Enhancing the Reliability of SERS Detection in Ampicillin Using Oriented Tetrahedral Framework Nucleic Acid Probes and a Long-Range SERS Substrate

Indirect surface-enhanced Raman scattering (SERS)-based methods are highly efficient in detecting and quantitatively analyzing trace antibiotics in complex samples. However, the poor reproducibility of indirect SERS assays caused by the diffusion and orientation changes of the probing molecules on SERS substrates still presents a significant challenge. To address this issue, this study reports the construction of a novel SERS sensing platform using tetrahedral framework nucleic acid (tFNA) as SERS probes in conjunction with a long-range SERS (LR-SERS) substrate. The tFNA was modified with sulfhydryl groups at three vertices and appended with a probing DNA at the remaining vertex, anchored on the substrate surface with a well-ordered orientation and stable coverage density, resulting in highly reproducible SERS signals. Owing to the weak SERS signal of tFNA inherited from its size being larger than the effective range of the enhancing electric field (E-field) of conventional SERS substrates, we utilized an LR-SERS substrate to enhance the signal of tFNA probes by capitalizing on its extended E-field. Correspondingly, the LR-SERS substrate demonstrated a 54-fold increase in the intensity of tFNA probes compared to the conventional substrate. Using this novel platform, we achieved a highly reliable detection of the antibiotic ampicillin with a wide linear range (10 fM to 1 nM), low detection limit (3.1 fM), small relative standard deviation (3.12%), and yielded quantitative recoveries of 97–102% for ampicillin in water, milk, and human serum samples. These findings, therefore, effectively demonstrate the achievement of highly reliable SERS detection of antibiotics using framework nucleic acids and an LR-SERS substrate.