Self-Referenced SERS Nanoprobe for Accurate and Real-Time Monitoring of Nitroreductase Activity in Hepatic Ischemia

Abstract Hepatic ischemia-reperfusion injury (HIRI) represents a severe complication in liver surgery and transplantation, thus early identification of ischemic damage, specifically the hallmark hypoxia, is pivotal for prognosis. However, the specific tools for detecting ischemic injury remain scarce. Herein, we present the first nitroreductase (NTR)-activatable, self-referenced surface-enhanced Raman scattering (SERS) nanoprobe (Au@MBN@Au@4-NTP, AMAN) for the quantitative monitoring of NTR activity, a key hypoxia biomarker, during hepatic ischemia. The nanoprobe integrates a physically shielded internal standard (4-mercaptobenzonitrile, 4-MBN) and an NTR-responsive reporter (4-nitrothiophenol, 4-NTP). Uniquely, the internal standard exhibits distinct vibrational fingerprints exclusively within the biologically Raman-silent window (1800–2800 cm–1), which substantially reduces spectral interference from complex endogenous biological matrices. Upon NTR activation, the reporter signal of 4-NTP at ∼1325 cm–1 diminishes, while the internal standard signal at 2217 cm–1 remains unchanged, yielding a robust ratiometric readout (I1325/I2217). This enables accurate quantification of NTR with a low detection limit of 0.406 μg/mL. Using this nanoprobe, we achieve accurate and sensitive quantification of hypoxia-induced NTR upregulation in living cells. More importantly, the nanoprobe realizes real-time monitoring of dynamic NTR fluctuations during hepatic ischemia in both 2D and 3D cellular models. This work establishes a novel, interference-free SERS strategy for accurate NTR quantification, providing a powerful tool for the real-time diagnosis and progression assessment of hypoxia-related pathologies.