Ligand-EngineeredMn-Cysteine as a Potent LaccaseMimic for CRISPR/Cas12a Electrochemical Biosensing of HepatocellularCarcinoma Biomarkers

Laccase is an environmentally friendly catalyst with water as the sole catalytic byproduct, yet its biomedical detection potential remains underexplored. Herein, a ligand engineering strategy was employed to synthesize Mn-cysteine nanoflowers (Mn-Cys NF) with laccase-mimicking activity via a one-pot method, using manganese (Mn) with rich valence variations as the active center and cysteine (Cys) as the ligand. Spectroscopic characterizations confirmed Cys-modulated Mn electronic structure, and theoretical calculations validated enhanced substrate adsorption and reduced reaction barriers. The specific activity of Mn-Cys NF is approximately 3.56 times that of natural laccase and exhibited excellent stability across pH, temperature, ionic strength, and organic solvent conditions. Leveraging this high-performance nanozyme, a CRISPR/Cas12a electrochemical biosensor was constructed with a DNA triangular prism interface, where a target-triggered catalytic hairpin assembly (CHA)–DNAzyme cascade regulated Cas12a cleavage to enable signal-on detection. This biosensor achieved quantification of hepatocellular carcinoma (HCC) biomarkers alpha-fetoprotein (AFP) and microRNA-122 (miRNA-122), with detection limits as low as 4.47 fg/mL and 6.21 aM, respectively. It also effectively discriminated HCC patients from healthy individuals in clinical serum samples. This work offers a ligand engineering strategy for designing high-performance laccase-mimicking nanozymes and expands the application scope of laccase nanozymes from environmental remediation to biomedical biosensing.