Liang Luo

Aflatoxin M1 (AFM1) in milk poses significant health risks due to its carcinogenicity and resistance to thermal processing. Here, we applied a computer-aided hapten design strategy integrating density functional theory, molecular docking, and molecular dynamics simulations to engineer four AFM1 haptens with tailored spacer arms (C2, C4, C6, and phenyl). A high-affinity monoclonal antibody (10C1, IC50 = 0.015 ng/mL) was generated. Based on this antibody, an icELISA (LOD = 0.0017 ng/mL, meeting EU standards of 0.05 ng/mL) and a gold nanoparticle-based LFIA (LOD = 0.3 ng/mL, meeting China/US/Russia standards of 0.5 ng/mL) were developed for laboratory and on-site detection, respectively. This strategy facilitates rational hapten optimization and efficient generation of high-affinity antibodies for AFM1 monitoring.

Chemical contaminants in food pose a serious threat to public health, driving the need for sensitive, rapid, and on-site screening methods. Lateral flow immunoassay (LFIA) is rapid and portable but suffers from single-signal readout and insufficient label stability. Nanozymes, nanomaterials with enzyme-like catalytic activity and excellent stability, have emerged as promising signal labels to address these limitations. Moreover, their diverse physiochemical properties enable multiplex signal readout, where two or more complementary signals (e.g., colorimetric, fluorescent, chemiluminescent, photothermal, and surface-enhanced Raman scattering) are generated simultaneously from a single test line. This multiplex strategy significantly enhances detection sensitivity, accuracy, and reliability through signal amplification and self-calibration. This review provides a systematic overview of the catalytic properties and their major types used in multiplex signal LFIA. The signal combination strategies employed in nanozyme-based multiplex signal LFIA were also summarized, and their applications in detecting veterinary drugs, mycotoxins, pesticides, and other food chemical contaminants are highlighted. Ultimately, current challenges and future prospectives in this field are discussed. This review offers guidance for designing high-performance, nanozyme-based multiplex signal LFIA platforms for food safety monitoring.

The enzyme-linked immunosorbent assay (ELISA) stands as one of the most frequently employed rapid detection techniques for both chemical and biological contaminants. Horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂), serving as signal generators, play indispensable roles throughout the entire ELISA process. However, HRP and H₂O₂ exhibit high sensitivity to elevated temperatures, hindering the broader utilization and transportation of ELISA. Hereby, being inspired by the cascade catalysis of glucose by glucose oxidase (GOx) and HRP as well as the advantageous properties of metal-organic frameworks (MOFs), we developed a cascade catalysis-based competitive fluorescent ELISA (CCF-ELISA) for mycotoxin detection in maize using zearalenone (ZEN) as a model. The results revealed the remarkable protective effects of MOFs on enzymatic activities. Based on upon characteristics, the built CCF-ELISA enabled the detection of ZEN with a half-maximal inhibitory concentration of 0.54 ng/mL and allowed a detection range from 0.19 to 1.51 ng/mL. When applied to naturally contaminated maize samples, the outcomes obtained from the CCF-ELISA showed a high level of comparability with high-performance liquid chromatography-tandem mass spectrometry, demonstrating the promising potential of the developed CCF-ELISA in the rapid detection for food security.

As the most widely used immunoassay, enzyme-linked immunosorbent assay (ELISA) relies on perishable enzymes and usually provides poor sensitivity and stability, limiting its application in detecting trace analytes in harsh environments. Herein, a new ratiometric fluorescence (RF) sensing platform enhanced by a nanozyme-enzyme cascade reaction composed of MnO₂ nanosheets (MnO₂ NSs) and alkaline phosphatase (ALP) was proposed. In this RF platform, the versatile MnO₂ NSs worked as a robust oxidase-like nanozyme to catalyze nonfluorescent Amplex Red (AR) into fluorescent resorufin and as a quencher to quench the fluorescence of carbon dots (CDs). Given that ALP could catalyze ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA), followed by AA reducing MnO₂ NSs into Mn²⁺, resulting in the fluorescence of AR decrease but fluorescence of CD recovery simultaneously. The regulation of MnO₂ NSs for the fluorescence intensity of AR (E_m at 585 nm) and CDs (E_m at 460 nm) could cascade with ALP-triggered reaction to construct an RF sensing platform based on RF signal (F585/F460) output. The proposed RF sensing platform could achieve a superior limit of detection (LOD) of 0.037 mU mL^-1 for ALP activity quantification. Inspired by the excellent performance of the RF sensing platform, a sensitive and accurate RF ELISA for ractopamine (RAC) detection was established with an LOD of 0.029 ng mL^-1, which was nearly 5.5 times more sensitive than traditional colorimetric ELISA. This RF sensing platform based on robust nanozyme-triggered enzymatic cascade signal amplification and excellent RF signal readout has offered a powerful and universal platform for RAC and other trace target detection.