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After optimizing the original aptamer sequence by truncation strategy, a magnetic separation-assisted DNAzyme-driven 3D DNA walker fluorescent aptasensor was developed for detecting the food-borne pathogen Cronobacter species. Iron oxide magnetic nanoparticles (MNPs) modified with a hybrid of truncated aptamer probe and DNAzyme strand (AP-E1) denoted as MNPs@AP-E1, were employed as capture probes. Simultaneously, a DNAzyme-driven 3D-DNA walker was utilized as the signal amplification element. The substrate strand (Sub) was conjugated with the gold nanoparticles (AuNPs), resulting in the formation of AuNPs@Sub, which served as a 3D walking track. In the presence of the target bacteria and Mg2+, E1-DNAzyme was activated and moved along AuNPs@Sub, continuously releasing the signal probe. Under optimized conditions, a strong linear correlation was observed for Cronobacter sakazakii (C. sakazakii) in the concentration range 101 to 106 CFU mL−1, with a low detection limit of 2 CFU mL−1. The fluorescence signal responses for different Cronobacter species exhibited insignificant differences, with a relative standard deviation of 3.6%. Moreover, the aptasensor was successfully applied to determine C. sakazakii in real samples with recoveries of 92.86%—108.33%. Therefore, the novel method could be a good candidate for ultra-sensitive and selective detection of Cronobacter species without complex manipulation. [ABSTRACT FROM AUTHOR]

A DNA tweezers fluorescence aptasensor based on split aptamer assisted-magnetic nanoparticles (MNPs) and magnetic separation was established for the detection of Enrofloxacin (ENR). The DNA tweezers' fluorescence aptasensor consists of two arms with fluorophores (FAM) and quenchers (BHQ 1) connected at both ends. Meanwhile, the high fluorescence signal can be generated with FAM and BHQ 1 being spatially separated. Split aptamer 1 (SPA 1) of ENR was annealed and incubated with avidin-coated MNPs. ENR and split aptamer 2 (SPA 2) were added to form a sandwich structure with two split aptamer strands (MNPS-SPA 1 /ENR/SPA 2). After magnetic separation, the supernatant did not interact with DNA tweezers, which made DNA tweezers a "turn-on" aptasensor to enhance the fluorescence signal. In the absence of ENR, SPA 2 was free in the supernatant to dramatically quench the fluorescence which made DNA tweezers a "turn-off" aptasensor. The linear range of ENR detection was 0.01–100 ng/mL with a limit of detection of 0.008 ng/mL. The prepared DNA tweezers fluorescence aptasensors were applied to detect ENR in spiked milk samples, which showed good recoveries between 90.27% and 106.62%. In addition, the present method showed great potential for the target, employing a split aptamer detection platform in practical application. • A novel fluorescence aptasensor assay for Enrofloxacin (ENR) detection was established. • The sandwich structure is formed by specific target recognition of split aptamer to achieve sensitive detection. • The "turn-on/turn-off" structure of DNA tweezers offers significant fluorescence signals. [ABSTRACT FROM AUTHOR]