Violet phosphorene nanosheets coupled with CRISPR/Cas12a in a biosensor with a low background signal for onsite detection of tigecycline-resistant hypervirulent Klebsiella pneumoniae

Abstract

CRISPR/Cas12a-based detection has been widely used for DNA targets, but always suffers from a relatively high background fluorescent signal, which potentially gives false positive results, and limits the accuracy and specificity of detection. To address this problem, violet phosphorene nanosheets (VPNSs) with excellent DNA adsorption and light absorption capacities were synthesized by liquid exfoliation from bulk violet phosphorus. The introduction of VPNSs into CRISPR/Cas12a-based detection decreased the relatively high background fluorescent signal by 32.7 %, and resulted in a background with the relative fluorescence unit of 2590.5 that was no longer visible via a visual readout, demonstrating that VPNSs are efficient fluorescence quenchers. By combining the detection with a smartphone-based fluorescence imaging system (FIS), a biosensor was developed for simultaneous detection of virulence genes iucA, iroB, peg-344, rmpA and rmpA2, and resistance gene tet(A) carried by tigecycline-resistant hypervirulent Klebsiella pneumoniae (HvKP). Upon optimization, the VPNSs-CRISPR/Cas12a-FIS biosensor detected 1 cfu of tigecycline-resistant HvKP in 60 min with a low background signal. The whole process was performed with portable devices including a block heater and an FIS, and the signal could be observed with either a smartphone or the naked eye. The biosensor provides new insights for exploration of low background, multiple target, and onsite analytical methods based on CRISPR/Cas12a.