Breaking the bottlenecks in nanozyme–antibody conjugate preparation: challenges, optimization strategies, and applications of in vitro diagnostics

Enzyme-labeled antibodies serve as the fundamental pillars of immunoassay-based biosensing, providing the necessary molecular recognition and signal amplification for the detection of trace analytes in complex matrices. As robust mimics of natural enzymes, nanozyme-antibody conjugates (NACs) represent a significant advancement in this field, offering superior physicochemical stability and cost-effectiveness. However, the practical transition of NACs from laboratory research to clinical and environmental commercialization is frequently hindered by critical preparation bottlenecks, particularly the loss of catalytic and biological activities, poor orientation control at the bio-nano interface, and significant batch-to-batch inconsistency. This review comprehensively details the advanced strategies developed to address these preparative challenges. The fundamental mechanisms governing signal attenuation and steric hindrance at the interface are systematically analyzed. The optimization strategies are highlighted, including the utilization of single-atom nanozymes for improved intrinsic activity and the implementation of site-specific conjugation methods to optimize antibody orientation. Furthermore, the review explores the diverse applications of optimized NACs across multiple platforms, achieving ultra-sensitive detection of oncology markers and pathogens. The discussion concludes with the requirements for clinical translation, emphasizing activity standardization and the development of integrated "sample-in-answer-out" point-of-care devices.