One-step litchi like bimetallic nanozyme-assisted indirect competitive immunoassay for the detection of acetamiprid in vegetables

The conventional enzyme-linked immunosorbent assay is limited in its ability to satisfy the increasing demand for advanced immunoassay due to its reliance on natural enzymes, low sensitivity, and narrow detection range. The development of nanozyme-assisted immunoassay is a viable approach to overcome this constraint. Therefore, a one-step litchi like nanozyme-assisted indirect competitive immunoassay was designed for the detection of acetamiprid. In this assay, litchi-like Au@Pt nanozyme (inexpensive, simple preparation, stable, and adjustable catalytic activity) were directly conjugated with antibodies as signal probes without using peroxidase-conjugated affinipure goat antimouse IgG (IgG-HRP). Acetamiprid competed with haptens to bind the Au@Pt probes. Subsequently, Au@Pt nanozymes, which exhibit peroxidase-like activity, were added to catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H ₂ O ₂ . The linear range, sensitivity, relative standard deviations (RSDs) ranged and limit of detection (LOD) were 1.85 ~ 327.19 µg/L, 25.58 µg/L, 0.46 ~ 10.35% and 0.78 µg/L, respectively, satisfying the requirement of immunoassay detection. In conclusion, the method exhibited high sensitivity, reproducibility and wide linear range for the detection of acetamiprid, while eliminating the need for IgG and simplifying the experimental procedure. The proposed indirect competitive immunoassay has great value for onsite sensitive detection of acetamiprid in vegetables.