Achievements and Prospects in Electrochemical-Based Biosensing Platforms for Aflatoxin M1 Detection in Milk and Dairy Products

Aflatoxins, which are mainly produced by Aspergillus flavus and parasiticus growing on plants and products stored under inappropriate conditions, represent the most studied group of mycotoxins. Contamination of human and animal milk with aflatoxin M1, the hydroxylated metabolite of aflatoxin B1, is an important health risk factor due to its carcinogenicity and mutagenicity. Due to the low concentration of this aflatoxin in milk and milk products, the analytical methods used for its quantification have to be highly sensitive, specific and simple. This paper presents an overview of the analytical methods, especially of the electrochemical immunosensors and aptasensors, used for determination of aflatoxin M1.


Occurrence and Toxicity
Mycotoxins represent natural substances which occur as secondary product of the development of parasitic fungi, such as Aspergillus, Fusarium, Penicillium, Claviceps and Alternania genus in plants and products stored and then used for human and animal nutrition [1,2]. Usually, Aspergillus and Penicillium species grow on food and feeds during drying and storage conditions. Infections of crops with Aspergillus species can also occur in the field, during growing season, while Fusarium species represent destructive plant pathogens, producing mycotoxins in growing crops (corn, wheat and barley), before or post-harvesting [1].
Most of the mycotoxins are cytotoxic, producing a breakdown of cell membranes and preventing or influencing the DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and protein synthesis, which poses significant risks to food safety [3]. Usually, they act by inhibition of protein synthesis at the ribosomes in cell, therefore cell division being inhibited too [4].
Contamination with these toxins is still considered unavoidable and unpredictable, even when good agricultural, storage and processing conditions are implemented. In order, to prevent the contamination of the crops, food and forage with aflatoxins, different strategies have been addressed. Chen et al. have reported the development of host resistance to aflatoxin producers, especially for maize crops [5].
Most of the time, human exposure to mycotoxins takes place through dermal contact, inhalation and ingestion. Mycotoxin ingestion may occur by eating contaminated food, direct via cereals or indirectly via animal products (e.g., eggs, milk and meat) [6]. Many mycotoxins present high A number of factors such as caloric deprivation, vitamin deficiency, alcohol excess and infectious diseases status strongly influences the severity of mycotoxin contamination in humans [3].
Nowadays, there are more than 300 substances named mycotoxins, but it is not only difficult to define them, they are also difficult to classify. Usually, the classification criteria tend to follow the specialization of the person who is doing this. For example, chemists classify them by their chemical structure (e.g., coumarins, lactones, etc.), biochemists by their biosynthetic origins, clinicians by the affected organ (hepatotoxins, nephrotoxins, neurotoxins and immunotoxins), mycologists according to the fungi that produce them (e.g., Aspergillus toxins, Penicillium toxins, etc.) and not last, cell biologists classify them in four generic groups: carcinogens, teratogens, mutagens and allergens [3,7,8]. The International Agency for Research on Cancer (IARC) has defined four groups for classification of mycotoxins as carcinogenic or potentially carcinogenic to humans [15], as follows: Food and Agriculture Organization (FAO) are responsible for the evaluation of mycotoxin-related contamination [3]. In the European Union (EU), the contamination of food with several mycotoxins is evaluated by European Food Safety Authority (EFSA) which has established maximum allowed limits for aflatoxins (AFs), ochratoxin A (OTA) and patulin (PAT) (EU regulation 466/2001) (European Commission, 2001) [21]. This regulation underwent several updates and was replaced in 2006 by EU regulation No. 1881/2006 [22], further updated in 2007 and 2010 by EU regulations No. 1126/2007and No. 165/2010 [23,24]. Table 2 highlights the most important mycotoxins, the fungi that produce them, the group of their carcinogenicity, as well as the contaminated food and feedstuffs and the maximum admissible limits (MLs) for these compounds given by US-FDA and EU(EC2006) [25]. Contamination by mycotoxins is considerably influenced by several environmental factors, the geographic position and seasonal factors, such as temperature, humidity, pH and oxygen concentration, the same factors that affect the growth of toxic fungi. Thus, cultivation, harvesting, storage and the transport conditions of the crops and cereals are strongly related with level of mycotoxin contamination [3,9,19].
The chemical control of fungal growth and of mycotoxin biosynthesis in stored grains or other production stages can be achieved by using fumigation with oxidized ethylene, bromomethane [27] and 0.2 to 0.4% of ammonia [28]; insecticide treatments to prevent grain injury through facilitating its infection with toxigenic fungi and fungicidal treatments using some "natural" fungicides (e.g., chitosan). Good control for the mycotoxigenic fungi has been achieved under laboratory conditions using plant product (e.g., essential oils and extracts) as environmentally-friendly fungicides [29][30][31]. The use of some bacteria like Bacillus sp., propionic acid bacteria and lactic acid bacteria, seems to be a new opportunity for biological control of fungal growth and production of mycotoxins [32].
Great concern has been raised by the use of aflatoxins, satratoxins, trichothecene and fusarium toxins as biological warfare agents, since they are cheap and easy to access [33,34]. Aflatoxins have been used as biological weapon by Iraq in so-called "cancer bombs" [35]. Many countries considered that these mycotoxins have biothreat potential because of their effects over the nervous system function (the effects are temporary and/or reversible), inducing also the damage of the cell membranes (the effects are not reversible), therefore sensitive methods for their detection and monitoring being absolutely necessary [36].
Aflatoxin M1 represents the principal hydroxylated metabolite of AFB 1 , which can be detected in animal tissues and fluids (urine and milk). In the milk of mammals, aflatoxin M 1 can be detected at 12 to 24 h after ingestion of contaminated food or feed with AFB 1 , its concentration which occur in excreted milk being correlated with the AFB 1 levels found in the raw feedstuffs [40]. Contamination with AFM 1 occurred also in dairy products from cow milk, especially in cheese, even at higher concentration than those found in raw milk. This is possible due to the stability of AFM 1 towards the heating treatment involved in milk processing, binding well to casein [25,40].
The aflatoxins were named B and G based on their fluorescence colors under ultraviolet light (UV, 365 nm), AFB 1 and AFB 2 produce blue color, whereas AFG 1 and AFG 2 green color. They are difurano-coumarins derivatives, with low molecular weight which are soluble in solvents, such as methanol, chloroform and acetonitrile, having a wide spectrum of toxicity. Their chemical structures are drawn in Figure 1. Aflatoxins are unstable to UV light, but highly stable to thermal treatments (e.g., baking, pasteurization, roasting) [41]. These mycotoxins can induce some types of cancer, hypoglycemia and elevated serum transaminase levels, being considered that aflatoxins are lipophilic molecules which are transported by blood stream and deposited in the hepatocytes [42].
After ingestion, the biodegradation of AFB1 takes place in liver, in an enzymatic system involving the hepatic microsomal cytochrome P450. The hydroxylated-AFB1 metabolite resulting from oxidation and demethylation of AFB1 is considered to be highly toxic, with mutagenic and Aflatoxins are unstable to UV light, but highly stable to thermal treatments (e.g., baking, pasteurization, roasting) [41]. These mycotoxins can induce some types of cancer, hypoglycemia and elevated serum transaminase levels, being considered that aflatoxins are lipophilic molecules which are transported by blood stream and deposited in the hepatocytes [42].
After ingestion, the biodegradation of AFB 1 takes place in liver, in an enzymatic system involving the hepatic microsomal cytochrome P450. The hydroxylated-AFB 1 metabolite resulting from oxidation and demethylation of AFB 1 is considered to be highly toxic, with mutagenic and carcinogenic effects [3,41,42]. A schematic representation of the aflatoxin B 1 metabolism and the adduct formation are shown in Figure 2.
The epoxide form of aflatoxin B 1 (AFBO), usually binds to proteins, to DNA and RNA, at the guanine base position in liver cells, modifying the genetic code that ensure the cell growth in the most active tissues (liver, intestine and bone marrow), and therefore leading to the appearance of genetic mutations and further to cancer [18,43].
The reduction of aflatoxin B 1 by cytosolic reductase leads to formation of the aflatoxicol, a detoxification product which can be re-oxidized back to aflatoxin B 1 by a microsomal dehydrogenase, increasing in this way the half-life physiological of AFB 1 [44].
Other naturally occurring aflatoxins and their hydroxylated metabolites are not involved in the epoxidation reactions, and thus are considered to be less mutagenic and carcinogenic. Some of these metabolites are excreted in the urine of the contaminated individuals, this being used as an indicator for the individual exposure to aflatoxin B 1 [18].  Up to now, the aflatoxin B1, found in high concentration in contaminated food and animal feed, is considered to be the most toxic compound among the aflatoxins. The level and duration of exposure to AFB1 are determinant factors for the toxic effects of this mycotoxin. Its lethal dose (LD50) for most species is considered to range from 1 to 50 mg/kg, with a critical toxicity level of less than 1 mg/kg for some highly susceptible species, such as poultry, rainbow trout and rats [45].
In humans, consumption of aflatoxin-contaminated food has been linked to different diseases, such as liver cancer, encephalopathy, pulmonary interstitial fibrosis, and effects on the reproductive and immune systems [2]. The incidence of hepatocellular carcinoma is directly related to aflatoxin Up to now, the aflatoxin B 1 , found in high concentration in contaminated food and animal feed, is considered to be the most toxic compound among the aflatoxins. The level and duration of exposure to AFB 1 are determinant factors for the toxic effects of this mycotoxin. Its lethal dose (LD 50 ) for most species is considered to range from 1 to 50 mg/kg, with a critical toxicity level of less than 1 mg/kg for some highly susceptible species, such as poultry, rainbow trout and rats [45].
In humans, consumption of aflatoxin-contaminated food has been linked to different diseases, such as liver cancer, encephalopathy, pulmonary interstitial fibrosis, and effects on the reproductive and immune systems [2]. The incidence of hepatocellular carcinoma is directly related to aflatoxin consumption in diet, however, a quantification of the lifetime individual exposure is very difficult to predict. Liver cancer represents the most common type of cancer in Thailand, China, Philippines and many African countries, the incidence of this disease variating from one country to another [3,46,47].

Aflatoxin M 1
Aflatoxin M 1 (AFM 1 ) represents the principal hydroxylated metabolite of AFB 1 , biotransformed in the liver and excreted in the milk produced by mammary glands of lactating humans and animals, feed with AFB 1 -contaminated food [45]. Due to the high stability of AFM 1 towards milk processing technologies, such as pasteurization and ultra-high temperature heating (UHT), and to other dairy product processing methods, this mycotoxin can be found not only in milk, but also in dairy products, usually at higher concentration than that found in raw milk [48,49].
Since milk, containing proteins, vitamins, minerals and fatty acids, is the most common nutrient, especially for infants and children, the presence of AFM 1 in milk and dairy products represents an important health risk factor. The occurrence of AFM 1 in human breast milk, milk and dairy products is of real public health concern, especially for infants and young children. It is considered that infants are more exposed to AFM 1 contamination by breast milk intake than that using infant formula [50]. Thus, a maximum level of AFM 1 in infant milk formula was set at 0.025 µg/kg by European Commission Regulation (EU 165/2010) [24], but the presence of AFM 1 in human breast milk is not yet limited.
It has been observed that AFM 1 can be detected in milk of lactating mammals after 12 h, with a peak of the concentration to 24 h from ingestion of AFB 1 -contaminated food. During 72 h from stopping the intake of AFB 1 -contaminated food, it has been observed a decreasing of the AFM 1 concentration to undetectable levels [51].
Due to its semi-polar characteristics, the AFM 1 binds strongly to casein, a phosphoprotein found in milk. Inactivation of AFM 1 is difficult to achieve, being observed that the milk processing induced dramatic effects on AFM 1 concentration [52].
Even if AFM 1 is considered to be less mutagenic and carcinogenic than AFB 1 , its cytotoxicity has been studied in vitro using human liver microsomes and human cell line expressing or not expressing human cytochrome P450 enzymes. These experiments demonstrated a high toxic potential of AFM 1 in the absence of metabolic activation, compared to AFB 1 [53]. Thus, taking into consideration the toxic effect of AFM 1 , through DNA damage inducing gene mutation, chromosomal anomalies and cell transformation [40], the International Agency for Research on Cancer has changed the classification of this aflatoxin in 2002 from Group 2B to Group 1 [15].
As a result of all this, different analytical techniques for the detection and quantification of aflatoxins have been developed and reported in the literature, such as chromatography, UV-absorption, spectrometry, fluorescence and immunochemical assays. The choice of the analytical method must take into account different aspects for detection of these mycotoxins, such as target molecule, complex matrix, chemical characteristics, time of analysis and that limits of detection or/and quantification must be below the specific regulatory limits.
Further, this review will highlight the strengths and weakness of the different analytical methods developed and reported over the last period for AFM 1 detection and quantification in milk and dairy products as a need for food safety monitoring and control.

Detection of AFM 1
As was specified previously, the aflatoxin M 1 presents high thermal stability to milk processing by sterilization, pasteurization or freezing. Therefore, the ingestion of milk and dairy products, the essential components of human diets, represents the first route of contamination with AFM 1 . The occurrence of AFM 1 in milk and milk products take place in the range of ng/g and an early detection of this metabolite could be a relevant indicator of the risk factor to human health. Thus, there is still a continuous need for more sensitive, feasible, fast and affordable analytical methods for its detection and quantification.
Usually, most of the analytical methods require several steps prior analysis, such as extraction of AFM 1 from its source, purification by removing other interference substances and quantification. Aflatoxins are soluble in organic polar solvents, such as methanol, acetonitrile, chloroform and acetone in different proportions, but in the case of immunoassay techniques special attention should be paid to the solvent used for extraction. A methanol-water mixture is preferred instead of acetone and acetonitrile, due to its less negative effect on antibodies [54].
Following the extraction, the purification and concentration of aflatoxin before its determination is performed by a clean-up step. The most common clean-up procedures used in aflatoxin analysis are solid phase extraction (SPE) and immunoaffinity column chromatography (IAC). Immunoaffinity chromatography involves high specificity and reversibility of the antibody-antigen binding and provides high selectivity and efficiency in separation and purification of the target analyte from complex matrices [55].

Conventional Methods for Aflatoxin M 1 Detection
Generally, the most common methods used for monitoring and detection of aflatoxins are: chromatographic and immunochemical methods, such as thin layer chromatography (TLC) [62], high-performance liquid chromatography (HPLC) [63], as well as enzyme-linked immunosorbent assay (ELISA) [56], sequential injection immunoassay and radioimmunoassay [64]. Immunochemical methods are based on specific antibodies and can be used with a good sensitivity for rapid screening of aflatoxins. Chromatographic methods are used for confirmation of the results obtained by rapid tests of screening, as well as for sensitive detection of the aflatoxins.

Chromatographic Methods
Chromatography encompasses the most widely used techniques for separating a large number of analytes. These techniques are based on the physical interaction between a mobile phase and a stationary phase. As mobile phase liquid, gas or supercritical fluids can be used, hence the name of the corresponding chromatographic methods. Thin layer chromatography, HPLC and liquid chromatography coupled with different detectors are the main methods employed for analysis of mycotoxins [65]. Coupling these chromatographic methods with fluorescence or mass spectrometry detection allows for the sensitive determination of aflatoxin M 1 in milk, cheese or other dairy products. Table 3 summarizes reported techniques coupled with different detectors used for AFM 1 quantification.

Thin Layer Chromatography (TLC)
TLC is a traditional method used for the separation and determination of aflatoxins, reported for the first time by de Iongh et al. [66]. Using TLC, it was possible to determine several types of mycotoxins (e.g., aflatoxins, ochratoxins, patulin, tremorgenic toxins, zearalenone, citrinin, sterigmatocystins, versicolorins, etc.) in one sample, aflatoxins being detected in a range as low as 1 to 20 ppb [67,68]. Usually, the quantification of AFM 1 is performed by its UV fluorescence, using an excitation at 360 nm and emission at 435 nm [67]. Even if this method is simple, fast and sensitive, it requires skilled personal, extensive sample pretreatment and expensive equipment [67,69]. Therefore, thin layer chromatography is no longer useful for the detection of AFM 1 , since its performance only allows determination at contamination levels too high or around the current regulatory limits of 0.05 ppb AFM 1 [70].
High-Performance Liquid Chromatography (HPLC) HPLC has been in under continuous development since the 1960s, being the most reported technique used for assessment of the aflatoxin status in contaminated food. HPLC coupled with different detectors, such as fluorescence, UV-Vis absorption or mass spectrometry represents the standard method used for quantification of the aflatoxin M 1 in milk (liquid or powdered) and milk products [71,72].
Reversed-phase C-18 HPLC columns with fluorescence detection are commonly used for AFM 1 determination, the fluorometric excitation and emission wavelengths being 360 and 435 nm, respectively [72,73]. Depending on the complexity of the matrix and in order to enhance the sensitivity for fluorescence determination of aflatoxins, usually a chemical derivatization is performed [73]. Since AFM 1 is a naturally fluorescent compound, with an unsaturated furan ring, it can be either pre-column or post-column derivatized. An increase of the AFM 1 sensitivity has been obtained by Chiavaro et al., by adding cyclodextrin to the methanol-water mobile phase. The authors managed to lower the detection limit for AFM 1 to 0.0005 µg·kg −1 , compared to detection limits (0.005-0.025 µg·kg −1 ) achieved by using official methods with pre-column derivatization and trifluoracetic acid [74].
However, these stages of derivatization have several drawbacks, including the use of toxic solvents, are time consuming due to the solvent evaporation, have limited stability and require daily maintenance.
Thus, to overcome the disadvantages of derivatization processes, HPLC was coupled with mass spectrometry, resulting efficient systems as HPLC-MS or HPLC-MS/MS for AFM 1 detection. These methods use small amounts of sample for generating structural information and exhibit lower detection limits [75]. HPLC chromatographic techniques coupled with mass spectrometry are specific and selective are able to identify molecules by means of fragmentation patterns of spectral mass, sometimes involving just a single liquid extraction without any clean-up step.
However, all chromatographic methods are time consuming, expensive, requiring several complex sample pre-treatments steps and specialized personnel. These methods are suitable only for laboratory applications and not for in situ determination of AFM 1 , because usually the milk industries and dairy farms require a real-time and cheap monitoring of AFM 1 in their products. 15 Flow-injection immunoassay 11 (raw milk) Amperometric detection [98] 16 Direct chemiluminescent enzyme immunoassay 1 (milk) Sensitivity improved by using 3-(10 -phenothiazinyl)-propane-1-sulfonate and 4-morpholinopyridine [99] 17 Immunochip 240 (added in drinking water) indirect competitive immunoassay [100] Fluorescence Spectrophotometric Methods Since aflatoxins are fluorescent, the absorption process is followed by the emission of light under different wavelengths. Fluorescence is an important characteristic in the analysis of some molecules that emit energy at a specific wavelength, and therefore has been used for the determination of aflatoxins in animal feed and food grains [101]. Aflatoxins can be quantified in a range from 5 to 5000 ppb using spectrofluorometric methods in a short time, but the detection limit is quite higher than 4 µg/kg, the maximum limit set by the European Committee for total content of aflatoxins.

Immunochemical Methods
Since the 1970s, the development of immunochemical methods for determination of aflatoxins has appeared as a solution to all the limitations of chromatographic and spectrophotometric methods. These methods, based on the specificity of antibody-antigen binding, are simple, sensitive, fast, less laborious and do not require highly trained personnel. Different immunochemical techniques were developed based not only on the high affinity and specificity of the antibodies for antigens, but rather using the affinity and specificity between receptors and ligands, too [101]. Usually, the enzymes, fluorophores and radioisotopes are used as labels for amplification of the signal recognition. Quantification of the antibody-antigen or receptor-ligand complexes formation is performed in correlation with the change in the absorbance of photons of light energy spectrophotometrically [102].
The use of aflatoxin-specific antibodies to form complexes with corresponding antigens has been applied for determination of aflatoxins, the high affinity and specificity of the antibody-antigen interaction leading to a high sensitivity and selectivity of the related assay [97]. Depending on the type of production, the antibodies can be divided into polyclonal (pAb), monoclonal (mAb) and recombinant (rAb) antibodies, but the monoclonal antibodies are the most frequent used for aflatoxin assays [103].
ELISA represents the most common method used for rapid screening of aflatoxins in medical diagnostic laboratories and research institutions. There are numerous commercially available ELISA test kits based on competitive enzyme immunoassay using alkaline phosphatase and horseradish peroxidase as labels [69,101,103]. In the last period, many publications have reported the analysis of aflatoxin M 1 in different samples of milk (breast, cow, goat, etc.) using ELISA kit tests for screening of AFM 1 , with confirmation by the official HPLC method [52,64,91,92,105,106].
The ELISA technique is cheap, simple and sensitive, being suitable for a large number of samples at the same time. However, it is still laborious, requiring multiple washing steps and long incubation time, thus, it is a time-consuming method.
Immunostrips or immunodipsticks have been developed for the rapid screening method of the low-molecular weight AFM 1 . The antibodies in this case are conjugated with gold nanoparticles which provide a visual red colored of binding zone [107][108][109][110][111]. The principle of the assay is based on the specific and sensitive interaction between liquid test sample containing aflatoxin and antibody-gold nanoparticle conjugates along membrane strips (Figure 3). The sample suspends the gold nanoparticles and aflatoxin binds to these particles coloring in red the binding-line. This method is simple and efficient for on-site detection of AFM1 in milk samples, providing results in less than 10 min. The main disadvantage of these immunostrips is that each one can only be used for a single sample and large amounts of AFM1-conjugates are required for each assay, increasing in this way the assay cost [111].

Immunosensors
Immunosensors represent another direction that has grown in the last decades in development of sensitive, selective, simple and reliable systems for aflatoxins detection. Different biosensors based on optical (surface plasmon resonance, chemiluminescence, evanescent wave-based fiber optics) [112], surface acoustic wave (quartz crystal microbalance) [90], piezoelectric and electrochemical principles have been reported for AFM1 detection [112,113]. For the development of an immunosensor usually an antigen or an antibody coupled with a physical transducer such as gold, carbon or graphite is used as biological recognition element, which allows the detection of the binding species [101].

Electrochemiluminescence
Electrochemiluminescent immunoassay (ECLIA) represents another highly sensitive and selective technique combining the analytical advantages of electrochemiluminescence, such as sensitivity, absence of the background optical signal and a facile control by changing the electrode potential with the specificity of the immunoassay. By using this technique, it is possible to detect ultratrace amounts of AFM1 in food. For the development of sensitive electrochemiluminescent immunoassay sensors, it is very important to define a signal tag labeled with the AFM1 antibody [104]. For this purpose, Ru(bpy)3 2+ and luminol, as well as strongly luminescent semiconductor quantum dots (CdS, PbS, CdTe and ZnS) QDs have been extensively used for applications in biological imaging and labeling, having excellent optoelectronic properties [114,115]. It has been reported that the electrochemiluminescent signal was considerably amplified by using carbon nanomaterials, more specifically, carbon nanotubes (CNT). The surface of hybrid particles (QDs-CNT) can be labeled with attached antibodies as tags [93]. Thus, monoclonal antibodies of AFM1 have been immobilized on CdTe QDs-CNT composites for the development of ECLIA-based biosensors. Graphene oxide was chosen as absorbent material for AFM1 and further to conjugate with AFM1-monoclonal antibody/CdT QD-CNT, in order to form a sandwich immunocomplex GO/AFM1/AFM1-Ab/CdTeQD-CNT, which can generate an electrochemiluminescence signal on the electrode. Graphene oxide can be used also as absorbent material for other aromatic organic compounds, but the signal tag labeled with AFM1-Ab can react only with absorbed AFM1, thus the electrochemiluminescent signal will reflect only the amount of AFM1 absorbed onto graphene oxide [116]. This method is simple and efficient for on-site detection of AFM 1 in milk samples, providing results in less than 10 min. The main disadvantage of these immunostrips is that each one can only be used for a single sample and large amounts of AFM 1 -conjugates are required for each assay, increasing in this way the assay cost [111].

Immunosensors
Immunosensors represent another direction that has grown in the last decades in development of sensitive, selective, simple and reliable systems for aflatoxins detection. Different biosensors based on optical (surface plasmon resonance, chemiluminescence, evanescent wave-based fiber optics) [112], surface acoustic wave (quartz crystal microbalance) [90], piezoelectric and electrochemical principles have been reported for AFM 1 detection [112,113]. For the development of an immunosensor usually an antigen or an antibody coupled with a physical transducer such as gold, carbon or graphite is used as biological recognition element, which allows the detection of the binding species [101].

Electrochemiluminescence
Electrochemiluminescent immunoassay (ECLIA) represents another highly sensitive and selective technique combining the analytical advantages of electrochemiluminescence, such as sensitivity, absence of the background optical signal and a facile control by changing the electrode potential with the specificity of the immunoassay. By using this technique, it is possible to detect ultratrace amounts of AFM 1 in food. For the development of sensitive electrochemiluminescent immunoassay sensors, it is very important to define a signal tag labeled with the AFM 1 antibody [104]. For this purpose, Ru(bpy) 3 2+ and luminol, as well as strongly luminescent semiconductor quantum dots (CdS, PbS, CdTe and ZnS) QDs have been extensively used for applications in biological imaging and labeling, having excellent optoelectronic properties [114,115]. It has been reported that the electrochemiluminescent signal was considerably amplified by using carbon nanomaterials, more specifically, carbon nanotubes (CNT). The surface of hybrid particles (QDs-CNT) can be labeled with attached antibodies as tags [93]. Thus, monoclonal antibodies of AFM 1 have been immobilized on CdTe QDs-CNT composites for the development of ECLIA-based biosensors. Graphene oxide was chosen as absorbent material for AFM 1 and further to conjugate with AFM 1 -monoclonal antibody/CdT QD-CNT, in order to form a sandwich immunocomplex GO/AFM 1 /AFM 1 -Ab/CdTeQD-CNT, which can generate an electrochemiluminescence signal on the electrode. Graphene oxide can be used also as absorbent material for other aromatic organic compounds, but the signal tag labeled with AFM 1 -Ab can react only with absorbed AFM 1 , thus the electrochemiluminescent signal will reflect only the amount of AFM 1 absorbed onto graphene oxide [116].
Gan and co-authors have reported an electrochemiluminescent immunoassay for AFM 1 detection in milk, by using magnetic Fe 3 O 4 -graphene oxide as absorbent for AFM 1 and AFM 1 antibody-labeled CdTe quantum dots as the signal tag based on a carbon screen-printed electrode. The authors reported a great enhancement of the electrochemiluminescent signal by using this immunocomplex, detection of AFM 1 from milk samples being possible with a detection limit of 0.3 pg/mL [93].

Electrochemical Immunosensors
In the development of electrochemical immunosensors devoted to AFM 1 detection different types of bioreceptors on sensor platforms have been used, including antibodies, nucleic acids, protein ligands, prokaryotic and eukaryotic living cells and aptamers. Several electrochemical detection methods, such as amperometry, potentiometry and conductometry have been used in designing AFM 1 -based immunosensors, the most commonly used being amperometric methods. The good sensitivity, the reduced cost and the possibility of miniaturization of the developed amperometric immunosensors have been reported [117]. In designing electrochemical immunosensors, different electrodes materials were used as sensor platforms, the most commonly encountered materials being platinum, gold and various forms of carbon [118].
The possibilities of mass fabrication, low cost and single drop assays have attracted increasing interest in screen-printed technology. Also, the miniaturization of electrochemical sensor platforms leads to considerable lower sample consumption, and moreover, the combination of the electrochemical detection methods with the progress in sensor technologies makes these electrochemical immunosensors suitable for integration in point-of-care and portable devices as well as for in situ applications [119].
Using electrochemical immunosensors, the detection of aflatoxins can be performed in a direct competitive assay, in which a specific antibody is immobilized on the electrochemical transducer and the competition is carried out between the labelled and non-labelled analytes to bind with the specific antibody.
Thus, an amperometric disposable immunosensor have been designed Micheli et al. for AFM 1 detection in raw milk, based on immobilization of a monoclonal antibody on a carbon screen-printed electrode (SPE) [97]. A direct competition between free AFM 1 and it's conjugate with peroxidase (AFM 1 -HRP) for the specific antibody was allowed to occur. 3,3 ,5,5 -Tetramethyl-benzidine was used as enzymatic substrate for evaluation of the AFM 1 -HRP amount which reacted with the immobilized antibody, and electrochemical detection of the electroactive product was performed by chronoamperometric measurements at −0.1 V. A detection limit of 25 ppt for AFM 1 was obtained using the disposable immunosensor, working in a range from 30 to 160 ppt. The authors proved that using electrochemical detection a better detection limit and shorter analysis time could be achieved [97].
Another immunosensor based on an antibody-modified carbon screen-printed electrode has been reported by Parker and Tothill, using a competitive ELISA assay constructed at the surface of a carbon paste electrode [109]. Using the competition between free AFM 1 from samples and an AFM 1 -horseradish peroxidase conjugate for a monoclonal AFM 1 -antibody and electrochemical detection based on 3,3 ,5,5 -tetramethylbenzidine (TMB)/hydrogen peroxide (H 2 O 2 ) for HRP, the authors obtained a limit of AFM 1 detection of 39 ng·L −1 , with a dynamic range up to 1000 ng·L −1 . The developed immunosensor was comparable in term of sensitivity with the common methods (ELISA, HPLC), but presented superior characteristics in terms of portability and cost [109].
An automated flow-injection immunoassay system has been developed for determination of AFM 1 in raw milk samples by Badea et al. [98]. This system is characterized by an off-line incubation of a mixture containing the antigen (AFM 1 ), fixed amounts of specific antibody to AFM 1 (anti-AFM 1 ) and peroxidase marked AFM 1 (AFM 1 -HRP) until the equilibrium was reached, and then injected into the flow system. A column containing Protein G was used for separation of the free tracer and of the antibody-conjugate, while antibody-antigen complex was retained in the column due to the high affinity of the Protein G for the constant region of immunoglobulins. The activity of the enzymatic label horseradish peroxidase (HRP) has been evaluated by amperometric measurements using 3,3',5,5'-tetramethylbenzidine. The flow-injection immunoassay system showed good reproducibility and short time of analysis, with low cost instrumentation, being easy to operate and the results being comparable with those obtained by HPLC [98].
A competitive immunoassay was used for development of an electrochemical sensor for detection of AFM 1 , based on magnetic nanoparticles (MNPs) coated with anti-AFM 1 antibody [120]. The samples containing AFM 1 were incubated with fixed amount of MNP-Ab and AFM 1 -HRP conjugate until equilibrium was reached, and afterwards the mixture was deposited onto the surface of screen-printed electrodes. The enzymatic response was amperometrically determined using 5-methylphenazinium methyl sulphate as mediator, the detection limit achieved using this immunosensor being 0.01 ppb. The system allowed determination of AFM 1 directly in milk, after a simple centrifugation step, without any dilution or pretreatment steps, the analysis time being considerably reduced [120].
Dinckaya et al. have used a DNA biosensor for AFM 1 detection in milk and dairy products, based on immobilization of a thiol-modified single strained DNA (ss-HSDNA) that bound specifically AFM 1 , using a self-assembled monolayer of cycotiamine and gold nanoparticles prepared onto a gold electrode [104]. The specific binding of AFM 1 to ss-HSDNA has been studied by cyclic voltammetry and electrochemical impedance spectroscopy (EIS), and using this biosensor the detection of AFM 1 was possible to be performed in a linear range of 1 to 14 ng·mL −1 [104].
Another impedimetric immunosensor for detection of AFM 1 in milk has been developed by Bacher et al. [121]. This immunosensor is based on functionalization of a silver (Ag) wire electrode with selective monoclonal antibody of AFM 1 using a self-assembled monolayer (SAM) of 11-marcaptoundecanoic acid (11-MUA). Electrochemical impedance spectroscopy was used for analyzing the electrical properties of the modified electrode, when an antibody coupled to the electrode reacts with its specific antigen. It was shown that the applied potential strongly influences the antibody-antigen interaction. The limit of detection obtained using this impedimetric immunosensor was 1 pg/mL, with short time of analysis of about 20 min, while the sensitivity was about 2.1% impedance change per decade. The authors reported a period of use of the bio-functionalized silver-wire sensor for up to two weeks [121].
Biosensors based on cells for detection of estrogenic toxins represent another important direction in analytical science evolution due to the high sensitivity, fast rate detection, low cost and the possibility of one target analyte detection [122,123]. Usually, for development of such biosensors, viable whole cells which are able to recognize a particular analyte or a group of analytes are recruited as sensing element. In this sense, bacteria, yeast or eukaryotic cells, including vertebrate or mammalian cells, can be used for development of cells-based biosensors [122]. The use of a genetically modified Saccharomyces cerevisiae strain for the detection of estrogenic mycotoxin residues in milk was reported by Valimaa et al. [123].
Larou et al. have developed a biosensor based on mammalian cells containing membranes engineered by artificial electro-insertion of AFM 1 -specific antibodies [124]. This biosensor provides an electric response of the membrane-engineered fibroblast cells suspended in an alginate gel matrix, due to the change of their membrane potential after the interaction between AFM 1 and its specific antibodies. Thus, detection of AFM 1 at concentrations as low as 5 ppt in just 3 min was possible, the assay being selective for AFB 1 and OTA [124].
Recently, the development of aptamer-based biosensors for mycotoxin detection has received considerable attention, having several advantages such as low cost, high stability and sensitivity, and the fact they can be easily synthetized and modified compared to antibodies. Aptamers are functional short oligonucleotides, reported for the first time in 1990 [125,126], selected in vitro from combinatorial libraries, which can bind with high affinity and specificity to a wide range of target molecules (proteins, toxins, drugs, organic or inorganic molecules, etc.) [127]. The process of in vitro selection by which these oligonucleotide ligands are obtained is called Systematic Evolution of Ligands by Exponential enrichment (SELEX) [127,128]. The high specificity of the aptamers is a result of the selective step in the SELEX process called "counter-SELEX" that eliminates the sequences that also bind to closely-related analogs of the target [127]. Malhotra and co-authors reported for the first time a specific aptamer to AFM 1 having a dissociation constant (Kd) of about 35 nmol·L −1 [129].
Detection of AFM 1 can be performed with aptasensors using electrochemical and impedance spectroscopy detection [94,128,130]. An impedimetric aptasensor has been designed by Istamboulier and co-authors for determination of AFM 1 in milk based on DNA-aptamer recognition element and electrochemical impedance spectroscopy detection [130]. The AFM 1 -aptamer (a 21-mer DNA oligonucleotide) was covalently immobilized on the surface of carbon screen-printed electrodes through carbodiimide immobilization procedure, after a previous activation of the electrode surface with diazonium salt. The interaction between aptamer and AFM 1 induced an increase of the electron transfer resistance at the electrode surface, allowing in this way determination of AFM 1 with a detection limit of 1.15 ng·L −1 . A simple preliminary treatment of the milk samples was carried out, by filtration through a 0.2 µm polytetrafluoroethylene (PTFE) membrane of the mixture containing milk sample, methanol and binding buffer [130].
Another aptasensor for AFM 1 detection was designed by Guo et al. using the interaction between a specific aptamer to AFM 1 with biotin-streptavidin and its complementary ssDNA as template for a real-time quantitative polymerase chain reaction amplification [94]. This aptasensor has been used for determination of AFM 1 in infant rice cereals and infant milk powder samples, showing a high selectivity to AFM 1 over other aflatoxins, the detection limit obtained being 0.03 ng·L −1 [94].
Nguyen and co-authors attempted to improve the sensitivity of AFM 1 detection by using covalent immobilization of specific aptamers on COOH-functionalized magnetic nanoparticles [128]. The magnetic nanoparticles incorporated in polyaniline film were polymerized on the surface of an interdigitated electrode as sensitive film for an AFM 1 -based electrochemical biosensor. Direct detection of AFM 1 was performed at the Fe 3 O 4 /polyaniline interface by cyclic and square wave voltammetry, with good sensitivity and a detection limit of 1.98 ng·L −1 . The developed aptasensor allowed the detection of AFM 1 below the legislative set limits, with several advantages over other common analytical methods, such as sensitivity, stability, label free format, low analysis time and cost effectiveness [128].

Optical Immunosensors
Optical Waveguide Light mode Spectroscopy based on amino functionalized integrated optical waveguide sensors was used for quantitative determination of AFM 1 in milk samples [131]. The covalent immobilization of AFM 1 -HRP conjugate was carried out on the surface of the amino functionalized SiO 2 -TiO 2 based sensor using glutaraldehyde. The specific antibody to AFM1 was added in the sample and further measured by the immobilized antigen. For regeneration of the sensor surface was used HCl 10 mM, acting for dissociation of the immunocomplexes. Milk samples were analyzed using three different types of pre-sampling preparation (filtration, centrifugation and size exclusion centrifugation), AFM 1 being determined in a dynamic range from 0.001 to 0.1 ng·mL −1 [131].
Lou and co-authors have developed a wave-based optofluidic biosensing platform for sensitive detection of aflatoxin M 1 in dairy products [132]. The portable, miniaturized device consisted in an optical fiber biosensor modified with AFM 1 -Ovalbumin (ovalbumin) embedded in a poly-methyl-methacrylate-based optofluidic cell and a pulse diode laser was used to excite the fluorescence-labelled antibody. The fluorescence signal was linearly dependent on AFM 1 concentration allowing its direct quantification. Using the developed device, a detection limit of 5 ng·L −1 was achieved for AFM 1 in dairy products [132].
Surface plasmon resonance (SPR) represents another optical technique used for immunoassay analysis of mycotoxins. The principle of detection using SPR platforms is based on measurements of changes in refractive index produced when the target analyte binds to its specific antibody immobilized on the sensor surface [101]. The SPR immunosensor has been used for detection and quantification of aflatoxin B 1 or for multiple detection of mycotoxins [133]. The SPR immunosensors obtained by immobilization of monoclonal antibodies encountered serious problems concerning the regeneration of the sensor surface, due to the high affinity binding of the monoclonal antibodies [133].

Conclusions
By presenting different analytical methods for determination of aflatoxin M 1 in milk (animal, human or powdered) and dairy products we have shown that electrochemical biosensing platforms offer highly sensitive and specific alternatives to the conventional methods.
While TLC, HPLC and enzyme-linked immunosorbent assay are considered the gold standard methods for AFM 1 determination, they are still more cumbersome, expensive and time-consuming techniques. Thus, electrochemical immunosensors represent a suitable alternative for AFM 1 detection, offering several advantages, such as versatility, high sensitivity, low production cost, easy modification and good stability. The use of screen-printed electrodes in combination with monoclonal antibodies or aptamers as bioreceptors leads to miniaturization of the system and to an improvement of the sensitivity, speed and low cost of analysis.
In addition, an oriented immobilization of biomolecules can be achieved due to their small size, simplicity and easy functionality, allowing in this way an increase of the binding efficiency and minimizing non-specific adsorptions on the biosensor surface.
Since aflatoxin B 1 contamination of feed and food products still remains a matter of increasing concern, aflatoxin M 1 contamination of milk and milk products will continue to be a risk factor for humans and especially for infants and young children. Taking into consideration the rapid and continuous development of the analytical methods and nanotechnology, different approaches for aflatoxins analysis will continue to be developed.