Dai, JiahuiWu, BeibeiAi, FengxiangYang, ZhugenLu, YanyanZinian, CaiZeng, KunZhang, Zhen2025-01-222025-01-222025-01-21Dai J, Wu B, Ai F, et al., (2025) Exploiting the potential of spherical PAM antenna for enhanced CRISPR-Cas12a: a paradigm shift toward a universal amplification-free nucleic acid test platform. Analytical Chemistry, Volume 97, Issue 2, January 2025, pp. 1236-12450003-2700https://doi.org/10.1021/acs.analchem.4c04871https://dspace.lib.cranfield.ac.uk/handle/1826/23417The CRISPR-Cas12a system has shown tremendous potential for developing efficient biosensors. Albeit important, current CRISPR-Cas system-based diagnostic technologies (CRISPR-DX) highly rely on an additional preamplification procedure to obtain high sensitivity, inevitably leading to issues such as complicated assay workflow, cross-contamination, etc. Herein, a spherical protospacer-adjacent motif (PAM)-antenna-enhanced CRISPR-Cas12a system is fabricated for universal amplification-free nucleic acid detection with a detection limit of subfemtomolar. Meanwhile, the clinical detection capability of this sensor was further verified using gold-standard real-time quantitative polymerase chain reaction through <i>Mycobacterium tuberculosis</i> measurement, which demonstrated its good reliability for practical applications. Importantly, its excellent sensitivity is mainly ascribed to high efficiency of target search induced by a localized PAM-enriched microenvironment and improved catalytic activity of Cas12a (up to 4 folds). Our strategy provides some new insights for rapid and sensitive detection of nucleic acids in an amplification-free fashion.pp. 1236-1245Print-ElectronicenAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/40 Engineering34 Chemical SciencesEmerging Infectious DiseasesInfectious DiseasesBiotechnology3 Good Health and Well BeingAnalytical Chemistry3205 Medical biochemistry and metabolomics4004 Chemical engineeringArticle1520-6882562148972