Development of an interference-free chemiluminescence method for monitoring acetylcholine and choline based on immobilized enzymes Original Research Article

A new detection method for measurement of acetylcholine (Ach) and choline (Ch) was developed, the H2O2 that is generated in enzymatic conversion action from Ach or Ch was detected through the luminol-hexacyanoferrate based chemiluminescence (CL) reaction. A new enzyme immobilization method was developed and an interference-free membrane was prepared to achieve a higher sensor response with good selectivity and stability for detection of Ach and Ch. A novel sample preparation method was developed to extract Ach and Ch from nerve tissue efficiently, and the Ach and Ch were detected by Ach and Ch sensors with very high sensitivity and reasonable precision. The Ch sensor has a detection limit (signal/noise=3) of 0.03 pmol (0.25 nM×120 μl) with a linear range of 4×10−8 to 1×10−4 M. The Ach sensor has a detection limit of 1.2 pmol (10 nM×120 μl) with linear range of 6×10−8 to 1×10−4 M. The sensitivity was improved more than 10-fold compared to the previous CL method.