Ultrafast Self-Assembly of Zeolitic Imidazolate Framework-8 Enables Antibody Orientation for Ultrasensitive Lateral Flow Immunoassays

Gold-nanoparticles-based lateral flow immunoassays (AuNPs-LFIAs) are widely used for point-of-care testing, yet their sensitivity remains fundamentally limited, likely because random antibody orientation on AuNP surfaces restricts Fab accessibility and antigen recognition efficiency. Precise control of antibody orientation is thus critical for maximizing probe activity, yet existing directional conjugation strategies are often multistep, technically complex, and poorly scalable. Here, we report an ultrafast biomimetic mineralization strategy that enables one-step, room-temperature self-assembly of zeolitic imidazolate framework-8 (ZIF-8) with antibodies and dual-ligand AuNPs, producing uniform hybrid nanoprobes within 5 min. ZIF-8 shell preserves the plasmonic properties of AuNPs while simultaneously enhancing colloidal stability and optical signal intensity. Molecular dynamics simulations suggest a two-step orientation model: (i) rapid electrostatic attraction between negatively charged Fc regions and Zn²⁺ ions initiates nucleation, followed by (ii) Fc-associated interfacial interactions that promote preferential Fc-oriented binding and anisotropic Fc/Fab distribution, thereby enhancing Fab exposure for efficient antigen recognition. This controlled assembly increases Fab accessibility more than 3-fold relative to conventional adsorption or co-precipitation strategies, minimizes nonspecific adsorption, and amplifies signal output. When applied to competitive LFIAs, the oriented nanoprobes enabled ultrasensitive detection of chloramphenicol down to 13 pg/mL, corresponding to approximately 1 order of magnitude higher sensitivity than AuNPs-based LFIA gold standard. By integrating mechanistic insight with operational simplicity, this work establishes a generalizable platform for ultrafast, scalable, and antibody-orientation-controlled nanoprobe fabrication for next-generation LFIAs in food safety, clinical diagnostics, and environmental monitoring.