Decoupled Signal Transduction via a Non-Copper Laccase Mimic for CRISPR-Based Electrochemical Detection of Hepatocellular Carcinoma

Abstract Laccase-mimicking nanozymes have been overwhelmingly copper-dependent, and largely confined to environmental contexts, with clinical translation remaining unrealized. Here, we depart from this paradigm by designing a non-copper nanozyme, Mn-Trp NR, assembled through manganese-tryptophan coordination. Spectroscopic and theoretical analyses show that tryptophan coordination finely modulates the Mn electronic structure, strengthening O2 adsorption and lowering the reaction barrier, thereby conferring laccase-like activity, substrate affinity, and stability superior to the native enzyme. To overcome broad-substrate promiscuity that precludes specific biomarker recognition, we decoupled signal transduction from target identification: Mn-Trp NR serves as a universal amplifier, whereas CRISPR/Cas12a confers high specificity. Accordingly, a cascaded electrochemical biosensor was built on a DNA triangular prism integrating catalytic hairpin assembly, hybridization chain reaction, and CRISPR/Cas12a activation. The platform detects hepatocellular carcinoma (HCC) biomarkers microRNA-122 (miRNA-122) and alpha-fetoprotein (AFP) with limits of 11.98 aM and 10.31 fg/mL, respectively, in a streamlined 160 min, two-step procedure. In clinical sera, the biosensor perfectly distinguishes HCC patients from healthy donors (AUC = 1.00) and agrees strongly with benchmark methods (RT-qPCR for miRNA-122, ELISA for AFP, R2 > 0.97). By expanding laccase-mimic synthesis beyond copper via ligand-field engineering and establishing a decoupled architecture that reconciles broad-spectrum catalysis with high-specificity recognition, this work provides a generalizable strategy for deploying nanozymes in precision diagnostics.