Programmable mesophilic argonaute-triggered SERS biosensor promotes amplification-free and multiplexed nucleic acid detection

Simultaneous on-site identification of multiple nucleic acids with rapidity and accuracy has consistently posed a significant challenge in the analytical chemistry field. This is primarily because current methods predominantly rely on nucleic acid amplification while lacking high-resolution multi-signal output strategies. Inspired by that argonaute (Ago) endonuclease coupled with guide DNA (gDNA) can specifically cleave nucleic acid target and exhibit stepwise endonuclease activity for signal amplification, as well as the ultrasensitive detection capability and high-resolution signal differentiation of surface-enhanced Raman scattering (SERS), we proposed a novel programmable mesophilic Clostridium perfringens argonaute (CpAgo)-triggered SERS sensing platform (CpAgo-SERS) for amplification-free and multiplexed nucleic acid detection. In this platform, two-round continuous cleavage mediated by CpAgo/gDNA ensures both specific DNA recognition and efficient signal amplification. To further enhance the detection sensitivity, controllable polystyrene (PS) nanoparticles, with high loading capacity for signal molecules, were utilized to encapsulate Raman reporters to further amplify signal after target activated cleavage of CpAgo. Besides, the optimal functioning of mesophilic CpAgo at room temperature promotes the development of on-site detection techniques. Through these designs, this platform was capable of sensitively and simultaneously identifying multiple nucleic acids in one tube, achieving a limit of detection (LOD) of 0.497 ng/μL without any pre-amplification. Subsequently, we applied this method to animal-derived food authentication, which poses severe challenges to trace target detection due to complex matrices. The proposed strategy demonstrated satisfactory feasibility, indicating its great potential as a versatile toolbox for nucleic acid screening even in complex sample matrices.