A Label‐Free Rapid Fluorescence Screening Approach for Identifying Cell‐Penetrating Peptides Using ANS as an Extrinsic Probe

ABSTRACT Labeling peptides with fluorophores remains the dominant approach for assessing their cellular uptake, yet this process is time‐intensive, costly, and can modify peptide structure and biological behavior. Here a label‐free fluorescence‐based screening method is presented that exploits the environmental sensitivity of 1‐anilino‐8‐naphthalene sulfonate (ANS) to monitor peptide–membrane interactions in real time. ANS shows negligible emission in water but undergoes a characteristic blue shift and intensity enhancement upon association with hydrophobic regions. These features were used to distinguish penetrating from non‐penetrating peptides in both plant protoplasts and mammalian HEK 293 T cells. Classical cationic cell‐penetrating peptides (CPPs), poly‐arginine (R9) and TAT (49–57), produced distinct ANS responses within minutes, while the non‐penetrating mutant mTAT showed no detectable effect. The ANS‐based assay provides a cost‐efficient, label‐free, and high‐throughput tool for screening native peptides and offers new insight into the hydrophobic transitions that accompany peptide internalization.