A Novel Two-Photon Ratiometric Fluorescent Probe for Imaging and Sensing of BACE1 in Different Regions of AD Mouse Brain

β-secretase (BACE1) is the vital enzyme in the pathogenic processes of Alzheimer’s disease (AD). However, development of a powerful tool with high selectivity and sensitivity for BACE1 determination in vivo is a challenge to understand 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 length 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 donor and acceptor, leading to the high selectivity of the present fluorescent probe. The fluorescent emission peaks of AF633mCyd probe were observed at 578 nm and 651 nm, and the emission ratio demonstrated a good linearity with the concentration of BACE1 varying from 0.1 to 40.0 nM with detection limit down to 65.3 ± 0.1 pM. Taken 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 depth greater than 300 μm. Using this powerful tool, it was clear that the level of BACE1 was different in the various brain regions of AD mouse such as S1BF, CPu, LD, and CA1. The up-regulation of BACE1 was observed especially in the regions of S1BF and CA1 in AD mouse brain. Moreover, BACE1 was also found to be closely related to AD pathogenesis caused by oxidative stress.