Integrated polymerase chain reaction and capillary electrophoresis microdevice for pathogen detection and cancer diagnostics

Abstract

Microfluidic technology has paved its way in a variety of chemical and biological applications due to the benefits such as small sample consumption, high throughput screening capability, fast analysis time and portability. DNA amplification based on polymerase chain reaction (PCR) and capillary electrophoresis (CE) DNA separation are the most indispensable technique for molecular diagnostics. In this study, we adopted microtechnology to miniaturize the PCR reactor in a nanoliter scale combined with an advanced micro-CE $(\mu u CE)$ system for highly sensitive and resolved pathogen detection and cancer diagnostics. As a first part, we developed a capillary electrophoretic microdevice for high-resolution multiplex pathogen detection by using single strand conformation polymorphism (SSCP) technique. In particular, we explored the capability of poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer as a sieving matrix in the CE microchannel to separate the single-stranded DNA molecules with high resolution. To demonstrate superior resolving power of PEO-PPO-PEO copolymers, 255-bp PCR amplicons obtained from the 16S rRNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, Neisseria meningitidis were analyzed in the PEO-PPO-PEO matrix in comparison with 5% linear polyacrylamide (LPA) and commercial GeneScanTM gels. Due to the enhanced dynamic coating and sieving ability, PEO-PPO-PEO copolymer displayed 4-fold enhancement of resolving power in the CE-SSCP to separate the same sized DNA molecules in about ~11 min. The produced peaks in the electropherogram were correlated well with the amount of genomic DNA templates, enabling correct quantitative analysis in the pathogen detection. Besides the high resolution sieving capability, a facile loading and replenishment of gels in the microchannel owing to the thermally reversible gelation property makes PEO-PPO-PEO triblock copolym...