A label-free fluorescent biosensor leveraging cascaded DNA dual-cycle amplification and HCR for ultrasensitive detection of circulating tumor DNA

High-sensitivity circulating tumor DNA (ctDNA) detection technology is critical for early cancer diagnosis and treatment. However, the extremely low abundance of ctDNA in bodily fluids, coupled with various interfering factors, makes its effective detection particularly challenging. Herein, a label-free fluorescence sensor for ultrasensitive ctDNA detection was developed by integrating enzyme-assisted dual-DNA-loop amplification with a hybridization chain reaction (HCR). The target ctDNA binds to a hairpin probe, initiating a catalytic hairpin assembly cycle that forms a complex containing recognition sites for the endonuclease Nb. Bbv CI. Upon binding its recognition site, Nb.BbvCI cooperates with KF polymerase to induce strand displacement amplification (SDA), forming a dual-cycle amplification process that generates an HCR trigger probe to initiate HCR and produce abundant complementary DNA sequences with multiple C-myc repeats. In the presence of Mg 2+ , these sequences fold into G-quadruplex structures. Subsequent intercalation of thioflavin T results in a dramatic enhancement of the fluorescence signal, enabling quantitative detection of ctDNA. The sensor demonstrates a linear response to ctDNA concentrations ranging from 10 fM to 1 nM, with a detection limit as low as 2.48 aM. This highly sensitive detection strategy offers promising potential for advancing early cancer diagnosis and personalized treatment.