A Label-Free Nanopore Biosensor for Rapid and Highly Sensitive Detection of Actinomycin in Human Serum

Actinomycin D (ActD), a potent anticancer drug, binds specifically to DNA at GpC-rich sites through intercalation and minor groove interactions. Detecting trace levels of ActD in complex biological fluids, such as human serum, is crucial for clinical, environmental, and pharmaceutical applications. Herein, we report a label-free detection method using an engineered α-hemolysin (α-HL) nanopore. In this assay, ActD binds to a ssDNA probe (GC₂), enriched with GpC sites, forming GC₂-ActD complexes that generate current modulations with distinct signatures from those of GC₂ alone. This sensor achieves high sensitivity, with a detection limit (LOD) of 1.20 nM, and a broad linear range of 2.5-250 nM. The sensor selectivity and the effects of metal ions and salt concentration on its performance were further evaluated. Moreover, this sensor enabled ActD detection in complex human serum samples with high selectivity. Overall, this DNA-based nanopore platform offers a powerful, label-free tool for drug screening and discovery.