Nanobody mediated dual-mode immunoassay for detection of peanut allergen Ara h 3

To improve the performance of peanut allergen Ara h 3 detection, depending on boron and nitrogen carbon dots (B/N-CDs), a nanobody (Nb) mediated dual-mode immunoassay was established, which combines the dominance of colorimetry with ratiometric fluorescence techniques. With the catalysis of Horseradish peroxidase (HRP), the oxidization of o-phenylenediamine (o-PD) in the presence of H₂O₂, leading to the production of yellow 2,3-diaminophenolazine (DAP) with an absorption peak at 431 nm. Owing to inner filter effect (IFE), DAP quenched the fluorescence of B/N-CDs at 426 nm, and it emerged a new emission peak at 549 nm. The fluorescence intensity ratio and absorption intensity can be utilized for quantitative analysis of Ara h 3 concentration. Under optimal conditions, the detection limits were 6.61 and 9.79 ng·mL^-1, respectively. The dual-mode immunoassay was assessed containing specificity, stability, reproducibility, and practicability. This method paved the way for sensitive detection of Ara h 3 without background Interference.