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

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.