Ultrasensitive fluorescent biosensor for prostate specific antigen detection based on magnetic separation and dual signal amplification via cell-free transcription coupled with DSN cleavage cycling

In this study, a fluorescence biosensor integrated a cell‑free transcription system, duplex‑specific nuclease (DSN)‑mediated cyclic amplification, and magnetic‑bead‑based background suppression to develop a novel cell‑free fluorescence biosensor for the highly sensitive detection of prostate specific antigen (PSA). Streptavidin magnetic beads conjugate with biotinylated PSA aptamers, which hybridize with transcription templates to form ternary complexes. The specific recognition of PSA by aptamers triggers strand displacement reaction and releases free transcription templates. Magnetic separation eliminates both aptamer–target complexes and unreleased templates to reduce background interference.The released template then initiates cell‑free transcription to produce RNA, which subsequently hybridizes with a TaqMan probe to form a DNA–RNA duplex. This duplex activates duplex‑specific nuclease (DSN), leading to cyclic cleavage of the probe and an enhanced fluorescence signal, thereby achieving primary amplification through transcription and secondary amplification through enzymatic cleavage cycling. The sensor exhibits a linear range from 100 fg/mL to 100 ng/mL, with a limit of detection of 35 fg/mL, and has been successfully applied to the analysis of real samples. Thus, this work provides a universal and efficient technical platform for the highly sensitive and specific detection of clinical protein biomarkers.