Ge Lihong, Liu Zhichao, Tian Yang
Department of Chemistry , School of Chemistry and Molecular Engineering , East China Normal University , Dongchuan Road 500 , Shanghai 200241 , China . Email:
Chem Sci. 2020 Jan 13;11(8):2215-2224. doi: 10.1039/c9sc05256a. eCollection 2020 Feb 28.
β-Secretase (BACE1) is the vital enzyme in the pathogenic processes of Alzheimer's disease (AD). However, the development of a powerful tool with high selectivity and sensitivity for BACE1 determination is a challenge in understanding the pathogenesis of AD. In this work, a novel two-photon ratiometric fluorescent probe (AF633mCyd) was first developed for imaging and sensing of BACE1 in live cells and deep tissues, in which the fluorescence resonance energy transfer (FRET) system was designed and synthesized by a novel two-photon donor, merocyanine derivative (mCyd), connected with an acceptor, Alexa Fluor 633 (AF633), through a peptide substrate (EVNL-DAEFRHDSGYK) with a length of less than 10 nm. The emission spectrum of mCyd possessed sufficient overlap with the absorption spectrum of AF633, resulting in the high sensitivity of the developed AF633mCyd probe. The peptide substrate which can be specifically cleaved by BACE1 was inserted between the donor and acceptor, leading to the high selectivity of the present fluorescent probe. The fluorescence emission peaks of the AF633mCyd probe were observed at 578 nm and 651 nm and the emission ratio demonstrated good linearity with the concentration of BACE1 varying from 0.1 to 40.0 nM with a detection limit down to 65.3 ± 0.1 pM. Considering the advantages of high selectivity and sensitivity, as well as long-term stability and good biocompatibility, the developed probe was successfully applied in imaging and sensing of BACE1 in different regions of AD mouse brain tissue with a depth greater than 300 μm. Using this powerful tool, it was clear that the level of BACE1 was different in various brain regions of AD mouse such as S1BF, CPu, LD, and CA1. The up-regulation of BACE1 was observed especially in the regions S1BF and CA1 in AD mouse brain. Moreover, BACE1 was also found to be closely related to AD pathogenesis caused by oxidative stress.
β-分泌酶（BACE1）是阿尔茨海默病（AD）致病过程中的关键酶。然而，开发一种对BACE1测定具有高选择性和高灵敏度的强大工具是理解AD发病机制的一项挑战。在这项工作中，首次开发了一种新型双光子比率荧光探针（AF633mCyd），用于活细胞和深部组织中BACE1的成像和传感，其中荧光共振能量转移（FRET）系统是通过一种新型双光子供体部花青衍生物（mCyd）与受体Alexa Fluor 6,33（AF633）通过长度小于10纳米的肽底物（EVNL-DAEFRHDSGYK）连接而设计合成的。mCyd的发射光谱与AF633的吸收光谱有足够的重叠，从而使所开发的AF633mCyd探针具有高灵敏度。可被BACE1特异性切割的肽底物插入供体和受体之间，导致本荧光探针具有高选择性。AF633mCyd探针在578纳米和在651纳米处观察到荧光发射峰，并且发射比率与BACE1浓度在0.1至40.0纳摩尔范围内变化呈良好线性关系，检测限低至65.3±0.1皮摩尔。考虑到高选择性和高灵敏度以及长期稳定性和良好生物相容性的优点，所开发的探针成功应用于深度大于300微米的AD小鼠脑组织不同区域中BACE1的成像和传感。使用这个强大的工具，可以清楚地看到AD小鼠的不同脑区如初级躯体感觉皮层-底部区域（S1BF）、尾壳核（CPu）、外侧隔核（LD）和海马体1区（CA1）中BACE1的水平不同。尤其在AD小鼠脑的S1BF和CA1区域观察到BACE1上调。此外，还发现BACE1与氧化应激引起的AD发病机制密切相关。
