Simplified transient isotachophoresis/capillary gel electrophoresis method for highly sensitive analysis of polymerase chain reaction samples on a microchip with laser-induced fluorescence detection.
We present a sensitive, simple and robust on-chip transient isotachophoresis/capillary gel electrophoresis (tITP/CGE) method for the analysis of polymerase chain reaction (PCR) samples. Using chloride ions in the PCR buffer and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the background electrolyte, respectively, as the leading and terminating electrolytes, the tITP preconcentration was coupled with CGE separation with double-T shaped channel network. The tITP/CGE separation was carried out with a single running buffer. The separation process involved only two steps that were performed continuously with the sequential switching of four voltage outputs. The tITP/CGE method showed an analysis time and a separation efficiency comparable to those of standard CGE, while the signal intensity was enhanced by factors of over 20. The limit of detection of the chip-based tITP/CGE method was estimated to be 1.1 ng/mL of DNA in 1x PCR buffer using confocal fluorescence detection following 473 nm laser excitation.
Liu
Dayu
D
Bioengineering Center, Improve Medical Instrument's Co., Ltd., 510370 Guangzhou, China. ruark@126.com
Ou
Ziyou
Z
Xu
Mingfei
M
Wang
Lihui
L
D004247:DNA / Q000737:chemistry*; D004586:Electrophoresis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D046209:Electrophoresis, Microchip / Q000379:methods*; D005453:Fluorescence; D007834:Lasers; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004247:DNA