title: Online transient isotachophoresis concentration by the pseudo-terminating electrolyte buffer for the separation of DNA-aptamer and its thrombin complex in poly(methyl methacrylate) microchip.
abstract: Online automatic transient isotachophoresis concentration of DNA-aptamer and its thrombin complex by using one kind of pseudo-terminating electrolyte buffer in a cross-channel poly(methyl methacrylate) microchip is reported. Sample injection, transient concentration and separation were done continuously and controlled by a sequential voltage switching program, time-consuming steps and complicated chip design were not required. Peak resolution between DNA-aptamer and its thrombin complex was influenced by this novel pseudo-terminating electrolyte buffer, which was prepared by the addition of chemical component with slow mobility into the same buffer as leading electrolyte buffer. 1100-fold signal enhancement of thrombin complex was achieved by this transient isotachophoresis on a standard cross-form microchip. The concentration effect or standing time of transient isotachophoresis was proved to be influenced by the concentration of leading electrolyte ion and the concentration of pseudo-terminating electrolyte buffer ion (glycine). The transient concentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution for the size-based separation. The detection limit, taken as the lowest thrombin concentration at threefold S/N, was determined to be 0.5 amol in mass by this method.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japan. wjzyy988@gmail.com
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Noritada
authors.initials: N
authors.lastname: Kaji
authors.forename: Manabu
authors.initials: M
authors.lastname: Tokeshi
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Baba
mesh_terms: D052157:Aptamers, Nucleotide / Q000032:analysis* / Q000302:isolation & purification; D002021:Buffers; D004573:Electrolytes / Q000737:chemistry*; D046209:Electrophoresis, Microchip / Q000379:methods*; D057625:Isotachophoresis / Q000379:methods*; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000032:analysis* / Q000302:isolation & purification
chemical_list: D052157:Aptamers, Nucleotide; D002021:Buffers; D004573:Electrolytes; D019904:Polymethyl Methacrylate; D013917:Thrombin