Design, Synthesis and Biological Evaluation of the Two-photon Fluorescence Probe for Acetylcholinesterase Detection

OBJECTIVE To design and synthesize a two-photon fluorescence probe for acetylcholinesterase(AchE) detection, and evaluate its imaging detection performance toward AChE. METHODS The probe was synthesized by esterification and substitution reaction and the structures of synthetic compounds were fully characterized by ¹H-NMR,¹³C-NMR and ESI-MS. The signal-to-noise ratio, sensitivity, specificity, enzyme kinetics and double correlation absorption cross-sectional area of fluorescence response of probe toward AChE were evaluated. Whether the probe could be used to screen AChE inhibitors in vitro was studied. One and two photon imaging experiments were conducted to investigate the ability of probes for detecting changes of AChE activity in cell and tissue levels. RESULTS The structures of synthetic products were confirmed by ¹H-NMR,¹³C-NMR and ESI-MS. The fluorescence signal-to-noise ratio of the probe toward AChE was 15 times and the detection limit was 0.23 U·mL^-1. The probe also exhibited strong specificity and excellent binding constant for AChE detection, and could be applied for screening AChE inhibitors. Meanwhile, the reaction solution of probe with AChE showed the optimal two-photon absorption was 820 nm. Moreover, the probe could be successfully used for imaging the changes of AChE activity in living cells and tissues, and the detection depth of tissue imaging could reach 110 μm. CONCLUSION In this study, a two-photon fluorescence probe for AChE detection has been successfully developed, and this probe has the potential to become a two-photon imaging reagent for detecting AChE activity in vivo.