Highly fluorescent copper nanoclusters as programmable reporters for CRISPR/Cas12a-based detection of bacterial DNA

Early and accessible pathogen detection is crucial for global health security and demands diagnostic assays that are rapid, affordable, and suitable for Point-of-Care use. This study presents a cost-effective, rapid, one-pot fluorescence assay for bacterial DNA detection that exploits the unique optical properties of DNA-templated copper nanoclusters (CuNCs). These nanoclusters offer a sustainable alternative to conventional fluorophores, thanks to their eco-friendly synthesis, high photostability, and large Stokes shift. The assay integrates CuNCs with the CRISPR/Cas12a system to achieve programmable and highly specific target recognition. Upon target binding, activation of the Cas12a/gRNA complex triggers collateral cleavage of rationally designed DNA templates that normally support CuNCs formation, resulting in a marked fluorescence decrease. A panel of hairpin and poly-thymine DNA structures was systematically evaluated to maximize both CuNCs fluorescence and responsiveness to Cas12a/gRNA trans-cleavage, ultimately identifying an AT-rich stem-loop reporter that provided strong signal intensity and complete signal shutdown upon target recognition. The final CRISPR-CuNCs assay achieved picomolar sensitivity, accurately detected E. coli DNA from reference strains, clinical isolates, and serum-spiked samples, and required no fluorophore-quencher probes or multistep procedures. Overall, this work demonstrated that combining the programmability of CRISPR/Cas12a with the versatility and low-cost of DNA-templated CuNCs enables a robust and accessible platform for molecular diagnostics, with strong potential for Point-of-Care deployment.