Non-immobilized graphene oxide-based screening of aptamers for non-small cell lung cancer (NSCLC) drug resistance biomarkers and development of a nano-detection platform.

This study aims to establish a technology for the dynamic monitoring and early identification of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in patients with non-small cell lung cancer (NSCLC). To achieve this, we first developed an osimertinib-resistant NSCLC cell model and employed a graphene oxide-based SELEX (GO-SELEX) strategy to screen for high-affinity single-stranded DNA aptamers. After multiple rounds of enrichment and validation, the selected Osi-1 aptamer exhibited optimal binding affinity to the supernatant of resistant cells. Subsequent pull-down immunoblotting analysis using Osi-1 identified calnexin as its specific target for the first time, with a dissociation constant (Kd) of 71.93 nM. Building on this, we developed a label-free gold nanoparticle (AuNP)-based aptamer sensing platform and systematically optimized key parameters, including NaCl concentration, aptamer dosage, and incubation time. The results demonstrated exceptional specificity in distinguishing drug-resistant from sensitive cells, exhibiting a highly linear response (R² = 0.9912) to calnexin concentrations ranging from 10 to 500 nM. The detection limit was 7.89 nM, with excellent stability and recoveries (97.43%-107.12%) in serum samples. In summary, this study not only identifies calnexin as a potential biomarker for osimertinib resistance but also establishes a highly efficient, sensitive, and clinically translatable label-free detection platform, providing a novel solution for early monitoring and precision intervention in NSCLC drug resistance.