An ultrasensitive aptamer-based fluorescent biosensor for luteinizing hormone with mutually orthogonal DNAzyme and self-replication CHA amplification.

Sensitive and specific detection of luteinizing hormone (LH) is critical for the early diagnosis of diseases and cancers, offering valuable insights for clinical strategies aimed at regulating physiological functions to maintain health. To this end, we developed a novel sensing platform for the ultrasensitive detection of LH, harnessing a mutually orthogonal cascade amplification composed of DNAzyme and self-replicating catalytical hairpin assembly (SR-CHA). Upon binding of LH to its aptamer, an LH/aptamer complex is formed, leading to the release of an initiator strand ("strand A") that triggers the assembly of Y-shaped DNA structures. Each Y-shaped structure comprises a modular DNAzyme-based amplification unit, which generates a one-to-many fluorescence signal, and an initiator mimic sequence capable of self-replicating to produce numerous new initiators for accelerating the formation of additional Y-shaped structures, thereby amplifying the detection signal. This synergistic design resulted in outstanding analytical performance, achieving a detection limit as low as 0.00038 mIU/mL in buffer and 0.0006 mIU/mL in real samples, with a broad dynamic range from 0.001 to 100 mIU/mL. In addition, the proposed method also demonstrated excellent selectivity, and a strong correlation with the standard ELISA method, which reveals its promising potential as a universal simple and sensitive methodology for the construction of various aptamer-based bioassays.