Dual CRISPR/Cas-driven amplification-free surface-enhanced Raman scattering biosensor combined with a smartphone for...

Simultaneous detection of total and live counts of target bacteria is significant but challenging. To address this challenge, here we proposed a dual CRISPR/Cas-driven amplification-free surface-enhanced Raman scattering (SERS) biosensor, termed cc-SERS. The biosensor was constructed based on the property that DNA remains stable while some RNA degrades rapidly after bacterial death. Briefly, the target DNA and RNA from live bacteria could activate both CRISPR/Cas12a and CRISPR/Cas13a, while dead bacteria could only activate CRISPR/Cas12a through the target DNA. In the absence of the target bacteria, neither CRISPR/Cas12a nor CRISPR/Cas13a could be activated. Therefore, the characteristic Raman signal at 1079 cm^-1 generated by the target DNA-activated CRISPR/Cas12a indicated the presence of the target bacteria (the sum of dead and live), while the characteristic Raman signal at 593 cm^-1 produced by the target RNA-activated CRISPR/Cas13a indicated the presence of the live target bacteria. With this unique signaling pattern, the biosensor is capable of detecting both total and live target bacteria in a single tube with a detection limit of ∼10 CFU/mL. The introduction of a rapid pre-processing procedure and a smartphone-assisted portable Raman spectrometer enabled the entire process to be completed in the field within 45 min. Thanks to the excellent programmability of CRISPR/Cas systems, the biosensor has been successfully applied to the detection of Staphylococcus aureus and Cronobacter sakazakii, respectively. As a proof-of-concept, this work opens a promising avenue for the simultaneous detection of total and live target bacteria.