检测乙酰胆碱酯酶的双光子荧光探针的设计、合成及生物学评价

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

关键词:  乙酰胆碱酯酶  有机荧光探针  细胞和组织成像  双光子成像

Key words:  acetylcholinesterase  organic fluorescent probe  cell and tissue imaging  two-photon imaging

摘要:

目的 设计并合成用于检测乙酰胆碱酯酶（acetylcholinesterase，AChE）的双光子荧光探针，并考察探针对AChE的成像检测性能。方法 经酯化、取代等多步有机反应合成探针，并通过¹H-NMR、¹³C-NMR和ESI-MS确认结构。考察探针与AChE发生响应后的荧光信噪比、灵敏度、特异性、酶动力学和双关子吸收截面积数值等指标；研究探针可否用于AChE抑制剂的体外筛选；利用单-双光子成像试验考察探针在细胞和组织水平上对AChE活力变化的检测能力。结果 通过¹H-NMR、¹³C-NMR和ESI-MS确认了产物的结构。体外试验表明探针与AChE响应后的荧光信噪比为15倍，检测限达到0.23 U·mL^–1，并具有很强的特异性和优良的酶亲合能力，在波长820 nm有最优的双光子吸收；探针可用于AChE抑制剂的体外筛选；探针通过单-双光子成像技术可对细胞和组织水平上的AChE活力变化进行成像检测，并且组织成像检测深度可达110 μm。结论 本研究成功开发了可用于AChE检测的双光子荧光探针，有潜力成为可用于活体中检测AChE活力的双光子成像试剂。

Abstract:

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.