# Dual-Mode Aptamer AP1-F Achieves Molecular–Morphological Precision in Cancer Diagnostics via Membrane NCL Targeting

## TL;DR

A new aptamer called AP1-F improves cancer diagnostics by targeting membrane-bound nucleolin with high specificity and accuracy.

## Contribution

AP1-F is a G4-structured aptamer that targets membrane NCL, achieving superior cancer cell discrimination compared to existing probes.

## Key findings

- AP1-F showed a >10-fold fluorescence signal ratio between malignant and normal cells in co-cultures.
- AP1-F achieved over 98.78% specificity in heterogeneous cell populations using dual-channel flow cytometry.
- AP1-F provided 40.5-fold fluorescence intensity ratio in tumor vs. normal tissue in pathological sections.

## Abstract

Nucleic acid aptamers leverage defined tertiary structures for precise molecular recognition, positioning them as transformative biomedical tools. We engineered AP1-F, a G-quadruplex (G4)-structured aptamer that selectively binds membrane-anchored nucleolin (NCL) non-permeabilizing, overcoming a key limitation of conventional probes. Microscale thermophoresis confirmed nanomolar affinity to NCL. By means of rigorous optimization, AP1-F attained a greater than ten-fold fluorescence signal ratio between malignant and normal cells in co-cultures, exceeding the extensively researched AS1411. Dual-channel flow cytometry demonstrated over 98.78% specificity at single-cell resolution within heterogeneous cell populations, owing to AP1-F’s unique membrane localization—unlike AS1411’s intracellular uptake, which elicited erroneous signals from cytoplasmic NCL. Competitive binding experiments and Laser Confocal Imaging confirmed that AP1-F specifically identifies cancer cells by binding to the NCL recognition site on the membrane. In pathological sections, AP1-F exhibited a 40.5-fold fluorescence intensity ratio between tumor and normal tissue, facilitating accurate tissue-level differentiation. Significantly, it delineated molecular subtypes by associating membrane NCL patterns with morphometric analysis: luminal-like MCF-7 displayed consistent staining in cohesive clusters, whereas basal-like MDA-MB-468 revealed sporadic NCL with irregular outlines—characteristics imperceptible to intracellular-targeted antibodies, thus offering subtype-specific diagnostic insights. This combination biochemical–morphological approach accomplished subtype differentiation with a single-step, non-permeabilized process that maintained lower cytotoxicity and tissue integrity. AP1-F enhances diagnostic accuracy by utilizing spatial confinement to eradicate intracellular interference, connecting molecular specificity to intraoperative margin evaluation or biopsy categorization.