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

Abnormal cellular Cu²⁺ level is closely associated with many various pathological conditions, including cancer, Menkes disease, and Wilson's disease. However, sensitive and accurate detection of intracellular Cu²⁺ remains challenging. To address this, we engineered an interference-free surface-enhanced Raman scattering (SERS) nanoprobe utilizing a target-responsive aggregation mechanism for selective Cu²⁺ detection. The nanoprobe (Au@MBN@Cys) was fabricated by conjugating gold nanoparticles (Au NPs) with both the Raman reporter 4-mercaptobenzonitrile (MBN) and the Cu²⁺-specific chelating ligand l-cysteine. The nanoprobe shows a distinct Raman peak in the silent region (1800-2800 cm^-1), minimizing cellular background. Upon binding with Cu²⁺, 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 Cu²⁺ 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 Cu²⁺ detection in living cells, including hepatic (L02) and neuronal (PC12) cells. Notably, the nanoprobe enabled real-time tracking of Cu²⁺ 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 Cu²⁺ dynamics during cellular processes, providing a powerful platform for investigating metal ion-related pathophysiology.