Title: Design and Synthesis of a Ratiometric Photoacoustic Probe for In Situ Imaging of Zinc Ions in Deep Tissue In Vivo

Abstract: As a noninvasive deep-tissue imaging technique, photoacoustic (PA) imaging has great application potential in biomedicine and molecular diagnosis. The zinc ion (Zn²⁺), which is a necessary metal ion in the human body, plays a very important role in the regulation of gene transcription and metalloenzyme function. The imbalance of Zn²⁺ homeostasis is also associated with a variety of neurological diseases. Therefore, it is critically important to accurately image the steady-state changes of Zn²⁺ in vivo. However, no PA imaging method is currently available for Zn²⁺. To this end, we designed and synthesized the first PA probe of Zn²⁺, namely, CR-1 for in situ ratiometric imaging of Zn²⁺ in deep tissue in vivo. The CR-1molecule, combined with Zn²⁺, weakened the conjugation system of the π-electron in the CR-1 molecule, which resulted in the blue shift of its absorption peak from 710 nm to 532 nm. The PA signal intensity decreased at 710 nm and increased at 532 nm, and the ratiometric PA signal at these two wavelengths (PA₅₃₂/PA₇₁₀) showed a good linear relationship with the concentration of Zn²⁺ in the range of 0–50 μM, with a detection limit as low as 170 nM. Furthermore, this probe exhibits extremely fast responsiveness, is highly selective, and has excellent biocompatibility. We have used the developed PA probe for the ratiometric PA imaging of Zn²⁺ in the thigh tissue of mice, and we still can accurately image Zn²⁺ after covering chicken breast tissue on the surface of mice thigh. In light of these outstanding features, the developed PA probe has high potential for imaging Zn²⁺ in deep tissues; thus, it will open up new avenues for the study of the complex biochemical processes involving Zn²⁺ in vivo.