A dual-targeting nanobiosensor for Gender Determination applying Signal Amplification Methods and integrating Fluorometric Gold and Silver Nanoclusters

A dual-targeting nanobiosensor has been developed for the simultaneous detection of AMELX and AMELY genes based on the different fluorescence signals emitted from gold and silver nanoclusters, AuNCs and AgNCs respectively. In our design, both catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) have been used as isothermal, enzyme-free and simple methods for signal's amplification. The working principle is based on the initiation of a cascade of CHA-HCR reactions when AMELX is present, in which AuNCs, synthesized on the third hairpin, are aggregated on the surface of the dsDNA product, performing the phenomenon of aggregation induced emission (AIE) and enhancing their fluorescence signal. On the other hand, the presence of the second target, AMELY, is responsible for the enhancement of the fluorescence signal corresponding to AgNCs by the same phenomenon, via hybridizing to the free end of the dsDNA formed and at the same time to the probe of silver nanoclusters fixing it closer to the surface of the dsDNA product. Such a unique design has the merits of being simple, inexpensive, specific and stable and presents rapid results. The detection limits of this assay for AMELX and AMELY are as low as 3.16 fM and 23.6 fM respectively. Moreover, this platform showed great performance in real samples. The design has great promise for the application of dual-targeting nanobiosensors to other biomarkers.