Self-assembled multiprotein nanostructures with enhanced stability and signal amplification capability for sensitive fluorogenic immunoassays

Fundamentally improving the sensing sensitivity of immunoassay remains a huge challenge, which limited further critical applications. Herein we designed a new immunoprobe by integrating biometric unit (antibody) and signal amplification element (enzyme) to form urease-antibody-CaHPO₄ hybrid nanoflower (UAhNF) via the biomineralization process. The dual-functional UAhNF enhances the stability of urease in NaCl (10 mmol L^-1) and high temperature (60 °C), and also maintains the ability of antibody recognition, fitting greatly well with the need for immunosensor. Using imidacloprid as a model target, the fixed coating antigens are competed with imidacloprid to capture primary antibodies, and the secondary antibody of UAhNF was linked to construct the competitive-type fluorogenic immunoassays. An in-situ etching process of copper nanoparticles initiated by urease is integrated with UAhNF-based immune response for further improving the detection sensitivity. The proposed immunosensor possessed a 50% inhibition concentration value of 0.72 ng mL^-1, which is 30-fold lower than conventional enzyme-linked immunosorbent assay. This presented approach provided a versatile sensing tool by varying building blocks, making it practically functional for a variety of bioassay applications.