A crRNA-optimized CRISPR/Cas12a-based aggregation - dispersion colorimetric biosensor using gold nanoparticles for ultrasensitive detection of Staphylococcus aureus

A highly sensitive colorimetric sensing platform was developed based on the trans-cleavage activity of CRISPR/Cas12a and the controllable aggregation of gold nanoparticles (AuNPs) for rapid and visual detection of the foodborne pathogen Staphylococcus aureus. Following PCR or RPA, the target DNA was specifically recognized by the Cas12a/crRNA complex, thereby activating its trans-cleavage activity. This process led to the degradation of linker single-stranded DNA (ssDNA) responsible for crosslinking AuNPs probes, resulting in significant changes in both UV–vis absorption signals and solution color. Three different crRNA sequences were systematically evaluated, revealing distinct efficiencies in target recognition and signal amplification. Among them, the crRNA-3 mediated system exhibited superior analytical performance, achieving an ultralow detection limit of 1.14 fM and a good linear response over a low concentration range. Furthermore, the proposed platform demonstrated excellent anti-interference capability in complex matrices, including serum, milk, bovine serum albumin (BSA), and phosphate-buffered saline (PBS), with recovery rates ranging from 83.2% to 125.9%. This work provides a robust and visual tool for the on-site detection of S. aureus and offers new insights into the design of portable CRISPR-based diagnostic platforms.