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 (λex = 570 nm, λem = 625 nm) and H2-AgNCs (λex = 470 nm, λem = 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 and H2-HP2 double-stranded DNA sequences (dsDNA) were formed, resulting in 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 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.

Dual-channel biosensor for simultaneous detection of S. typhimurium and L. monocytogenes using nanotags of gold nanoparticles loaded metal-organic frameworks.

Simultaneous detection of multiple foodborne pathogens is of great importance for ensuring food safety. Herein, we present a sensitive dual-channel electrochemical biosensor based on copper metal organic frameworks (CuMOF) and lead metal organic framework (PbMOF) for simultaneous detection of Salmonella typhimurium (S. typhimurium) and Listeria monocytogenes (L. monocytogenes). The MOF-based nanotags were prepared by functionalizing gold nanoparticles loaded CuMOF (Au@CuMOF) and PbMOF (Au@PbMOF) with signal DNA sequences 1 (sDNA1) and sDNA2, respectively. By selecting invA of S. typhimurium and inlA gene of L. monocytogenes as targe sequences, a sandwich-typed dual-channel biosensor was developed on glassy carbon electrodes (GCE) through hybridization reactions. The sensitive detection of S. typhimurium and L. monocytogenes was achieved by the direct differential pulse voltametric (DPV) signals of Cu2+ and Pb2+. Under optimal conditions, channel 1 of the biosensor showed linear range for invA gene of S. typhimurium in 1 × 10-14-1 × 10-8 M with low detection limit (LOD) of 3.42 × 10-16 M (S/N = 3), and channel 2 of the biosensor showed linear range for inlA gene of L. monocytogenes in 1 × 10-13-1 × 10-8 M with LOD of 6.11 × 10-15 M (S/N = 3). The dual-channel biosensor showed good selectivity which were used to detect S. typhimurium with linear range of 5-1.0 × 104 CFU mL-1 (LOD of 2.33 CFU mL-1), and L. monocytogenes with linear range of 10 - 1.0 × 104 CFU mL-1 (LOD of 6.61 CFU mL-1).

Electrochemical Detection of ompA Gene of C. sakazakii Based on Glucose-Oxidase-Mimicking Nanotags of Gold-Nanoparticles-Doped Copper Metal-organic Frameworks.

The present work developed an electrochemical genosensor for the detection of virulence outer membrane protein A (ompA, tDNA) gene of Cronobacter sakazakii (C. sakazakii) by exploiting the excellent glucose-oxidase-mimicking activity of copper Metal-organic frameworks (Cu-MOF) doped with gold nanoparticle (AuNPs). The signal nanotags of signal probes (sDNA) that biofunctionalized AuNPs@Cu-MOF (sDNA-AuNPs@Cu-MOF) were designed using an Au-S bond. The biosensor was prepared by immobilization capture probes (cDNA) onto an electrodeposited AuNPs-modified glassy carbon electrode (GCE). AuNPs@Cu-MOF was introduced onto the surface of the GCE via a hybridization reaction between cDNA and tDNA, as well as tDNA and sDNA. Due to the enhanced oxidase-mimicking activity of AuNPs@Cu-MOF to glucose, the biosensor gave a linear range of 1.0 × 10-15 to 1.0 × 10-9 mol L-1 to tDNA with a detection limit (LOD) of 0.42 fmol L-1 under optimized conditions using differential pulse voltammetry measurement (DPV). It can be applied in the direct detection of ompA gene segments in total DNA extracts from C. sakazakii with a broad linear range of 5.4-5.4 × 105 CFU mL-1 and a LOD of 0.35 CFU mL-1. The biosensor showed good selectivity, fabricating reproducibility and storage stability, and can be used for the detection of ompA gene segments in real samples with recovery between 87.5% and 107.3%.