Hierarchical triple-mode immunosensing of aflatoxin B1: From visual screening to precise confirmation

This work establishes a hierarchical triple-signal immunoassay based on a multifunctional nanocomposite of Prussian blue nanoparticle@carbon dot (PBNPs@CDs), aiming to address material complexity and system integration issues in multimodal sensing. PBNPs@CDs possess peroxidase-like activity, efficient photothermal conversion, and strong fluorescence emission. These intrinsic properties enable signal generation and hierarchical utilization within a single platform. Aflatoxin B1 (AFB1) is selected as the proof-of-concept target. An AFB1-specific aptamer is covalently coupled to PBNPs@CDs to form the detection probe. The probe is anchored to 96-well plates via AFB1 and an immobilized antibody, producing proportional colorimetric, photothermal, and fluorescence signals. The signals are organized into a coherent workflow that transitions from on-site screening to laboratory confirmation. Specifically, the colorimetric readout for rapid, instrument-free qualitative screening. The photothermal readout for on-site semi-quantitative risk assessment with strong resistance to optical interference. The fluorescence readout for laboratory-grade ultra-sensitive quantification. Detection limits are 0.190 fg/mL (colorimetric), 0.893 fg/mL (photothermal), and 0.023 fg/mL (fluorescence), respectively. The system demonstrates high accuracy, selectivity, and strong agreement with commercial ELISA in complex food matrices. This work establishes a generalizable sensing paradigm based on functional matching and hierarchical synergy, offering an effective strategy for constructing integrated detection systems.