Ultra-sensitive detection of ampicillin via dual-enzyme mediated cascade-signal amplified aptasensor

•A dual-enzyme mediated cascade- signal amplified ampicillin aptasensor is developed.•The homogeneous process ameliorated the DNA interaction in enzymatic reaction.•The proposed aptasensor is of a LOD of aM..•The detection of ampicillin in milk samples is successfully performed. Herein, an ultra-sensitive aptasensor based on dual-enzyme mediated cascade- signal amplification (dubbed Demcsa aptasensor) for ampicillin (AMP) detection is proposed. The sensor’s activity involves two major steps, i.e., DNA ligase-assisted Exonuclease III (Exo III) catalyzed degradation reaction in a homogeneous solution, signal generation/amplification through the biotin-streptavidin linkage and hybridization chain reaction (HCR) on a 96-well polystyrene plate. Firstly, a detection probe was formed via the hybridization reaction between biotinylated AMP aptamer (bio-AP), HCR initiator strand (H strand) and complementary DNA (cDNA). As a result of the dual-enzyme mediated degradation, a long APH strand was formed via the DNA ligase-catalyzed ligation of bio-AP and the H strand, which served as integrated initiator to trigger the HCR reaction, thereby producing a long double-stranded DNA polymer. Subsequently, the SYBR Green I, as fluorescent dye for DNA, was added and a strong fluorescence signal was generated. In the presence of AMP, the function of DNA ligase was effectively suppressed due to the higher affinity of target-AP strand complex. As a result, the HCR reaction could not be triggered without generating a fluorescence signal, because the bio-AP cannot ligate to the H strand even when captured on the plate. The cascade- signal amplification of biotin-streptavidin linkage and the HCR reaction endowed the Demcsa aptasensor with a limit of detection (LOD) of aM, which was comparable or superior to those of the previously reported sensors. The Demcsa aptasensor has been used for the screening of AMP in food samples with favorable recoveries. The results of this work pave a new, universal way for developing efficient sensors for the detection of other trace food hazards.