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

A dual-emission fluorescent biosensing method was developed for simultaneous determination of CaMV35S and NOS in genetically modified (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 Fe 3 O 4 modified graphene oxide (Fe 3 O 4 @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 and H2-HP2 double-stranded DNA sequences (dsDNA) were formed, resulting in the desorption of dsDNA from the surface of Fe 3 O 4 @GO due to weak π-π interaction between dsDNA and Fe 3 O 4 @GO and leading to the fluorescence recovery of AgNCs. Under optimal conditions, the linear ranges 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. Graphical Abstract