Endogenous peroxynitrite-responsive bioorthogonal SERS nanoprobe for ratiometric Raman imaging assessment of drug-induced liver injury.

Peroxynitrite (ONOO-) plays a critical role in drug-induced liver injury (DILI), making it essential to elucidate its relationship with disease progression to improve early diagnosis and treatment. Nevertheless, the accurate and reliable detection of ONOO- in biological systems with surface-enhanced Raman scattering (SERS) nanoprobes remains challenging. In this study, inspired by the specific reaction between α-ketoamide and ONOO-, we designed the first endogenous ONOO--responsive bioorthogonal SERS nanoprobe to investigate drug-induced liver injury through ratiometric SERS imaging in the Raman-silent region. The SERS nanoprobe is composed of an alkyne-tagged internal standard (4-TEB) and a cyano-based ONOO- responsive unit (DPCO), which shows a remarkable ratiometric SERS change (I2233/I2153) in the Raman-silent region for ONOO- with high selectivity and sensitivity, and detection limits as low as 21.5 nM. We successfully applied the probe to track ONOO- dynamics at single-cell resolution during its induction and inhibition in living cells. Subsequently, it was used to monitor APAP-induced ONOO- fluctuations in HepG2 cells and to map ONOO- distribution in the livers of DILI mice, thereby revealing insights into drug-induced hepatotoxicity and its remediation. It is anticipated that the nanoprobe holds significant potential for elucidating DILI mechanisms and advancing clinical diagnostics.