Sujeong Shin

Investigation of the signaling mechanism and verification of the performance of an electrochemical real-time PCR system based on the interaction of methylene blue with DNA

The operation of an electrochemical real-time PCR system, based on intercalative binding of methylene blue (MB) with dsDNA, has been demonstrated. PCR was performed on a fabricated electrode-patterned glass chip containing MB while recording the cathodic current peak by measuring the square wave voltammogram (SWV). The current peak signal was found to decrease with an increase in the PCR cycle number. This phenomenon was found to be mainly a consequence of the lower apparent diffusion rate of the MB-DNA complex (D(b) = 6.82 × 10(-6) cm(2) s(-1) with 612 bp dsDNA) as compared to that of free MB (D(f) = 5.06 × 10(-5) cm(2) s(-1)). Utilizing this signal changing mechanism, we successfully demonstrated the feasibility of an electrochemical real-time PCR system by accurately quantifying initial copy numbers of Chlamydia trachomatis DNA templates on a direct electrode chip. A standard calibration plot of the threshold cycle (C(t)) value versus the log of the input template quantity demonstrated reliable linearity and a good PCR efficiency (106%) that is comparable to that of a conventional TaqMan probe-based real time PCR. Finally, the system developed in this effort can be employed as a key technology for the achievement of point-of-care genetic diagnosis based on the electrochemical real-time PCR.

Mismatch DNA-specific enzymatic cleavage employed in a new method for the electrochemical detection of genetic mutations

Utilizing enzymatic mismatched DNA-specific cleavage and electrocatalytic signaling, a new electrochemical method for the detection of DNA mutations was developed and successfully applied to detect various mutations in the BRCA1 gene.

Fabrication of conductive oxidase-entrapping nanocomposite of mesoporous ceria–carbon for efficient electrochemical biosensor

A conductive nanocomposite containing an immobilized oxidative enzyme in the pores of mesostructured ceria (CeO2)–carbon was developed as an efficient electrochemical biosensing platform. The construction of the nanocomposite began with the incorporation of CeO2 in a carbon matrix by the co-assembly of cerium nitrate, resol, and triblock copolymer via a facile evaporation-induced self-assembly method, which resulted in the formation of mesoporous ceria–carbon (denoted as Meso-CeO2/C). Glucose oxidase (GOx) was subsequently immobilized in the vacant pores of the Meso-CeO2/C by using glutaraldehyde crosslinking to prevent enzyme leaching from the matrix. H2O2 generated by the catalytic action of GOx in proportion to the amount of target glucose was rapidly converted into hydroxyl radicals by the catalytic activity of CeO2, which induced subsequent anodic oxidation of Ce3+ into Ce(OH)22+ or Ce(OH)4 with the anodic current. The constructed Meso-CeO2/C exhibited higher resolution in electrochemical detection of H2O2 than pure mesoporous carbon without ceria owing to the catalytic activity of ceria. The anodic current responses by the nanocomposite containing GOx in Meso-CeO2/C resulted in a linear increase in the concentration of target glucose (0.25–5 mM), which is suitable to measure the serum glucose, with excellent storage stability of over two months at room temperature. The biosensor also exhibited a high degree of precision and reproducibility when employing real human blood samples. Based on these results, we anticipate that this novel biosensing format can be readily extended to other oxidative enzymes for the detection of various clinically important target molecules.