Microchip electrophoresis with hydrodynamic injection and waste-removing function for quantitative analysis.
Quantitative analysis is problematic for microchip electrophoresis for several reasons including chip-to-chip variation, discontinuous sample re-loading, channel reconditioning, and electrokinetic injection bias. In this study, the capability for quantitative analysis on a flow-through based microchip electrophoresis, which provides continuous sample re-loading, channel washing, reconditioning and hydrodynamic injection as well as waste removing is demonstrated to be more quantifiable and more reproducible compared to manual electrokinetic injection method. Using the flow-through microchip with waste-removing function, FITC-labeled estrogen or Rhodamine B could be continuously analyzed without significant changes (R.S.D. < 6.6%) in signal intensity for over 3 h, which is sufficient for a complete set of quantitative analysis. With the use of a phosphorylated kinase substrate as the model, a calibration curve for quantitative analysis of phosphopeptides were constructed and results indicate that both R2 value of the linearity and R.S.D. values of the peak intensity were around 0.9961 and 3.16%, respectively, without the use of an internal standard. These values were slightly improved to be around 0.9986 and 2.27%, respectively, with the use of a non-phosphopeptide counterpart as the internal standard. The potential of this flow-through device for the development of a kinase phosphorylation assay based on the quantitative method was also briefly discussed.
Lin
Chun-Che
CC
Department of Chemistry, National Cheng Kung University, Tainan 70101, Taiwan.
Chen
Cheng-Chuan
CC
Lin
Ching-Erh
CE
Chen
Shu-Hui
SH
D000595:Amino Acid Sequence; D004586:Electrophoresis / Q000379:methods*; D046208:Microchip Analytical Procedures; D010748:Phosphopeptides / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results
D010748:Phosphopeptides