Detection Of Acetylcholine By Luminol-H₂O₂-Laccase Chemiluminescence System

Acetylcholine is one of the most crucial neurotransmitters and distributed widely in prokaryotic and eukaryotic cells. As the main component of cholinergic system, acetylcholine is of significance in the functions of central nervous system (CNS) and non-neuronal cells. In the central nervous system (CNS) and peripheral nervous system(PNS), acetylcholine plays a role of mediating effective information exchange between neurons and effector cells. The content of acetylcholine not only modulates the plasticity of cynapse, but also is related to the cognition involving memory and learning. Researchers have found that neurological disorders, such as Schizophrenia, Parkinson’s and Alzheimer’s diseases, were resulted from the abnormal concentrations of acetylcholine in CNS and PNS. Therefore, it is urgent to establish a convenient and sensitive method for acetylcholine detection in fundmental theory research and clinical diagnosis.Laccase, as a member of multicopper oxidase family, has active centre contains four copper atoms and attracted much attention. After been found and purified, studies on applications of laccase occurred gradually. Up to now, laccase has been used for biopulping in paper industry, baking and wine stabilization in food industry, biobleaching in texile industry and bioremediation in environmental protection. In addition, advantages of low specificity on its substrates and broadly distribution of laccase also make it have potential development prospectives.In this study, we built a simple, low-cost and convenient method for acetylcholine determination based on the traditional luminol-H2O2 chemiluminescence system, in which laccase was selected as the catalyst. In this procedure, acetylcholine was degraded into H2O2 by acetylcholine esterase and choline oxidase, H2O2 then toke part in the luminol chemiluminescence. After optimization of conditions, we obtained good working curve in which the concentrations of acetylcholine ranged from 2.5μM to 250μM with the detection limit of 2μM (S/N=3). Meanwhile, the reproducibility of the method was performed with 75μM acetylcholine, the RSD (3.2%) indicated that this method has excellent reproducibility. In addition, glutamic acid, aspartic acid, glycine, glucose, ascorbic acid, dopamine and lactic acid were used for the specificity and interference study and got ideal results.