[Show abstract][Hide abstract] ABSTRACT: Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE-SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer as a sieving matrix for CE-SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO-PPO-PEO copolymers, 255-bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO-PPO-PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan gel. Due to enhanced dynamic coating and sieving ability, PEO-PPO-PEO copolymer displayed fourfold enhancement of resolving power in the CE-SSCP to separate same-sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high-resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO-PPO-PEO triblock copolymer an excellent matrix in the CE-SSCP analysis on the microdevice.
[Show abstract][Hide abstract] ABSTRACT: Here we present telomerase activity screening using a capillary electrophoretic (CE) microdevice for human cancer diagnostics.
The telomerase enzyme, a contributor to the maintenance of telomere length in cancer cells, was extracted from various human
cells including MCF-7 (Human breast cancer cells), A549 (Human lung cancer cells), and SK-N-SH (Human neuroblastoma cells),
and then telomeric repeat amplification protocol (TRAP)-based genetic analysis was performed to evaluate the activity of telomerase
enzyme. The resultant 6-bp tandem repeat PCR amplicons derived from cancer cells were separated and detected on the 6 cm-long
CE microdevice within 5 min. A limit of detection of 5 cells was achieved to analyze the telomerase activity on a CE microchip.
In comparison with a conventional PAGE method, micro-fabricated CE-based analysis has many advantages in terms of high speed,
low sample consumption, and high sensitive detection. The successful demonstration of telomerase activity screening on a chip
implies great potential of a complete integration of a sample preparation and PCR unit in a single format for high-performance
cancer diagnostics in a clinical arena.
KeywordsTelomerase-Telomeric repeat amplification-Polymerase chain reaction-Capillary electrophoretic microdevice-Cancer diagnostics