CRISPR-Programmed CuO Nanocatalyst Release for Ultrasensitive Detection of Pathogens in Sterile Body Fluids

Treatment of sterile body fluids (SBFs) infections is delayed by conventional methods that require up to 72 h to detect pathogens. Here, we present a CRISPR-associated protein 12a (Cas12a)-programmed nanocatalyst release (CNR) method for culture-free diagnostics. To enhance both sensitivity and coverage, three starter DNA (sDNA)-complementary DNA (cDNA) probe pairs were designed for conserved regions and additional three pairs for variable regions of bacterial 16S or fungal 18S rRNA. Upon target recognition, cDNA undergoes strand displacement, releasing sDNA to activate Cas12a. The activated Cas12a cleaves copper oxide nanoparticle (CuONPs)-loaded magnetic probes, releasing tandem CuONPs. Upon acid dissolution, each CuONP generates Cu²⁺ ions that catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), producing a visible colorimetric signal. This quadruple signal amplification strategy integrates high-copy rRNA targets, multi-cDNA recognition, Cas12a-mediated continuous release of tandem CuONPs, and Cu²⁺-driven chromogenic amplification. This nucleic acid amplification-free assay detects pathogens at 0.69 CFU mL^-1 in original SBFs samples (after 10-fold centrifugation) within 70 min. In 64 clinical samples, it achieved 100% sensitivity and 100% specificity versus culture. Notably, one culture-negative but clinically confirmed case was correctly identified. Overall, the CNR method offers a rapid, ultrasensitive, and accessible diagnostic solution for resource-limited settings.