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

Fluorescent bioimaging is a key technique for real-time and <i>in situ</i> visualization of bioactive species in living organisms. However, its utility for <i>in vivo</i> 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, <b>Mito-PFTTA-Eu</b>^<b>3+</b><b>/Tb</b>^<b>3+</b>, and a ¹⁹F magnetic resonance (MR) probe, <b>Mito-PFTTA-Gd</b>^<b>3+</b>, for the highly sensitive and <i>in situ</i> 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) (<b>Mito-PFTTA</b>), 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, <b>Mito-PFTTA-Eu</b>^<b>3+</b><b>/Tb</b>^<b>3+</b>, were developed as a ratiometric TGL probe for H₂O₂ detection. In parallel, its Gd³⁺ complex, <b>Mito-PFTTA-Gd</b>^<b>3+</b>, 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 <b>Mito-PFTTA-Eu</b>^<b>3+</b><b>/Tb</b>^<b>3+</b> to serve as a ratiometric TGL probe for H₂O₂ detection using the <i>I</i>₅₄₀/<i>I</i>₆₁₀ ratio as a signal output. Thanks to its excellent mitochondrial targeting capability and excellent biocompatibility, <b>Mito-PFTTA-Eu</b>^<b>3+</b><b>/Tb</b>^<b>3+</b> 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 <b>Mito-PFTTA</b> was effectively quenched upon complexation with Gd³⁺ ions, but was restored upon H₂O₂-triggered PFBS cleavage. This behavior allowed <b>Mito-PFTTA-Gd</b>^<b>3+</b> to function as a ¹⁹F MRI probe for detection of H₂O₂ <i>in vitro</i> and <i>in vivo</i>.