Application Study On Capillary Electrophoresis-Electrochemiluminescence Detection In Bioanalysis

CE-ECL is a combination of capillary electrophoresis(CE) and electrochemiluminescence(ECL), which has the advantages of high separation efficiency and high detection sensitivity. This article applied CE-ECL to study the interactions of protein and drug and enzyme reactions. The main research work was summarized as follows:Chapter one: It Outlined the capillary electrophoresis(CE)-electrochemiluminescence(ECL) technology and its application in pharmaceutical analysis, drug and protein analysis, enzyme activity assay and so on.Chapter two: Graphite electrode, surface of which was modified with Ru(bpy)32+-doped silica nanoparticles(Ru DS)-chitosan film, was used as a working electrode. The ECL signal was further increased in the presence of azithromycin, which could be used for end-column detection of azithromycin concentration. The binding constant between azithromycin and human serum albumin(HSA) was estimated to be 7.46×104 L/mol by nonlinear regression analysis. Warfarin and ketoprofen were used as probes to study their interaction mechanism and the result showed that azithromycin binds site I of HSA.Chapter three: N, N-dimethylethanolamine(DMEA) could enhance the Ru(bpy)32+ ECL signals efficiently, based on this, a new method to assess the activity of butyrylcholinesterase(BCh E) was built. In this paper, the main factors affecting the activity of BCh E were inspected, including magnesium ions and calcium ions as BCh E activator of enzyme, incubation p H, incubation temperature, incubation time, as well as substrate concentration. The detection limit for DMEA was 1.98×10-8 M(S/N = 3), which was equal to that 6.6×10-10 units of BCh E. The Michaelis constant Km(1.16×10-3 mol/L) and the maximum reaction velocity Vmax(2.71×10-7 mol/L/min) of BCh E for tetracine hydrochloride were reported.Chapter four: Oxybuprocaine hydrochloride eye drops is also known as Benoxil, its chemical name is 2-(diethylamino)ethyl-4-amino-3-butoxy-benzoate hydrochloride salts, mainly used in topical anesthesia within the field of ophthalmic surgery. In this paper, we study the content changes of oxybuprocaine hydrochloride in eye drops with different p H values under illumination. In the presence of butyrylcholinesterase(BCh E), Oxybuprocaine hydrochloride as substrate was hydrolyzed into 4-amino-3-butoxy-benzoic acid and Diethylamino ethanol. This paper studies the pharmacokinetics of oxybuprocaine hydrochloride in the presence of butyrylcholinesterase. The Michaelis constant Km(1.28×10-3 mol/L) and the maximum reaction velocity Vmax(3.3×10-6 mol/L/min) of BCh E for oxybuprocaine hydrochloride were reported.