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Triblock copolymer matrix-based capillary electrophoretic microdevice for high-resolution multiplex pathogen detection

Kim, Se Jin, Shin, Gi Won, Choi, Seok Jin, Hwang, Hee Sung, Jung, Gyoo Yeol, Seo, Tae Seok
Electrophoresis 2010 v.31 no.6 pp. 1108-1115
Haemophilus ducreyi, Neisseria meningitidis, Proteus mirabilis, Pseudomonas aeruginosa, coatings, composite polymers, electrophoresis, gelation, gels, genes, microbial detection, pathogens, patients, polyacrylamide, polymerase chain reaction, quantitative analysis, ribosomal RNA, sieving, single-stranded DNA
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[trade mark sign] 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.