Dual-gold nanoprobe system enables highly sensitive SERS detection of glycosylated CEs1 in HepG-2 cell culture supernatant.

Carboxylesterase 1 (CEs1) is crucial in liver injury and hepatocellular carcinoma (HCC) progression, necessitating rapid, sensitive detection methods for its glycosylated form. This study developed a surface-enhanced Raman scattering (SERS) biosensor using a dual-gold nanoprobe system for quantifying specifically glycosylated CEs1 in HepG-2 cell culture supernatant. A glycan probe was constructed from 10 nm AuNPs modified with Raman reporter DTNB and diphenylcyclooctyne (DIBO) to recognize and couple metabolically labelled, glycosylated CEs1 via a copper-free click reaction. A protein probe, comprising 40 nm AuNPs conjugated with anti-CEs1, was used for specific capture of glycosylated CEs1. The proximity of these two probes induces a spatially controllable SERS effect, generating an enhanced SERS signal. This method achieved quantitative detection of glycosylated CEs1 in cell culture supernatants, demonstrating excellent linearity from 0.1 nM to 6.0 μM, with a correlation coefficient (R) of 0.992 and a detection limit of 16 pM. It was successfully applied to monitor glycosylated CEs1 release after HepG-2 cell stimulation by hepatotoxic drugs, confirming a positive correlation with both drug concentration and incubation time. This strategy is rapid, sensitive, and highly selective, enabling accurate measurement in complex matrices. It provides a novel approach for early warning of liver injury and HCC and for exploring the biological significance of glycosylated CEs1.