Method for the Sequential Online Analysis of Enzyme Reactions Based on Capillary Electrophoresis

Abstract

We have developed an easy-to-operate and effective method for performing the sequential online analysis of enzyme reactions based on capillary electrophoresis (CE). The system was constructed by passing two capillaries through a sample vial at a distance of 5 μm between the capillary ends. Direct online sample injection and sequential CE analysis were achieved by periodically switching the high-voltage power supply off and on, without any physical disturbance of the capillary inlet. The sample was injected via concentration diffusion with in-column derivatization of the amino acids occurring at the interface of the capillaries. High reproducibility of the sequential injections was demonstrated with relative standard deviation values (n = 20) of 1.01%, 1.25%, and 0.80% for peak height, peak area, and migration time, respectively. Sequential online CE enzyme assay of a glutamate pyruvate transaminase catalyzed enzyme reaction was carried out by simultaneously monitoring the substrate consumption and the product formation every 30 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. Our method has great potential for usage in sequential online CE analysis of chemical reactions with in-column chemical derivatization of the analytes for ultraviolet or laser-induced fluorescence detection.

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…While the sample is injected into the capillary by either hydrodynamic or electrokinetic injection following the precapillary enzyme reaction, the group of Yang developed a set-up for sequential online analysis of enzymatic reactions [40]. As schematically shown in Fig.…”

“…For further explanations see text. Reproduced with permission from Ref [40],. © 2012 American Chemical Society…”

Theoretical and experimental studies on sequential two-diffusional sample injection for capillary electrophoresis

“…1 presents a schematic diagram of the SDI CE system. The detail description about the system has been presented in our previous study [31]. Briefly, the system was simply constructed by coaxially aligning two capillaries and passing them through a sample vial.…”

“…Recently, our group developed a simple and easy-to-operate sequential CE analysis method [31,32]. The system was constructed by coaxially aligning two capillaries through a sample vial with a distance of 5 m between the capillary ends.…”

“…First, how is rapid and accurate online sample injection achieved, considering that it generally requires long time for the sample to diffuse into the capillary? Second, is it a versatile method for sequential CE analysis, or only applicable in the case that rapid derivatization reaction at the capillary interface occurs (as in our previous papers [31,32])? Therefore, the purpose of this paper is to carry out theoretical and experimental studies to reveal the mechanism of the SDI in our method.…”