Interference-free SERS tags for copper ion sensing upon hypoxia by in situ hot-spot generation.

Abnormal cellular Cu level is closely associated with many various pathological conditions, including cancer, Menkes disease, and Wilson's disease. However, sensitive and accurate detection of intracellular Cu remains challenging. To address this, we engineered an interference-free surface-enhanced Raman scattering (SERS) nanoprobe utilizing a target-responsive aggregation mechanism for selective Cu detection. The nanoprobe (Au@MBN@Cys) was fabricated by conjugating gold nanoparticles (Au NPs) with both the Raman reporter 4-mercaptobenzonitrile (MBN) and the Cu-specific chelating ligand l-cysteine. The nanoprobe shows a distinct Raman peak in the silent region (1800-2800 cm), minimizing cellular background. Upon binding with Cu, the nanoprobe undergo aggregation, leading to a significant enhancement of the SERS signal from MBN. The nanoprobe achieved a highly sensitive detection limit of 0.055 μM for Cu with a wide linear range (1 μM-10 mM) and excellent selectivity. Beyond its robust performance in aqueous solution, we successfully applied the nanoprobe for highly sensitive Cu detection in living cells, including hepatic (L02) and neuronal (PC12) cells. Notably, the nanoprobe enabled real-time tracking of Cu fluctuations under hypoxic conditions in both 2D monolayers and 3D multicellular spheroids, highlighting its versatility in complex biological environment. This work establishes a novel background-suppressed SERS strategy for monitoring Cu dynamics during cellular processes, providing a powerful platform for investigating metal ion-related pathophysiology.