Single-Atom Nanozyme Integrated Double-Clasp Transition Probe-Driven CRISPR/Cas12a Cascade for Preamplification-Free Detection of Non-Nucleic Acid Targets

Abstract

CRISPR/Cas12a diagnostics have greatly advanced nucleic acid testing, yet their extension to non-nucleic acid targets remains constrained by the intricate preamplification process and by inefficient signal transduction. Herein, we present a non-nucleic acid detection platform that integrates a CRISPR/Cas12a positive-feedback cascade with a single-atom nanozyme. In this strategy, cleavage of the designed double-clasp transition probe (DCTP) activates additional Cas12a ribonucleoprotein (RNP) complexes, enabling cascade signal amplification without a preamplification treatment. Meanwhile, the created single-atom Pt-engineered PdCeB nanozyme (Pt₁/PdCeB SAN) functions as an efficient electrochemiluminescence (ECL) nanolabel in the luminol/O₂ system, converting dissolved oxygen (O₂) into reactive oxygen species (ROS) to produce a robust ECL response without an external coreactant. Using human epidermal growth factor receptor 2 (HER2) as a clinically relevant non-nucleic acid target, the DCTP-driven Cas12a positive-feedback system shortened the cleavage time from 33 min in the conventional Cas12a system to 18 min and achieved a wide linear range from 10^–3 to 10⁴ ng/mL, with a low detection limit of 0.513 pg/mL. Furthermore, preliminary analysis of 31 clinical serum samples supported the feasibility of the strategy for quantitative serum HER2 (sHER2) determination, although validation in larger independent cohorts is required.