| Summary: | This paper presents a novel nano-material composite membrane for detecting aflatoxin B<sub>1</sub> (AFB<sub>1</sub>). The membrane is based on carboxyl-functionalized multi-walled carbon nanotubes (MWCNTs-COOH) @ antimony-doped tin oxide (ATO)-chitosan (CS). To prepare the immunosensor, MWCNTs-COOH were dissolved in the CS solution, but some MWCNTs-COOH formed aggregates due to the intertwining of carbon nanotubes, blocking some pores. ATO was added to the solution containing MWCNTs-COOH, and the gaps were filled by adsorbing hydroxide radicals to form a more uniform film. This greatly increased the specific surface area of the formed film, resulting in a nano-composite film that was modified on screen-printed electrodes (SPCEs). The immunosensor was then constructed by immobilizing anti-AFB<sub>1</sub> antibodies (Ab) and bovine serum albumin (BSA) on an SPCE successively. The assembly process and effect of the immunosensor were characterized using scanning electron microscopy (SEM), differential pulse voltammetry (DPV), and cyclic voltammetry (CV). Under optimized conditions, the prepared immunosensor exhibited a low detection limit of 0.033 ng/mL with a linear range of 1 × 10<sup>−3</sup>–1 × 10<sup>3</sup> ng/mL. The immunosensor demonstrated good selectivity, reproducibility, and stability. In summary, the results suggest that the MWCNTs-COOH@ATO-CS composite membrane can be used as an effective immunosensor for detecting AFB<sub>1</sub>.
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