**1. Introduction**

Aflatoxins (AFs), secondary metabolites produced by *Aspergillus Flavus* and *Aspergilluspara‐ siticus*, are a numerous group of chemically related compounds characterised by high toxici‐ ty. Among these, aflatoxin B1 (AFB1) is the most potent known carcinogen for liver and, together with aflatoxins B2 (AFB2), G1 (AFG1) and G2 (AFG2) is the most frequently found and the most toxic of the group [1]. Therefore, maximum residue levels (MRLs) for AFB1 and for the sum of the four AFB1 + AFB2 + AFG1 + AFG2 (total aflatoxins) in food and feed have been set by the European Union [2-4] and all over the world [5-7].

The occurrence of aflatoxins (AFs) has been widely reported in a variety of crops (including maize, wheat, barley, rice, groundnuts, nuts, pistachios, cottonseed, and spices) which can be infected pre-, during and post-harvest. Moreover, due to the relative stability of AFs to thermal and chemical stresses, they are found on commodities despite the elimination of mould, after long periods of storage, and also after the transformation of raw materials; therefore the presence of AFs has also been ascertained in commodities such as composite feed, flour, bakery products, and roasted peanuts.

In addition, products of the animal metabolism of aflatoxins could retain toxicity, such as in the case of AFB1, which, once ingested, is rapidly absorbed and transformed into a hydroxy‐ lated metabolite. The latter is secreted into the milk and thus has been named aflatoxin M1 (AFM1). The hepatotoxicity and carcinogenic effects of AFM1 have also been demonstrated and IARC have included this toxin in the group I human carcinogens as well as the parent AFB1[1]. Milk and derived products can consequently also be implicated in the spreading of aflatoxins. Therefore, most countries have also set up MRLs of AFM1 in milk, which varies

from the 50 ng kg-1 established by the EU to the 500 ng kg-1 established by US FDA [2, 8]. More restrictive MRLs have been decided by the EU for the presence of AFM1 in baby food [2].

Numerous immunosensors have been described [27] as well, and research is constantly evolving in this area, particularly for the development immunosensors for the selective de‐

Lateral Flow Immunoassays for Aflatoxins B and G and for Aflatoxin M1

http://dx.doi.org/10.5772/51777

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In parallel, strategies aimed at avoiding the use of antibodies in the development of rapid methods for aflatoxin detection have also been reported, such as those based on the prepara‐ tion of polymers with molecular recognition properties towards AFB1as capture systems [36-37] or those based on the exploitation of its natural fluorescence for the detection [38]. A combination of the surface plasmon resonance phenomenon and fluorescence has been ex‐ ploited in the work of Wang et al and permitteda very sensitive determination of AFM1, though the proposed assay took almost an hour to be accomplished and couldn't be consid‐ ered as a truly rapid method [39]. A fancy and cunning approach for the rapid quantifica‐ tion of AFB1 have been described in the work of Arduini et al, who exploited the inhibiting effect of the toxin towards the enzyme acetylcholinesterase. The measurement of the enzy‐ matic activity was demonstrated to directly allow AFB1 quantification in 3 minutes and

Among the rapid methods for screening of food contaminants, the'lateral flow immunoas‐ say" (LFIA) (also known as immunochromato graphic assayorimmuno-colloid gold immu‐ noassay, ICG) has recently attracted the interest of researchers and industry. This technology has long been known in medical fields for diagnosing blood infections and fail‐ ure of internal organs, disclosing drug abuse or ascertaining pregnancy and combines a ser‐ ies of benefits, including extreme simplicity, rapidity, and cost effectiveness [41]. These features make it ideally suited for screening large number of samples, for being conducted by non-trained personnel and practically everywhere, thus enabling the effective possibility

Since the early 2000's, scientific papers and commercial devices aimed at measuring myco‐ toxinsin food and feed have appeared, and recentlya certain amount of literature on this topic has become available, including comprehensive reviews [42-44]. In particular, some LFIAs for the qualitative and semi-quantitative detection of aflatoxins in food and feed have been described and will be discussed below. At the same time, commercial LFDs for the de‐ tection of aflatoxinsin various commodities have become available and some of them have

As aflatoxins are low-molecular-mass compounds, immunoassays in competitive formats should be conceived to measure them. The same principles and reagents as in the micro‐ well-type immunoassays could be applied, except for the fact that,in LFIA, the separation of bound and unbound antibody sites is obtained by means of the lateral flow on a suitable support (the membrane). The liquid flow transports immunoreagents along the membrane

termination of AFB1 [28-32] and for AFM1 detection [33-35].

of food safety assessment at all stages of food and feed production.

**2. Lateral flow immunoassays for aflatoxins**

also been validated by USDA-GIPSA [45].

**2.1. Principle of the method**

within the 10-60 µg l-1 range [40].

A part from safety issue, food contamination caused by AFs also strongly affects economic interests; so much effortis devoted to the development of analytical methods for detecting these contaminants. Newly developed methods of analysis are intended both for screening purposes (rapid, economic and simple methods) and for the accurate, reproducible and sen‐ sitive quantification by confirmatory methods.

Numerous chromatographic methods to detect AFs in foods have been developed, coupled to fluorescent or mass spectrometric detection [9-11]. Likewise, several methods for aflatoxin M1 determination in milk based on high-performance liquid chromatography associated to fluorescence or mass spectrometric detection have been developed [12-13]. However, chro‐ matographic techniques are mainly used in confirmatory analyses and are usually not ap‐ plied to routine controls owing to the necessity to use expensive equipment and extensive clean-up steps.

The first rapid methods of analysis for AFs were based on Thin Layer Chromatography [14]; this technique is still used today even though in a significant lesser extent compared to methods based on the use of antibodies. Immunochemical methods of analysis are widely employed as screening methods for measuring AFs in food and feed [9, 14-18] and also for AFM1 quantification in milk and dairy products [19-21] thanks to their rapidity, selectivity and sensitivity. Several ELISA kits are commercially available, whose performances are gen‐ erally adequate to meet legal requirements, and are routinely employed for aflatoxin moni‐ toring. Some of these methods have also been validated [17-18]. However, even immunoassays need to be run in a laboratory, use a minimum of equipment and occasional‐ ly require some sample treatments, which may also involve the use of hazardous chemicals. Instead, affordable monitoring of food contaminants requires the highest-through put and more economical methods of detection and, possibly, little or no sample treatment, userfriendliness, employment of non-hazardous chemicals, in situ applicability. Additional requisites in aflatoxin detection would be low detection limits (especially for aflatoxin M1) and adaptability to very differing commodities (for aflatoxins B and G).

Several innovative strategies have been proposed for the rapid, qualitative, semi-quantita‐ tive or quantitative detection of aflatoxins, also based on the use of specific antibodies with‐ out constraints of classical immunoassays [22]. For example, an interesting qualitative approach has been described for the detection of AFM1 in milk [23-24]. The proposed meth‐ od is based on a flow-through immunoassay with visual detection. Main advantages are represented by the high sensitivity and by the on site applicability of the assay which does not require any equipment for the treatment of the sample, norfor the analysis. In addition, it allowed the possibility of obtaining sample pre-concentration and/or clean-up in the same device used for the analysis [25]. Nevertheless, this method implies several subsequent steps to be carried out, thus limiting simplicity and rapidness of use. Very recently, the same ap‐ proach has also been demonstrated for the multi-detection of different mycotoxins, thus in‐ creasing its potentiality of utilization [26].

Numerous immunosensors have been described [27] as well, and research is constantly evolving in this area, particularly for the development immunosensors for the selective de‐ termination of AFB1 [28-32] and for AFM1 detection [33-35].

In parallel, strategies aimed at avoiding the use of antibodies in the development of rapid methods for aflatoxin detection have also been reported, such as those based on the prepara‐ tion of polymers with molecular recognition properties towards AFB1as capture systems [36-37] or those based on the exploitation of its natural fluorescence for the detection [38]. A combination of the surface plasmon resonance phenomenon and fluorescence has been ex‐ ploited in the work of Wang et al and permitteda very sensitive determination of AFM1, though the proposed assay took almost an hour to be accomplished and couldn't be consid‐ ered as a truly rapid method [39]. A fancy and cunning approach for the rapid quantifica‐ tion of AFB1 have been described in the work of Arduini et al, who exploited the inhibiting effect of the toxin towards the enzyme acetylcholinesterase. The measurement of the enzy‐ matic activity was demonstrated to directly allow AFB1 quantification in 3 minutes and within the 10-60 µg l-1 range [40].

Among the rapid methods for screening of food contaminants, the'lateral flow immunoas‐ say" (LFIA) (also known as immunochromato graphic assayorimmuno-colloid gold immu‐ noassay, ICG) has recently attracted the interest of researchers and industry. This technology has long been known in medical fields for diagnosing blood infections and fail‐ ure of internal organs, disclosing drug abuse or ascertaining pregnancy and combines a ser‐ ies of benefits, including extreme simplicity, rapidity, and cost effectiveness [41]. These features make it ideally suited for screening large number of samples, for being conducted by non-trained personnel and practically everywhere, thus enabling the effective possibility of food safety assessment at all stages of food and feed production.
