A CRISPR/Cas12a-responsive DNA hydrogel-δ-MnO₂ colorimetric biosensor for multiplex detection of uropathogens

A CRISPR-responsive DNA hydrogel gates δ-MnO 2 nanozyme release. • RPA–Cas12a recognition enables detection of five common uropathogens. • The assay achieved LODs of 2.44–45.47 copies/μL across targets. • Parallel channels showed specificity, reproducibility, and 20-day stability. • KernelRidge-RBF provided the lowest prediction error among nine evaluated regression models. Urinary tract infection (UTI) requires rapid and accurate pathogen identification, yet conventional culture-based methods are time-consuming and many molecular assays remain dependent on sophisticated instrumentation. Here, we developed a multiplex visual biosensing platform for uropathogen detection by integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a recognition, a CRISPR-responsive DNA hydrogel gate, and a δ-MnO 2 nanozyme-mediated colorimetric module. In this system, target-derived amplicons activate pathogen-specific Cas12a, which degrades the DNA hydrogel and releases encapsulated δ-MnO 2 , thereby catalyzing the TMB/H 2 O 2 reaction to produce a visible and spectrophotometrically measurable signal. The platform was applied to five representative uropathogens, including Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa , Enterococcus faecium , and Enterococcus faecalis . Under optimized conditions, the assay showed good linearity for all five targets, with R 2 values of 0.9955, 0.9958, 0.9948, 0.9974, and 0.9956, respectively, and limits of detection of 2.60, 3.15, 2.44, 20.40, and 45.47 copies/μL, respectively. The platform also exhibited satisfactory specificity, multiplexing capability, reproducibility, and storage stability. To improve quantitative interpretation, an image-recognition- and machine-learning-assisted workflow was further introduced, in which KernelRidge-RBF showed the best overall performance among the tested models. Clinical urine sample testing further supported the applicability of the platform, with AUC values of 1.000, 0.976, 0.994, 0.947, and 0.939 for the five target pathogens, respectively. Collectively, the RPA-Cas12a-DNA hydrogel-δ-MnO 2 platform enables visual and parallel multi-target detection of uropathogens within approximately 3 h and provides a proof-of-concept framework for the further development of portable nucleic acid biosensors. Download: Download high-res image (194KB) Download: Download full-size image