Nanoscale mapping of nucleolin–aptamer interactions on lung cancer cells reveals binding affinity hierarchy and spatial heterogeneity

Nucleolin, a protein overexpressed on the surface of cancer cells, has emerged as a promising therapeutic target due to its high affinity interactions with aptamers. This study localized nucleolin on lung cancer and normal cells at single-molecule resolution using the single molecule recognition imaging mode of Atomic Force Microscopy (AFM) with three aptamers: 9FU-AS1411, AS1411, and CRO. The results revealed abundant nucleolin expression on lung cancer cells, while minimal levels were detected on normal cells. The binding affinities and interaction dynamics of these aptamers were systematically evaluated. Flow cytometry and AFM-based force spectroscopy demonstrated that 9FU-AS1411 exhibited the strongest unbinding forces (piconewton level) and higher dissociation activation energy compared to AS1411, indicating enhanced complex stability. In contrast, CRO showed negligible binding, confirming its lack of specificity. Further analysis <i>via</i> Kelvin Probe Force Microscopy (KPFM) revealed distinct surface potential decrements after aptamer interactions: 24.4 mV (9FU-AS1411), 11.7 mV (AS1411), and 2.5 mV (CRO), correlating with their binding strengths. These findings quantitatively rank aptamer affinity as 9FU-AS1411 > AS1411 >> CRO, supported by molecular-level mechanistic insights into electrostatic and structural interactions. This work pioneers high-resolution spatial mapping of nucleolin-aptamer interactions, offering novel methodologies for studying protein-aptamer binding kinetics and electrical properties at unprecedented precision (0.1 mV resolution). The approaches established here not only advance nucleolin-targeted cancer therapy but also provide a framework for investigating other protein-aptamer systems in biomedical research.