Microscale separation methods for enzyme kinetics assays

Abstract

Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.

Screening bioactive compounds from natural product and its preparations using capillary electrophoresis

“…HPCE has various modes including CZE, MEKC, ACE, CGE, CIEF, CEC, integrated capillary electrophoresis chips (ICEC) etc. Currently, the study of different separation modes (including on‐line and off‐line) for screening active ingredients in CE is being concentrated in the field of screening enzyme activity . We can develop effective methods that are suitable for screening active ingredients from TCMs and controlling the quality by means of referencing the existing screening enzyme activity approaches using CE.…”

Ultrasensitive assay of O‐GlcNAc transferase using capillary electrophoresis‐laser induced fluorescence

“…Although some progresses have been made, further studies toward a universal, robust, and high-sensitive method are still needed for detection of OGT activity. Owing to its advantages of high separation efficiency and sensitivity, extremely small sample requirement, and rapid analysis, capillary electrophoresis (CE) is a promising and powerful analytical technique to be widely applied in various enzyme assays [22][23][24][25], including evaluation of enzymatic activity and kinetics [26][27][28], screening of inhibitors [29,30], investigation of enzyme-mediated metabolic pathways [31,32], and proteome analysis [33]. However, to the best of our knowledge, there is no report to date using CE for assaying OGT activity in real bio-samples.…”

Prototyping of poly(dimethylsiloxane) interfaces for flow gating, reagent mixing, and tubing connection in capillary electrophoresis

“…Online derivatization is often preferred to dynamically monitor the concentration variation. Similarly, any analyses involving two or more reagents to form detectable species such as antibody-antigen binding studies and enzyme kinetics analyses also prefer online pre-column mixing and curing before being injected for electrophoretic separations [11]. Therefore, reagents mixers and tube-to-tube connectors are essential to interconnect multiple flow modules.…”