Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual emission silver nanoclusters and catalytic hairpin amplification strategy

Reliable, rapid and cost-efficient tools for the inspection and discrimination of genetically modified (GM) ingredients in food and food-related products are highly demanded to enforce relevant regulations in many countries. Herein, a dual-emission fluorescent biosensing method was developed for simultaneously quantitative analysis of CaMV35S and NOS in GM plants. Two designed hairpin DNA (H1, H2) sequences were used as templates to synthesize H1-AgNCs (λ_ex = 570 nm, λ_em = 625 nm) and H2-AgNCs (λ_ex = 470 nm, λ_em = 555 nm). By using H1-AgNCs and H2-AgNCs as dual-signal tags, combined with signal amplification strategy of magnetic separation to reduce background signal and an enzyme-free catalytic hairpin assembly (CHA) signal amplification strategy, a novel multi-target fluorescent biosensor was fabricated to detect multiple targets based on FRET between signal tags (donors) and magnetic Fe₃O₄ modified graphene oxide (Fe₃O₄@GO, acceptors). In the presence of the target NOS and CaMV35S, the hairpin structures of H1 and H2 can be opened respectively, and the exposed sequences will hybridize with the G-rich hairpin sequences HP1 and HP2 respectively, displacing the target sequences to participate in the next round of CHA cycle. Meanwhile, H1-HP1, H2-HP2 double-stranded DNA sequences (dsDNA) were formed, resulting the desorption of dsDNA from the surface of Fe₃O₄@GO due to weak π-π interaction between dsDNA and Fe₃O₄@GO, and leading to the fluorescence recovery of AgNCs. Under optimal conditions, the linear range of this fluorescence sensor were 5 ~ 300 nmol L^{− 1} for NOS and 5 ~ 200 nmol L^{− 1}CaMV35S, and the LODs were 0.14 nmol L^{− 1} and 0.18 nmol L^{− 1}, respectively. In addition, the fluorescence sensor has good selectivity for the detection of NOS and CaMV35S in GM soybean samples, showing the potential applications in GM screening.