a series of truncated pyrone products. When combined with variations in the ratio of acyl-CoA and malonyl-CoA (extender substrate) feed rates, high yields of the pyrone products could be achieved, which is further structurally diversified from self- and cross-coupling in the SBP microchannel. The ability to rapidly evaluate the effects of reaction conditions and synthetic multienzyme pathways on a microfludic platform provides a new paradigm for performing metabolic pathway engineering, namely, the reconstruction of pathways for use in new compound discovery.

Catalysis↗