Aflatoxin B1 (AFB1) is the most potent genotoxic and carcinogenic aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus. AFB1 is considered a major concern in food safety because of its high toxicity and possible contamination of a variety of food commodities such as cereals, groundnuts, tree nuts, and milk. To guarantee the food safety and address health concerns of consumers, it is important to monitor the contamination of the food chain with this toxin. Screening and regular testing for aflatoxin are mainly done by reference laboratories from the European Union (EU) and the United States (US), using sensitive but low-throughput technologies, thus limiting the frequency of product testing. The problem is still more serious in developing countries, where contamination is often high and rapid field methods are needed. New technologies are needed to provide broader, more thorough and efficient screening for these mycotoxins. In this context, we present the development of a lateral flow immunoassay (LFIA) for the detection of AFB1 as a semiquantitative approach. We have combined the advantages of engineered nanobodies specific to AFB1 with an easy fluorescence read-out system of green fluorescence protein (GFP) fused as a label to a specific nanobody. Optimized LFIA reached a limit of detection (LOD) of 4.80 ng/mL in undiluted almond milk. Because of lifestyle changes, there is an increasing demand for almond milk compared to dairy products. Almond milk is susceptible to contamination with aflatoxin and thus was selected as a food matrix for AFB1 monitoring. The U.S. Food and Drug Administration (FDA) and the European Commission established an action level or maximum residue level of 20 and 8 ng/mL for almond milk, respectively. The developed LFIA has been optimized to distinguish between compliant/noncompliant samples taking into consideration the signal provided by the control line (CL). The CL signal has been set as a threshold of the maximum residue level (MRL) signal (TL). A validation study has been performed and blind samples have been measured successfully.
