Catalytic Hairpin Assembly-Aided CRISPR/Cas-SERS for Nucleic Acid Detection Enhanced by Multiple Catalytic...

This study established a surface-enhanced Raman scattering (SERS) detection method based on the catalytic hairpin assembly (CHA) reaction combined with clustered regularly interspaced short palindromic repeats and its associated proteins (CRISPR/Cas), integrated with the guanine-quadruplexes/Hemin (G4/Hemin) catalytic amplification effect, which is available for identifying a typical indicator for genetically modified crops, the 35S promoter from the cauliflower mosaic virus (CaMV35S). Target CaMV35S-derived RNA (CaMV35S RNA) triggers the CHA reaction, yielding a double-stranded DNA product (C1/C2) that is recognized by CRISPR RNA (crRNA), thereby activating the trans-cleavage activity of Cas12a. A magnetic bead-based nanoprobe was fabricated by linking the hybridization chain reaction (HCR) product to the magnetic bead. This probe can fold into repeated G4 units under a specific potassium ion concentration to enable a high loading of Hemin, thereby providing strong peroxidase-like catalytic activity for 3,3',5,5'-tetra-methyl-benz-idine (TMB). In a positive trial, the 35S RNA initiates CHA, and the CHA product activates Cas12a, disrupting the HCR products on the magnetic bead probe, preventing the G4 structure formation, accordingly yielding a weak SERS signal from TMB. In contrast, a negative trial fails to activate Cas12a, and the probe retains its nanoenzyme activity, resulting in a strong SERS signal from the produced oxTMB. Owing to the collateral trans-cleavage activity of Cas12a and the high peroxidase-like nanozyme activity of G4/Hemin, this method achieves the highly sensitive SERS detection of genetically modified samples without target gene preamplification.