A Comparative Review of Enzyme and Nanozyme Biosensors: Design Methodology, Design Tool, and Prospects.

Natural enzymes and nanozymes represent complementary paradigms for catalytic biosensing: the former provides evolutionarily refined molecular recognition and exceptional catalytic efficiency, while the latter offers tunable, robust, and manufacturable nanomaterial-based catalysis. Recent advances span catalytic theory, design and fabrication, and application-driven integration - illustrating how immobilization chemistries, protein engineering, and nanostructured transducer interfaces can preserve or recapitulate active-site function, and how defect engineering, single-atom centers, and two-dimensional materials deliver enhanced activity and environmental resilience. Persistent technical bottlenecks include enzyme fragility and cold-chain dependence, as well as nanozyme issues of biocompatibility, substrate selectivity, and batch reproducibility. These limitations are being addressed through convergent innovations: microfluidic platforms and scalable synthesis routes improve sampling and manufacturability, while machine-learning-assisted design and advanced signal-processing restore effective selectivity and enable rapid candidate triage. Emphasis on integrated toolchains - curated databases, multiscale simulation, and AI models - facilitates cross-domain knowledge transfer between protein and materials design. Finally, translational readiness will depend on standardized metrics, regulatory validation, and interdisciplinary consortia; near-term impact is expected in targeted applications (clinical biomarkers, antibiotic residues, environmental pollutants) where hybrid enzyme-nanozyme systems and data-driven workflows can deliver field-ready, robust sensors.