A fluorometric assay for zinc ion is described that relies (a) on the use of an isothermal cycle to amplify the fluorescence signal, and (b) of magnetic beads (MBs) to completely remove unreacted DNA detection probes. Biotin and fluorophore-labeled substrate (Zn-Sub) strands acting as detection probes were first assembled on MBs. Next, Zn(II)-specific DNAzyme (Zn-Enz) strands were hybridized with the Zn-Sub strands. In the presence of Zn(II), the Zn-Sub strands are cleaved. This results in the release of the shorter DNA fragments (containing fluorescent label) and in the dissociation of Zn-Enz strands. The dissociated Zn-Enz strands then hybridize with the residual Zn-Sub strands and cleave them in a similar fashion. This leads to a target recycling amplification mechanism and in a cumulative signal amplification process. A strongly amplified signal is thus obtained in the presence of Zn(II). The use of MBs warrants that unreacted Zn-Sub strands can be magnetically separated from the solution. The method has a detection limit as low as 33 fM at a signal-to-noise ratio of 3 and a linear response in the 100 fM to 11 nM Zn(II) concentration range. It was applied to the determination of Zn(II) in spiked tap water and seawater samples, and the results compared well with data obtained by ICP-MS analysis. The method was also applied to the determination of Zn(II) in infant milk powder and breast milk. Graphical abstract Magnetic beads (MBs) carrying fluorescein-labeled substrate (Zn-Sub) strands were hybridized with Zn(II)-specific DNAzyme (Zn-Enz) and cleaved in the presence of Zn(II). After recycling, the unreacted Zn-Sub strands were removed with MBs and the released fluorescein tags are measured.
Keywords: Breast milk; Fluorescence; Infant milk powder; Isothermal cycle; Seawater; Signal amplification; Target recycling; Zinc detection.