CeO₂ nanozyme mediated RPA/CRISPR-Cas12a dual-mode biosensor for detection of invA gene in Salmonella

This study reports a novel biosensing system that leverages recombinase polymerase amplification (RPA) in conjunction with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology, integrated with a nanozyme (NZ) based on cerium dioxide (CeO₂). With the integration of CeO₂ NZ, a dual-mode detection platform could be developed for Salmonella detection using fluorometric and colourimetric assays. The CRISPR/Cas12a system, when activated in the presence of target DNA, could cleave the FAM-labelled probe to lead to a fluorometric response. Also, when the CeO₂ NZ was introduced in the presence of H₂O₂, a colourimetric response was generated, directly proportional to the concentration of target DNA present. We hypothesise that adding highly reactive H₂O₂ within the post-CRISPR/Cas12a reaction system allows for increased release of hydroxyl free radicals within the mixture. Thus, the double recognition through NZ and the CRISPR/Cas12a system provided enhanced selectivity and sensitivity to the method. The proposed biosensor could successfully detect Salmonella at concentrations as low as 0.88 pg/μL and 1.28 pg/μL for fluorometric and colourimetric responses, respectively. Furthermore, the developed biosensor could be applied in real sample analysis of raw food samples (chicken, egg, and beef) to give a good recovery in the spiked food samples with varying concentrations of cultured bacterial DNA.