Functional Zonation Strategy of Heterodimer Nanozyme for Multiple Chemiluminescence Imaging Immunoassay

Although nanozymes with intrinsic enzyme-like characteristics have aroused great interest in the biosensing field, the challenge is to keep high enzyme-like activity of the nanozyme after the modification of biomolecules onto nanozymes. Herein, a functional zonation strategy of a heterodimer nanozyme was proposed to tackle the challenge and further construct a multiple chemiluminescence (CL) imaging immunoassay. Here Fe₃O₄-Au as a heterodimer nanozyme model was divided into two zones, in which Fe₃O₄ nanoparticles (NPs) were regarded as a nanozyme zone and AuNPs were defined as an antibody immobilization zone. A signal amplification probe (Fe₃O₄-Au-Ab₂) was prepared by modifying the secondary antibody (Ab₂) on AuNPs of the Fe₃O₄-Au heterodimer owing to the Au-S bond. The exposed Fe₃O₄ of the Fe₃O₄-Au-Ab₂ probe shows very high peroxidase-like activity and can efficiently catalyze H₂O₂-luminol to produce strong CL imaging signals for multiple antigens detection. Using chicken interleukin-4 (ChIL-4) and chicken gamma interferon (ChIFN-γ) as models, the proposed CL imaging immunoassay shows wide linear ranges (0.005-0.10 ng/mL for both ChIL-4 and ChIFN-γ) and low detection limits (0.58 pg/mL for ChIL-4, 0.47 pg/mL for ChIFN-γ) with the characteristics of high sensitivity, high specificity, and good stability. This work provides a promising functional zonation concept for nanozymes to construct new types of nanozyme probes for immunoassay of multiple biomolecules.