CRISPR/Cas12a has emerged as a powerful platform for developing next-generation biosensors, yet achieving rapid, instrument-free visual detection remains a challenging. Conventional CRISPR-AuNP assays rely on pre-functionalized, thiol-modified gold nanoparticles (AuNPs), which introduce steric hindrance that slows Cas12a kinetics and increases cost and complexity. Here, we developed a rapid, label-free colorimetric platform that integrates a mismatched catalytic hairpin assembly (MCHA) for low-background amplification with CRISPR/Cas12a trans-cleavage, coupled to a microwave-assisted dry-heating method for in-situ probe conjugation. Unlike conventional approaches where CRISPR acts on pre-conjugated probes, our method allows Cas12a to cleave free, non-thiolated ssDNA probes. Intact probes rapidly conjugate to unmodified AuNPs under microwave irradiation via their high-affinity domain, forming a protective corona that prevents salt-induced aggregation. Conversely, probes that have been cleaved by target-activated CRISPR/Cas12a lose this conjugation ability, rendering AuNPs susceptible to aggregation and producing a visible color change. To address the conflicting ionic requirements between the CRISPR reaction and AuNP stability, a Ba(OH)-mediated ionic reset step is introduced to remove excess Mgprior to probe conjugation. This platform enables the sensitive detection of Escherichia coli O157:H7 within 2 h a limit of detection of 5 CFU/mL and robust performance in complex food and clinical matrices. Combined with smartphone-based RGB analysis, the instrument-minimized strategy eliminates the need for chemical modification of AuNPs and specialized operators, offering a versatile, low-cost, and user-friendly platform well-suited for pathogen detection in resource-limited settings. 2 2+
