A thiol-tolerant Au-Se anchored fluorescent aptamer nanosensor for GP73 detection with anti-interference validation...

Golgi protein 73 (GP73) is a clinically relevant biomarker associated with hepatocellular carcinoma (HCC). However, fluorescence-based quantification and intracellular imaging are susceptible to interference and signal instability in thiol-rich biological environments. Herein, we report a fluorescent aptamer nanosensor based on Au-Se anchoring, which integrates a mechanistically guided anti-interference design with a calibratable, buffer-based quantitative workflow for GP73 detection. The sensing interface translates GP73 recognition into fluorescence recovery via target-induced aptamer conformational switching and increased fluorophore-gold nanoparticle distance, while the Au-Se bond mitigates thiol-driven false activation. Under optimized conditions, the assay shows a linear response in the range of 0-800 ng/mL with a detection limit of 1.51 ng/mL. Stability and tolerance studies demonstrate its robust performance across a pH range of 6.2-8.0 and in the presence of physiologically relevant concentrations of glutathione. Notably, in 5 mM glutathione, the signal increases by less than 1.5-fold compared to the untreated control, and its performance is significantly superior to that of the conventional Au-S anchored probe. Specificity experiments confirm that the probe responds significantly only to GP73, showing negligible responses to a panel of non-target proteins and a scrambled aptamer control. Preliminary serum experiments further demonstrate feasibility in a 10× diluted human serum matrix, where the probe preliminarily differentiates GP73-positive HCC patient sera from sera of other cancer patients using a ΔF strategy to reduce serum background fluorescence; however, we emphasize that the cohort size is limited and larger clinical validation is required for definitive clinical claims. Cellular experiments reveal GP73-correlated fluorescence contrast between HepG2 and normal liver (THLE-2) cells, and the thiol-depletion control (N-ethylmaleimide) supports that intracellular signal activation is thiol-independent. In summary, this work provides a robust Au-Se-stabilized nanosensor for quantitative GP73 readout in buffer and cellular imaging under thiol-rich conditions. The control experiments clearly define its specificity and anti-interference advantages, delineating key practical operating boundaries and providing a foundation for future expanded validation in complex clinical matrices.