CRISPR‐Responsive Reprogrammable Label‐Free Fluorescent Nanoclusters for ML‐Assisted Pathogenic Genome Detection on Solid Substrates

The development of a paper-based genome detection assay using target-responsive DNA-templated silver nanoclusters (DFN-1) is presented. The reported nanoclusters exhibit intrinsic fluorescence, which is regulated by the cleavage of the DNA template surrounding the silver core. To enable the nanoclusters to respond to a specific genome, CRISPR-Cas12a is employed for highly specific and programmable digestion of the nanoclusters. Upon detection of the target, the DNA template is cleaved by the CRISPR-Cas12a complex, leading to a reduction in fluorescence. This assay successfully demonstrates for the detection of the Salmonella genome in the liquid phase and on 2 mm solid filter paper discs. By altering only the crRNA in the CRISPR complex, the assay is programmed to detect two different Salmonella serotypes. The selectivity of the assay is evaluated in DNA mixtures with and without the target genomic fragments. The assay also demonstrates the detection of as little as 33 copies of the full Salmonella genome by incorporating an isothermal amplification step. Furthermore, 60 unknown samples with different target content in standard 344 well plates are evaluated. The results are analyzed using custom-developed machine-learning algorithms, successfully detecting the presence of the target with 100% prediction accuracy.