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In this paper, a novel and ultrasensitive lateral flow assay (LFA) based on aptamer-magnetic separation, and multifold Au nanoparticles (AuNPs) was developed for visual detecting Salmonella enterica ser. Typhimurium ( S. Typhimurium). The method realized magnetic enrichment and signal transduction via magnetic separation and achieved signal amplification through hybridizing AuNPs-capture probes and AuNPs-amplification probes to form multifold AuNPs. Two different thiolated single-strand DNA (ssDNA) on the AuNPs-capture probe played different roles. One was combined with the AuNPs-amplification probe on the conjugate pad to achieve enhanced signals. The other was connected to transduction ssDNA1 released by aptamer-magnetic capture of S. Typhimurium, and captured by the T-line, forming a positive signal. This method had an excellent linear relationship ranging from 8.6 × 10 2 CFU/mL to 8.6 × 10 7 CFU/mL with the limit of detection (LOD) as low as 8.6 × 10 0 CFU/mL in pure culture. In actual samples, the visual LOD was 4.1 × 10 2 CFU/mL, which did not carry out nucleic acid amplification and pre-enrichment, increasing three orders of magnitudes than unenhanced assays with single-dose AuNPs and no magnetic separation. Furthermore, the system showed high specificity, having no reaction with other nontarget strains. This visual signal amplificated system would be a potential platform for ultrasensitive monitoring S. Typhimurium in milk samples.

An Enhanced Lateral Flow Assay Based on Aptamer–Magnetic Separation and Multifold AuNPs for Ultrasensitive Detection of Salmonella Typhimurium in Milk