Development of a ratiometric two-photon fluorescent probe for imaging of hydrogen peroxide in ischemic brain injury

Abstract

Hydrogen peroxide (H₂O₂) plays crucial roles in immunological processes and has been revealed to be closely linked to the hypoxic–ischemic process. Thus, it is important to develop a reliable method for monitoring H₂O₂ in hypoxic–ischemic brain injury (HIBI). To achieve this, we report on a rationally designed fluorene-based ratiometric two-photon fluorescent probe for H₂O₂, i.e., FH₂O₂. The probe and fluorophore were tested under either one- or two-photon modes, respectively. After reacting with H₂O₂, the relative emission intensity ratio at wavelengths of 390–465 (F_blue) and 500–550 nm (F_yellow) of FH₂O₂ had a 6.5-fold increase (F_yellow/F_blue) within 40 min, and the maximal two-photon active cross-section value was detected as 66 GM at a wavelength of 750 nm. The probe exhibited high selectivity, low cytotoxicity, high sensitivity with a detection limit of 0.57 μM, and adequate photostability. After confirming satisfactory sensing performance of the probe in vitro, we were able to monitor H₂O₂in situ in mice with HIBI via two-photon microscopy, which could provide a potential method for clinical diagnosis during the neonatal HIBI process.