Electroosmotic Sampling and Its Application to Determination of Ectopeptidase Activity in Organotypic Hippocampal Slice Culture when coupled with HPLC-EC

[摘要] Sampling through extracellular space has always been a challenge. Traditional methods, such as push-pull perfusion, microdialysis and direct sampling, are not suitable for sampling through thin slice cultures; since they all involve probe implantation. Through this research we developed a novel sampling method based on electroosmosis. By taking advantage of the natural ζ-potential in an organotypic hippocampus slice culture, an electroosmotic flow was successfully invoked within the extracellular space by applying an electric field. This flow was used to draw the physiological fluid from the slice culture. We have characterized this method by examining the flow rate, sampling bias, internal standard and damage caused during sampling. Coupled with a capillary high performance liquid chromatography (HPLC) and an electrochemical detection, this novel sampling method was applied to study enkephalin hydrolysis by ectoenzymes distributed in the hippocampus area. The results showed not only the feasibility and efficiency of the electroosmotic sampling technique in extracting extracellular material, but also its potential for studying ectopeptidase activity. A capillary Taylor reactor was employed to perform post-column reactions in the electrochemical detection. This reactor shares pressure with the capillary column and adds band-spreading, which reduce separation efficiency. We quantitatively evaluated this effect and gave a set of optimal and practical conditions.

[发布机构] the University of Pittsburgh