**2.3. LFIAs for aflatoxin M1**

tracted in methanol/water (typically 70/30 or 80/20 v/v) followed by dilution of the extract before LFIA analysis to reduce the proportion of the organic solvent, which is hardly com‐ patible with materials composing LFDs. However, a methanol amount lower than 15-20% has been demonstrated by most authors to be compatible with LFD materials and further

Most recent contributes to the topic are due to the group of Zhang and co-workers, who de‐ scribed two LFDs, the first highly selective towards AFB1 and the second able to measure total aflatoxins [57-58]. Both devices have been applied to visually detect target toxins in peanuts (the highly selective one could also be exploited to detect AFB1 in pu-erh tea, vege‐ table oil and feed). Both methods allowed reliable results (agreeing with HPLC determina‐ tion) to be obtain in 15 minutes. In addition, the LFIA aimed at measuring total AFs was extremely sensitive, with VDL in peanut extracts as low as 0.03, 0.06, 0.12, and 0.25 µg l-1for

In addition to papers aimed at describing actually functioning devices for measuring AFs, those targets have often been chosen as system models for the development of original devi‐ ces which exploited non-traditional signal reporters to label antibodies. Besides the above mentioned approach of Ho and Wauchope, based on the use of dye-encapsulating lipo‐ somes, Liao and Li described a visual device which exploited nanoparticles with a silver core and a gold shell as the reporters in the construction of a LFD for AFB1. The toxin was determined in cereals and nuts and performances were compared to those of a GNP-based LFIA and to results obtained through a classic microwell-based immunoassay. The authors demonstrated that the newly developed LFD was comparable to the GNP-LFD in terms of stability of components and reproducibility of signals. On the other hand, it allowed a great enhancement in sensitivity so that values as low as 0.1 µg l-1AFB1 could be measured [59].

With the expectation of increasing the useful signal, therefore being able to reduce immu‐ nore agents for the benefits of the competition, magnetic nanogold microspheres with a Fe2O3 core and a shell of multiple GNPs have also been proposed. The magnetic core of par‐ ticles allowed authors to simplify separation steps during the labelling of antibodies and their micro- dimensions to enhance colour during the test itself. A three-fold increase in sen‐ sitivity was stated for the visual detection of AFB2 compared to the use of simple gold col‐

A major concern in the development of LFDs for aflatoxins is the unpredictable effects due to food components co-extracted from the sample beyond the target and which affect not only the antigen-antibody interaction on which the immunoassay is based, but also the me‐

Some authors experienced the apparently inexplicable failure of recovery experiments con‐ ducted on fortified materials and the incongruity of results attained for artificially and natu‐ rally contaminated samples, which necessitate matrix-matched calibrations and recommended the use of naturally contaminated samples blended in varying proportions

more not to affect immunoassay performance.

322 Aflatoxins - Recent Advances and Future Prospects

AFB1, AFB2, AFG1, and AFG2, respectively.

loid nanoparticles [60].

chanics of the device itself.

*2.2.1. Application of LFIA for aflatoxins B and G in food analysis*

The development of LFIAs for AFM1 is one of the most challenging goals in this field of re‐ search because of the extreme sensitivity required by legislation for this contaminant (partic‐ ularly in the European Union).

The first paper dealing with the subject reported a validation study on a commercial device which was conceived for meet US regulations and did not described any preparation proto‐ cols and methods. The ROSA Charm Aflatoxin M1™ aimed at quantitatively measuring AFM1 in milk was validated as the result of an inter-laboratory trial, which involved 21 par‐ ticipants, at four levels above and two below the declared LOD of the assay (400 ng l-1) [64]. Less than 5% of false negative (n=83) and no false positive below 300 ng l-1 were found. For contaminations between 350 and 450 ng l-1 false positivity increased from 21 to 93%.

More recently, Wang et al first described the development of a LFD for the detection of AFM1 [65]. The cut-off level (0.5-1 µg l-1) is just above the eligible value required by the US regulation [8] and far beyond the more severe limits imposed by the European Un‐ ion for this contaminant [2]. However, it is an effectively sensitive and rapid assay, provid‐ ed that the whole analytical procedure can be completed in 10 minutes, as no sample treatment is required.

A visual device has also been developed by Zhang et al which showed a VDL for AFM1of 0.3 µg l-1 [66]. Although the sensitivity improvement respect to the work of Wang et al, the obtained VDL remains far away from the detectability demand imposed by EU MRLs for this contaminant.
