Lanthanide Complex-Based Probes for Ratiometric Time-Gated Luminescence and ¹⁹ F Magnetic Resonance Imaging of Hydrogen Peroxide In Vitro and In Vivo

Fluorescent bioimaging is a key technique for real-time and in situ visualization of bioactive species in living organisms. However, its utility for in vivo applications is often limited by a low tissue penetration depth. Herein we report novel lanthanide complex-based probes, a ratiometric time-gated luminescence (TGL) probe, Mito-PFTTA-Eu3+/Tb3+, and a ¹⁹F magnetic resonance (MR) probe, Mito-PFTTA-Gd3+, for the highly sensitive and in situ detection of hydrogen peroxide (H₂O₂) in biological samples. To construct the probe, we first designed and synthesized a multifunctional ligand, (4'-pentafluorobenzenesulfonate-2,2':6',2''-terpyridine-6,6''-diyl) bis(methylenenitrilo)tetrakis(acetic acid) (Mito-PFTTA), by integrating a mitochondria-anchoring motif, triphenylphosphonium cation and a H₂O₂-responsive pentafluorobenzenesulfonate (PFBS) moiety into a terpyridine polyacid framework. Then a mixture of its Eu³⁺ and Tb³⁺ complexes, Mito-PFTTA-Eu3+/Tb3+, were developed as a ratiometric TGL probe for H₂O₂ detection. In parallel, its Gd³⁺ complex, Mito-PFTTA-Gd3+, was constructed as an ¹⁹F NMR probe for H₂O₂ sensing. Upon reaction with H₂O₂, the PFBS group is cleaved from the complex, leading to opposite changes in the emission intensity: an increase in the Tb³⁺ emission at 540 nm and a decrease in the Eu³⁺ emission at 610 nm. These changes allow Mito-PFTTA-Eu3+/Tb3+ to serve as a ratiometric TGL probe for H₂O₂ detection using the I₅₄₀/I₆₁₀ ratio as a signal output. Thanks to its excellent mitochondrial targeting capability and excellent biocompatibility, Mito-PFTTA-Eu3+/Tb3+ enabled the ratiometric TGL imaging of mitochondrial H₂O₂ in living cells and in the livers of mice with drug-induced liver injury. Additionally, the ¹⁹F NMR signal of Mito-PFTTA was effectively quenched upon complexation with Gd³⁺ ions, but was restored upon H₂O₂-triggered PFBS cleavage. This behavior allowed Mito-PFTTA-Gd3+ to function as a ¹⁹F MRI probe for detection of H₂O₂ in vitro and in vivo.