Nanobody-based electrochemical competitive immunosensor for the detection of AFB through AFB-HCR as signal amplifier.

A novel nanobody (Nb)-based voltammetric immunosensor coupled with horseradish peroxidase concatemer-modified hybridization chain reaction (HRP-HCR) signal amplifying system is described to realize the rapid and ultrasensitive detection of AFB. To design such an immunoassay, anti-AFB Nbs with smaller molecular size were coated densely onto the surface of Au nanoparticle-tungsten disulfide-multi-walled carbon nanotubes (AuNPs/WS/MWCNTs) functional nanocomposites as an effective molecular recognition element, whereas AFB-streptavidin (AFB-SA) conjugates were ingeniously bound with biotinylated HCR dsDNA nanostructures as the competitor, amplifier, and signal report element. In the presence of AFB targets, a competitive immunoreaction was performed between the analyte and AFB-SA-labeled HCR (AFB-HCR) platform. Upon the addition of SA-modified polyHRP (SA-polyHRP), AFB-HCR nanostructures containing abundant biotins were allowed to cross-link to a quantity of HRP by streptavidin-biotin chemistry for signal amplification and signal conversion. Under optimal conditions, the immunosensor displayed a good linear correlation toward AFB ranging from 0.5 to 10 ng mL with a sensitivity of 2.7 μA • (mL ng) and an ultralow limit of detection (LOD) of 68 fg mL. The specificity test showed that the AFB immunosensor had no obvious cross-reaction with OTA, DON, ZEN, and FB. The signal of this sensor decreased by 10.18% in 4 weeks indicating satisfactory stability, and its intra- and inter-laboratory reproducibility was 3.42~10.35% and 4.03%~12.11%, respectively. This biosensing system will open up new opportunities for the detection of AFB in food safety and environmental analysis and extend a wide range of applications in the analysis of other small molecules. Graphical abstract.