Abstract
Excessive streptomycin (STR) residues pose significant risks to dairy product quality and safety. Thus, developing highly sensitive detection methods for STR is essential to safeguard dairy safety and public health. In this study, we present a dual signal amplification lateral flow strip (LFS) using enzyme-assisted cycling and composite probe composed of multiple gold nanoparticles (AuNPs) for STR detection. Through targeted mutation, an optimized aptamer (Apt52-0) with both high affinity for STR and recognition by nicking endonuclease was developed, exhibiting enhanced binding to STR with a Kd value of 6.87 nM, compared to the original Apt52. Enzyme-assisted amplification was achieved using the hairpin structure of the Apt52-0, Vent (exo-) DNA polymerase, and Nt.Alwl nicking endonuclease, enabling dual cycling of the target and double-stranded DNA (dsDNA). The cyclic products were then isothermally amplified, and composite probe composed of multiple AuNPs was utilized to construct a dual signal amplification lateral chromatography sensor for STR detection. This aptamer-based LFS achieved a visual limit of detection (LOD) of 25 nM, and a LOD of 17.6 nM analyzed via ImageJ software. The test strip demonstrated promising sensing performance in practical application, with STR recovery rates of 95.8%–103.9% in milk samples. This dual signal amplification-based LFS offers an inexpensive, rapid and sensitive approach to STR detection.