An aptamer-based fluorometric zearalenone assay using a lighting-up silver nanocluster probe and catalyzed by a hairpin assembly.

An enzyme-free fluorometric assay is described for the determination of zearalenone (ZEN). The method combines (a) catalyzed hairpin assembly (CHA), (b) ultrahigh fluorescent light-up G-rich DNA sequences in proximity to silver nanoclusters (Ag NCs), and (c) the use of aptamers (Apt). In the presence of ZEN, the inhibit sequence (Inh) is released from the aptamer-trigger sequence (Apt-T) via the binding of ZEN and the aptamer of Apt-T. The free Apt-T acts as a switch that opens the hairpins H1 and H2 to generate H1-H2 complex. The released Apt-T is available to trigger the next round of CHA between H1 and H2. Finally, the hybridization between H1 and the Ag NCs probe (P) causes the G-rich sequence to be in close proximity to the dark Ag NCs encapsulated by P. This leads to highly efficient lighting up of the Ag NCs and the production of amplified fluorescence with excitation/emission peaks at 575/628 nm. Under the optimized conditions, a linear correlation was observed with concentrations ranging from 1.3 pg mL-1 to 100 ng mL-1, and the limit of detection was 0.32 pg mL-1 (at S/N = 3). The method was successfully validated by analyzing maize and beer for levels of ZEN after a simple sample preparation procedure. Graphical abstractSchematic of the assay. The inhibit sequence (Inh) is released from aptamer-trigger sequence (Apt-T) via binding of ZEN and aptamer. The free Apt-T triggers catalyzed hairpin assembly (CHA).G-rich DNA is in proximity to silver nanoclusters (Ag NCs) and fluorescence intensity increases to detect ZEN.