DNAzyme Walkers Empowered Self-Priming Isothermal Amplification for Label-Free and Sensitive Detection of Interleukin-6 in Senescence Cell Models

Abstract

Accurate quantification of interleukin-6 (IL-6) in complicated biological matrices is essential to clarifying its roles in inflammation and cellular senescence. However, matrix interference and resultant false-positive signals remain a major challenge for existing analytical methods. Herein, we developed a novel IL-6 biosensor by coupling the DNAzyme walker with a terminal phosphorothioate modification-assisted hairpin formation and self-priming (PS-THSP) amplification strategy. To assemble the DNAzyme walker, the DNAzyme/blocker duplexes and substrate probes were simultaneously immobilized on the magnetic beads (MBs) via biotin-streptavidin interaction. Hybridization of the blocker with the DNAzyme strand keeps the DNAzyme walker catalytically inactive. When the target IL-6 is present, it can trigger the disassembly of the DNAzyme/blocker duplex on the MBs, activating the DNAzyme walker to autonomously cleave substrate strands. The released substrate fragments then hybridize separately with H1 and H2 hairpin probes, prompting them to form dumbbell-shaped templates, which in turn facilitate highly efficient PS-THSP amplification in a self-priming manner. Compared with traditional loop-mediated isothermal amplification (LAMP) and rolling circle amplification (RCA) techniques, the PS-THSP system features simplified oligonucleotide design and markedly lowers the risk of non-specific amplification. Furthermore, the integration of magnetic separation reduces interference from complex biological matrices, thereby suppressing false-positive signals and enhancing the signal-to-noise ratio. The proposed cascade amplification strategy generates abundant double-stranded DNA products, yielding a strong fluorescent signal upon SYBR Gold staining without complex probe modifications. This method demonstrates exceptional sensitivity, achieving a limit of detection of 0.0315 pg/mL, and has been successfully implemented to monitor IL-6 secretion in LPS-induced premature senescence models as well as in human serum samples. Notably, this method provides a universal platform that enables the detection of various biomarkers by simply changing the aptamers, offering promising prospects for clinical diagnostic applications.