Study And Application Of Capillary Electrophoresis-based Enzyme Assay

Capillary electrophoresis(CE)has become an important tool for enzyme assay due to its unique advantages of fast analysis speed,high separation efficiency,low consumption of samples and reagents,and its ability in combination with a variety of detection methods.Focused on the application of CE in enzyme assay,we studied the L-lactate dehydrogenase(L-LDH)immobilized enzyme microreactors(IMERs)and the activity assay of recombinant human O-GlcNAc transferase(OGT)in this thesis.1.CE-integrated IMERs with graphene oxide(GO)as support: Immobilization of negatively charged L-LDH via hydrophobic interactivity.We report the first application of hydrophobic interaction between GO and negatively charged enzymes to fabricate CE-integrated IMERs by a simple and reliable immobilization procedure based on layer by layer assembly.L-LDH,which is negatively charged during the enzymatic reaction,is selected as the model enzyme.Various spectroscopic techniques,including SEM,FTIR,and UV-vis are used to characterize the fabricated CE-IMERs,demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface.The IMER exhibits excellent repeatability with RSDs of inter-day and batch-to-batch less than 3.49 and 6.37%,respectively,and the activity of immobilized enzymes remains about 90% after five-day usage.The measured K_m values of pyruvate and NADH of the immobilized L-LDH are in good agreement with those obtained by free enzymes.The results demonstrate that the hydrophobic interaction and/or ?-? stacking is significant between the GO backbone and the aromatic residues of L-LDH and favorable to fabrication of CE-integrated IMERs.Finally,the method is successfully applied to the determination of pyruvate in beer samples.2.Rapid and sensitive enzyme assay of OGT based CE-laser induce fluorescence(LIF)detection.OGT is a vital intracellular enzyme which catalyzes the transfer of single O-linked N-acetylglucosamine(O-GlcNAc)moiety to a serine or threonine residues of proteins,this process termed O-GlcNAcylation.And the deregulation of OGT activity has also been linked to a range of diseases such as alzheimer's,diabetes and cancer.We designed an AF488 modified peptide with a serine active site that can be glycosylated by OGT.The AF488 modified peptide was used as the substrate of OGT and a universal nonradioactive UDP-GlcNAc was used as the sugar donor.In the presence of OGT,it catalyzes the glycosylation reaction to generate a glycosylated peptide.The produced glycosylated peptide and unreacted peptide are analyzed by CE-LIF,and the corresponding relationship between the intensity of fluorescence signal and the concentration of OGT were obtained.The limit of detection of OGT this method is 1.71 nM.This method could be applied in the determination of OGT enzyme activity,the study of enzyme reaction kinetics,the screening of enzyme inhibitors and the determination of intracellular OGT activity,which provides a potential therapeutic target for the diagnosis of diseases.