A sensitive electrochemical immunosensing interface for label-free detection of aflatoxin B.sub.1 by attachment of nanobody to MWCNTs-COOH@black phosphorene

A label-free electrochemical immunosensor has advantages of real-time and rapid detection, but it is weak in detection of small molecular toxins such as aflatoxin B.sub.1 (AFB.sub.1). The greatest obstacle to achieving this is that small molecules bound to a common immunosensing interface cannot interfere with electron transfer effectively and the detection signal is so weak. Therefore, a sensitive electrochemical immunosensing interface for small molecules is urgently needed. Here, we employed functionalized black phosphorene (BP) as electrode modification materials and anti-AFB.sub.1 nanobody (Nb) as a biorecognition element to construct a very sensitive immunosensing interface towards small molecular AFB.sub.1. The BP functionalized by carboxylic multi-walled carbon nanotubes (MWCNTs-COOH) via P-C bonding behaved with a satisfactory stability and good catalytic performance for the ferricyanide/ferrocyanide probe, while the small-sized Nb showed good compatibility with the functionalized BP and also had less influence on electron transfer than monoclonal antibody (mAb). Expectedly, the as-prepared immunosensing interface was very sensitive to AFB.sub.1 detection by differential pulse voltammetry (DPV) in a redox probe system. Under optimized conditions, a linear range from 1.0 pM to 5.0 nM and an ultralow detection limit of 0.27 pM were obtained. Additionally, the fabricated immunosensor exhibited satisfactory stability, specificity, and reproducibility. The strategy proposed here provides a more reliable reference for label-free sensing of small molecules in food samples. Graphical abstract