Surface-Confined Ratiometric SERS Sensing Enables Quantitative Imaging of Cellular Malondialdehyde

Quantitative imaging of lipid peroxidation-derived biomarkers in living cells remains challenging because signal fluctuations and probe heterogeneity often compromise the reliability of cellular surface-enhanced Raman spectroscopy (SERS) measurements. Here, we report a biocompatible ratiometric SERS nanoprobe for quantitative detection and cellular imaging of malondialdehyde (MDA), a key biomarker of oxidative stress. The probe integrates a plasmonic core-shell architecture with a surface-confined chemical reaction, in which 4-aminothiophenol (4-ATP) reacts with MDA through a Schiff-base condensation to generate a characteristic Raman band at 1657 cm^-1. By using the invariant Raman band at 1080 cm^-1 as an internal reference, a ratiometric readout (I₁₆₅₇/I₁₀₈₀) enables self-calibrated detection and effectively compensates for variations in laser excitation and nanoprobe distribution. Importantly, the probe demonstrates high chemical specificity toward MDA, showing negligible cross-reactivity with structurally related aldehydes, ketones, and common cellular biomolecules. The silica shell enhances structural stability and significantly reduces Ag-associated cytotoxicity, allowing reliable operation in biological environments. The developed probe exhibits a linear range (0.25-12.5 μM) and a detection limit of 0.5 nM for MDA. In cellular studies, the nanoprobe enables dose-dependent visualization of exogenous MDA and quantitative imaging of endogenous MDA generated during AAPH-induced lipid peroxidation. The ratiometric SERS imaging clearly differentiates oxidative stress levels among treatment groups. This ratiometric SERS platform provides a robust strategy for the mapping of cellular oxidative stress and offers a versatile tool for evaluating antioxidant interventions and neurodegenerative processes.