Bioluminescence readout lateral flow immunoassay using nanobody targeting aflatoxin B1

Multiple signal detection methods are known for lateral flow immunoassays (LFIAs), with colorimetric approaches dominating the field. However, their limited sensitivity is a remaining challenge. Fluorescence-based signaling is regarded as a more sensitive method, but it comes at the cost of partial sacrifice of the user-friendliness of LFIAs due to the requirement of an excitation light source. In this context, bioluminescence providing an inherently high signal to noise ratio without the need of excitation light could be an attractive alternative. But only a few studies have demonstrated the application of bioluminescence signaling in LFIAs. This work aimed at the development of a simple bioluminescence-based LFIA for the detection of aflatoxin B1 (AFB1), used as a model target in a competitive LFIA format. Signal transduction was achieved by nanobody-nanoluciferase (Nluc) fusion proteins. These small-sized recombinant heavy-chain-only antibodies derived from the camelidae family directly linked with the Nluc enzyme produce high intensity glow-type bioluminescence in combination with the furimazine substrate. LFIA devices consisting of a sample pad, nitrocellulose membrane and absorbent pad with AFB1-BSA conjugate deposited at the test line on the nitrocellulose membrane, achieved an LOD of 0.26 ng mL^-1 for aqueous AFB1 solutions pre-mixed with Nanobody-Nluc and bioluminescence emission observed on an imaging system. More user-friendly LFIA devices with integrated conjugate pad and pre-deposited Nanobody-Nluc provided clear AFB1 concentration-dependent bioluminescence signals with low background and enabled readout with a standard digital camera, resulting in an LOD of 1.12 ng mL^-1. Finally, the LFIA strips have been applied in AFB1-spiked oat milk samples. The LOD of 4.09 ng mL^-1 achieved in the real sample matrix is well below the maximum allowable residual concentration of AFB1 in the U.S. (20 ng mL^-1).