Mycotoxins In Food Commodities Research Articles

Fungal mycotoxins in food commodities: present status and future concerns

Mycotoxins are toxic secondary metabolites produced by fungi when they colonies the foodstuffs. These are potent toxins having severe health consequences in people, being mutagenic, teratogenic, and carcinogenic. In agricultural commodities, the contamination of mycotoxins is more prevalent. Several fungi can produce mycotoxins on agricultural products during harvest or in postharvest, and they have significant adverse effects on both animal and human beings. The most prevalent mycotoxins found in food commodities are aflatoxins and ochratoxins produced by Aspergillus species, ochratoxins and patulin produced by Penicillium, as well as fumonisins, deoxynivalenol, and zearalenone produced by Fusarium species. Worldwide, fumonisins, patulin, aflatoxins, and ochratoxins, among others, are responsible for numerous acute and chronic diseases in people and domestic animals. In food commodities, mycotoxins have been quantified and detected using various analytical methods. Besides, mycotoxins occurrence in food commodities were decontaminated through many potential approaches, such as physical, chemical, and biological methods. This review summarizes the findings of 30 years of research into mycotoxins in major commercial food crops including wheat, maize, sorghum, pearl millet, peanut, oat, pulses, barley, oilseeds, rice, and fruits and fruit juices. We also discuss the detection methods of major mycotoxins, available decontamination strategies along with their disadvantages and knowledge gaps. It is anticipated that data from meticulous studies on mycotoxins in food commodities will help in the development of safer food and in setting priorities for future research.

Preliminary screening of toxigenic fungi and mycotoxin contamination: A case of agricultural products in Ivory Coast

Toxigenic fungi are generally known to contaminate agricultural products; the causes, rate, and extent of its effects on common products have less emphasis, especially in sub-Saharan African countries. This lack of emphasis could be attributed to the state of research, storage, and accountability that could have intimated the public with necessary measures to curb the menace.For the purpose of the food safety control, this study investigates the fungal presence and mycotoxins in major food crops such as cereals (millet, rice, white and yellow maize), vegetables (chili, gnangnan, haricot, melon, and okra), legumes (haricot), and root products (attieke, cassava flakes) in Abidjan, Ivory Coast. Each sample is serially diluted and inoculated onto Czapek yeast agar (CYA), malt extract agar (MEA) and potato dextrose agar (PDA), to screen for fungal contamination. Colony color, colony edge, mycelia color, conidia shape, and microscopic features are used to identify the isolates. In addition, thin layer chromatography (TLC) was used to identify seven mycotoxins in food commodities. It is revealed that, Aspergillus species are found in 64 (92%) of the samples, Penicillium species in 29 (41%), and Fusarium species in 20 (29%) of the samples. A significant incidence rate of aflatoxin B1 (AFB1) is found in vegetables (gnangnan, haricot, and melon), roots products (cassava flakes), cereals (rice, and white maize). According to this investigation, each sample contains one or more fungus and mycotoxins.

Underreported Human Exposure to Mycotoxins: The Case of South Africa.

South Africa (SA) is a leading exporter of maize in Africa. The commercial maize farming sector contributes to about 85% of the overall maize produced. More than 33% of South Africa’s population live in rural settlements, and their livelihoods depend entirely on subsistence farming. The subsistence farming system promotes fungal growth and mycotoxin production. This review aims to investigate the exposure levels of the rural population of South Africa to dietary mycotoxins contrary to several reports issued concerning the safety of South African maize. A systematic search was conducted using Google Scholar. Maize is a staple food in South Africa and consumption rates in rural and urban communities are different, for instance, intake may be 1–2 kg/person/day and 400 g/person/day, respectively. Commercial and subsistence maize farming techniques are different. There exist differences influencing the composition of mycotoxins in food commodities from both sectors. Depending on the levels of contamination, dietary exposure of South Africans to mycotoxins is evident in the high levels of fumonisins (FBs) that have been detected in SA home-grown maize. Other potential sources of exposure to mycotoxins, such as carryover effects from animal products and processed foods, were reviewed. The combined effects between FBs and aflatoxins (AFs) have been reported in humans/animals and should not be ignored, as sporadic breakouts of aflatoxicosis have been reported in South Africa. These reports are not a true representation of the entire country as reports from the subsistence-farming rural communities show high incidence of maize contaminated with both AFs and FBs. While commercial farmers and exporters have all the resources needed to perform laboratory analyses of maize products, the greater challenge in combatting mycotoxin exposure is encountered in rural communities with predominantly subsistence farming systems, where conventional food surveillance is lacking.

Determination and detoxification of aflatoxin and ochratoxin in maize from different regions of Pakistan.

The contamination of food commodities withmycotoxinscould be a serious health threat to humans and animals. Therefore, identification, quantification and reduction of mycotoxins in food commodities, particularly of aflatoxins (AFs) and ochratoxin A (OTA) in grain foods, is essentially required to guarantee safe food. This study determined the levels of AFs and OTA in 135 maize grains samples belonging to eight salient maize varieties cultivated in Pakistan, and evaluated the usefulness of radiations and adsorbents to reduce their levels. High performance liquid chromatography (HPLC)-based method was validated for the determination of AFs and OTA in maize grains. The results showed that 69 and 61% samples were positive for AFs and OTA, respectively and 54 and 22% of the respective samples had AFs and OTA above the permissible limits set by Pakistan Standards and Quality Control Authority. The concentration of AFs, AFB1and OTA in grains ranged from 14.5 to 92.4, 1.02 to 2.46 and 1.41 to 53.9μgkg-1, respectively. Among the varieties, Pearl had the highest level of total AFs and OTA, whereas YH-5427 had the highest AFB1 level. The lowest concentration of AFs and OTA was found in Malaka and 30Y87, respectively. The use of 15kGy gamma irradiation for 24h, sunlight-drying for 20h and UV irradiation for 12h almost completely degraded the mycotoxins. The microwave heating for 120s resulted in 9-33% degradation of mycotoxins. Moreover, the treatment of grains' extract with activated charcoal (5% w/w) removed > 96% of total AFs and AFB1, and up to 43% of OTA. The use of bentonite at the same rate removed OTA, total AFs and AFB1 by 93, 73 and 92%, respectively. Thus, it is concluded that contamination of maize grains with mycotoxins was fairly high in the collected maize grain samples in Pakistan, and treatment with radiations and adsorbents can effectively reduce mycotoxins contamination level in maize grains.

Alternaria mycotoxins in food commodities marketed through e-commerce stores in China: Occurrence and risk assessment

The natural occurrence of mycotoxins like Alternaria mycotoxins in food commodities is concerning in terms of health risks for humans. Currently, detailed investigations of Alternaria mycotoxins, such as alternariol (AOH), alternariol methyl ether (AME), tenuazonic acid (TeA), tentoxin (TEN), and altenuene (ALT) in food commodities marketed through e-commerce stores in China remains scarce. In addition, not many studies have focused on the associated dietary risk of these mycotoxins. This study conducted a survey (486 samples) for five Alternaria mycotoxins using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods. Among five Alternaria mycotoxins, TeA had the highest occurrence rate (51.0%) and concentration (average concentration of 40.6 μg/kg) in food commodities, followed by TEN, AOH, AME, and ALT. Millet and tomato products were contaminated with higher levels of TeA than other types of food commodities. The combination of TeA + TEN and AME + TeA + TEN were the predominant two contamination patterns detected in cereal and fruit products. Chronic dietary exposures to Alternaria mycotoxins were performed by the threshold of toxicological concern (TTC) approach. The mean and 95th percentile chronic dietary exposures to AOH and AME indicated different risk levels for consumers in China, with the highest exposure observed in the 2–7 years group, indicating potential dietary health concern. The mean chronic dietary exposures to TeA and ALT did not indicate a risk for the consumers, but the exposure to TeA through cereal product consumption exceeded the TTC value in the 2–12 years group at 95th percentile evaluation. Therefore, strategies should be continuously adopted to decrease or eliminate the occurrence of Alternaria mycotoxins in food commodities in order to reduce the dietary risks for humans.

Electrochemical aptasensing for the detection of mycotoxins in food commodities

Electrochemical aptasensing for the detection of mycotoxins in food commodities

The Existing Methods and Novel Approaches in Mycotoxins' Detection.

Mycotoxins represent a wide range of secondary, naturally occurring and practically unavoidable fungal metabolites. They contaminate various agricultural commodities like cereals, maize, peanuts, fruits, and feed at any stage in pre- or post-harvest conditions. Consumption of mycotoxin-contaminated food and feed can cause acute or chronic toxicity in human and animals. The risk that is posed to public health have prompted the need to develop methods of analysis and detection of mycotoxins in food products. Mycotoxins wide range of structural diversity, high chemical stability, and low concentrations in tested samples require robust, effective, and comprehensible detection methods. This review summarizes current methods, such as chromatographic and immunochemical techniques, as well as novel, alternative approaches like biosensors, electronic noses, or molecularly imprinted polymers that have been successfully applied in detection and identification of various mycotoxins in food commodities. In order to highlight the significance of sampling and sample treatment in the analytical process, these steps have been comprehensively described.

Aspergillus flavus and Aspergillus ochraceus inhibition and reduction of aflatoxins and ochratoxin A in maize by irradiation

Grains are susceptible to contamination by molds; some cause spoilage and others produce certain mycotoxins that cause a serious health threat to humans and animals. Aspergillus flavus and Aspergillus ochraceus and their mycotoxins, aflatoxins and ochratoxin A, are natural contaminants of various agricultural commodities. Control of these molds and their mycotoxins in food commodities is of utmost importance; therefore, the target of this research was to explore the effects of gamma irradiation doses on the growth of A. flavus and A. ochraceus in artificially inoculated yellow maize as well as on the production of aflatoxin B1, ochratoxin A, and the formation of toxins in maize. The irradiated dose of 6.0 kGy was found to completely inhibit the growth of the two molds, while a dose of 4.5 kGy reduced the production of their mycotoxins. Maximum degradation of the formed aflatoxins and ochratoxin A in maize occurred at 20 kGy, with best reduction rates of 40.1%, 33.3%, and 61.1% observed for aflatoxin B1, aflatoxin B2, and ochratoxin A, respectively. We recommend grains irradiation by gamma radiation at 6.0 kGy to decontaminate mycotoxin-producing molds before they produce mycotoxins. The study represents a proactive, efficient, and potent method for avoiding potential contamination of fungus during grains storage and transfer for one to two months.

Quantification of multi-mycotoxin in cereals (maize, maize porridge, sorghum and wheat) from Limpopo province of South Africa

ABSTRACT Mycotoxins are secondary metabolites that are produced by filamentous mycotoxigenic fungi belonging to the Alternaria, Aspergillus, Fusarium and Penicillium genera amongst others. Multi-class mycotoxins were extracted from 55 cereal samples and analysed using liquid chromatography-tandem mass spectrometry. The adopted extraction method for maize, maize porridge, sorghum and wheat was comprehensively validated. This method was further tested to determine the natural occurrence of mycotoxins in foodstuffs. Twelve (12) out of 22 mycotoxins were detected in maize, maize porridge, sorghum and wheat, including α-zearalenol (α-ZEL) (89%), fumonisin B3 (FB3) (84%), fumonisin B1 (FB1) (80%), tenuazonic acid (TeA) (78%), ochratoxin B (42%), deoxynivalenol (DON) (12%), ochratoxin A (11%), 3-acetyldeoxynivalenol (7%), sterigmatocystin (STG) (6%), 15-acetyldeoxynivalenol (2%), cyclopiazonic acid (CPA) (2%) and aflatoxin B2 (2%). The data revealed high incidence rate of α-ZEL (range: 6.5–70.5 µg kg−1) in all matrices. Maize samples had high mycotoxin co-occurrence compared to other matrices. All recovered mycotoxins in food commodities were within the maximum regulatory limits, with the exception of fumonisins (FB1 and FB3) exceeded the South African and European Commission regulation, and the highest concentration was 2153 µg kg−1 in maize. It is essential to monitor the level of emerging mycotoxins in food commodities from rural areas as trace amount of CPA (< limit of quantification), STG (range: 0.30–0.74 µg kg−1) were detected and high concentration of TeA (292.7 µg kg−1) was detected in sorghum. The occurrence of these mycotoxins further encourages frequent analyses, their co-occurrence in the samples poses a significant threat to public health and more emphasis should thus be placed on reducing the contamination levels of these toxins in staples.

Occurrence of relevant mycotoxins in food commodities consumed in Chile.

The aims of this study were to analyse the occurrence of aflatoxins, aflatoxin M1 (AFM1), fumonisins, ochratoxin A (OTA), patulin (PAT), zearalenone (ZEN) and deoxynivalenol (DON) in foodstuffs consumed in Chile between 2008 and 2017 and to estimate the contribution of main contaminated foodstuff in human exposure by the probable daily intake (PDI) estimation. In 9years of surveillance, 2020 food samples were analysed with an occurrence of 18.2% and with 2.7% of the samples being over the Chilean regulation. The occurrence of mycotoxins in food were 16% for aflatoxins, 6% for AFM1, 30% for OTA, 12% for DON, 7% for PAT, 21% for fumonisins and 2% for ZEN. The estimated median PDI of DON because of bread consumption was 129.2ng/kg bw/day for children and 96.0ng/kg bw/day in adults. Median PDI because of capsicum consumption was 0.006ng/kg bw/day for OTA and 0.005ng/kg bw/day for aflatoxins. Median PDI of aflatoxins was estimated at 0.02ng/kg bw/day for spices and 0.04ng/kg bw/day for nuts consumption. In children, the median PDI of AFM1 for dairy consumption was 0.07ng/kg bw/day. The derived margin of exposure (MoE) values ranged from 1133 to 8500 suggested that aflatoxins would be of public health concern. The PDI of the other mycotoxins did not show a health risk. This is the first survey of mycotoxins in food made in Chile; further research is needed to improve surveillance and guidelines based on national risk assessments and considering sensitive population groups.

Fluorescent competitive aptasensor for detection of aflatoxin B1.

Aflatoxin B1 (AFB1 ) is one of the most commonly found mycotoxins in food commodities, particularly cereals, oilseeds, spices and tree nuts. In the past decade, aptamers have come into limelight and emerged as a new biosensing element replacing antibodies in various detection formats. Herein we report a faster, more sensitive, high throughput method for the detection of AFB1 using AFB1 -specific aptamers. The assay format was based on a competitive reaction of the fluorescent tagged aptamer specific to AFB1 with the aflatoxin conjugate. Under optimal conditions, a linear range of detection (50ng to 50pg) was achieved with a limit of detection (LOD) of 10pg/mL in the buffer system. Results of inter- and intra-assay revealed that the assay was repeatable with standard deviation in acceptable range. The assay was also validated in food samples such as dried red chilies, groundnut and whole pepper with recovery in the range of 92 to 102% at 10ng/mL and 100pg/mL levels. The aptasensor assay was also compared with standard analytical method of HPLC and was found to be more sensitive. This detection technique has the potential to be developed into a biosensor platform for AFB1 detection.

Toxicological interactions between the mycotoxins deoxynivalenol, nivalenol and their acetylated derivatives in intestinal epithelial cells.

In case of mycotoxin contaminations, food and feedstuff are usually contaminated by more than one toxin. However toxicological data concerning the effects of mycotoxin combinations are sparse. The intestinal epithelium is the first barrier against food contaminants and this constantly renewing organ is particularly sensitive to mycotoxins. The aim of this study was to investigate the effects of deoxynivalenol (DON) and four other type B trichothecenes (TCTB), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX) alone or in combination on intestinal epithelial cells. Proliferating, non-transformed IPEC-1 cells were exposed to increasing doses of TCTB, alone or in binary mixtures and mycotoxin-induced cytotoxicity was measured with MTT test. The toxicological interactions were assessed using the isobologram-Combination index method. The five tested mycotoxins and their mixtures had a dose-dependent effect on the proliferating enterocytes. DON-NIV, DON-15-ADON and 15-ADON-3-ADON combinations were synergistic, with magnitude of synergy for 10% cytotoxicity ranging from 2 to 7. The association between DON and 3-ADON also demonstrated a synergy but only at high doses, at lower doses antagonism was noted. Additivity was observed between NIV and FX, and antagonism between DON and FX. These results indicate that the simultaneous presence of mycotoxins in food commodities and diet may be more toxic than predicted from the mycotoxins alone. This synergy should be taken into account considering the frequent co-occurrence of TCTB in the diet.

The loss of the inducible Aspergillus carbonarius MFS transporter MfsA leads to ochratoxin A overproduction

Ochratoxin A (OTA), a nephrotoxic compound produced by certain Aspergillus and Penicillium species, is one of the most abundant mycotoxins in food commodities. Aspergillus carbonarius is the main source of OTA in wine, grape juice and dried vine fruits. Although many studies have focused on OTA production by A. carbonarius, little is known about the genes related to OTA production and transport. We have found a transporter that belongs to the major facilitator superfamily (MfsA) which is highly expressed with a 102-fold induction in an ochratoxigenic A. carbonarius strain compared to a low OTA producer strain. The encoding mfsA gene shows similarity to the multidrug efflux transporter flu1 from Candida albicans. A high number of putative transcription factor binding sites involved in the response to stress were identified within the promoter of mfsA. Phenotypical analysis of ΔmfsA deletion mutants revealed that the loss of mfsA leads to a slight growth reduction and increased OTA production. We therefore hypothesize that MfsA could be a stress response transporter whose disruption could cause an increase in oxidative stress together with a stimulation of mycotoxin production.

New insights into mycotoxin mixtures: The toxicity of low doses of Type B trichothecenes on intestinal epithelial cells is synergistic

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. DON is often present with other type B trichothecenes such as 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). Although the cytotoxicity of individual mycotoxins has been widely studied, data on the toxicity of mycotoxin mixtures are limited. The aim of this study was to assess interactions caused by co-exposure to Type B trichothecenes on intestinal epithelial cells. Proliferating Caco-2 cells were exposed to increasing doses of Type B trichothecenes, alone or in binary or ternary mixtures. The MTT test and neutral red uptake, respectively linked to mitochondrial and lysosomal functions, were used to measure intestinal epithelial cytotoxicity. The five tested mycotoxins had a dose-dependent effect on proliferating enterocytes and could be classified in increasing order of toxicity: 3-ADON<15-ADON≈DON<NIV≪FX. Binary or ternary mixtures also showed a dose-dependent effect. At low concentrations (cytotoxic effect between 10 and 30–40%), mycotoxin combinations were synergistic; however DON–NIV–FX mixture showed antagonism. At higher concentrations (cytotoxic effect around 50%), the combinations had an additive or nearly additive effect. These results indicate that the simultaneous presence of low doses of mycotoxins in food commodities and diet may be more toxic than predicted from the mycotoxins alone. Considering the frequent co-occurrence of trichothecenes in the diet and the concentrations of toxins to which consumers are exposed, this synergy should be taken into account.

Mycotoxins and their effect on human health

Health risks associated with the consumption of products contaminated with mycotoxins are worldwide recognized and depend on the extent to which they are consumed in diversified diet. To some extent, the presence of small amount of mycotoxins in cereals and related food products is unavoidable; this requires risk assessments which are to be carried out by regulatory bodies in several countries to help establish regulatory guidelines for the protection of public health. By assessing the levels at which these substances in food may pose a potential risk to human health, it is possible to devise appropriate risk management strategies. However, several important factors have to be taken into account in making a rational risk management decision, such as adequate toxicological data and information concerning the extent of exposure, availability of technically sound analytical procedures (including sampling), socioeconomic factors, food intake patterns and levels of mycotoxins in food commodities which may vary considerably between countries.

Co-exposure of Fusarium mycotoxins: In vitro myelotoxicity assessment on human hematopoietic progenitors

Mycotoxins such as beauvericin (BEA), deoxynivalenol (DON), enniatin B (ENB), fumonisin B1 (FB1), T-2 toxin and zearalenone (ZEA) can co-occur in food commodities.This aim of this study was to assess the myelotoxicity of these mycotoxins in couple using in vitro human granulo-monocytic (Colony Forming Unit-Granulocyte and Macrophage, CFU-GM) hematopoietic progenitors. Clonogenic assays have been performed in the presence of the following couples of fusariotoxins: DON + BEA, DON + FB1, DON + T-2, DON + ZEA, T-2 + ZEA and BEA + ENB.Co-exposure of human CFU-GM to DON + BEA resulted in synergic myelotoxic effects. The combination of DON + T-2 presented additive or synergic myelotoxic effects. The couples DON + ZEA, T-2 + ZEA and BEA + ENB had additive myelotoxic effects, while the combination of DON + FB1 showed antagonist myelotoxic effects.These in vitro results suggested that the simultaneous presence of mycotoxins in food commodities and diet may be more myelotoxic than the presence of one mycotoxin alone. Diminution of hematopoietic progenitors could give rise to a decrease number of mature blood cells, inducing agranulocytosis and/or thrombocytopenia and in severe cases aplastic anemia.

High throughput cellular biosensor for the ultra-sensitive, ultra-rapid detection of aflatoxin M1

The aim of this study was the development of a rapid novel biosensor system based on the Bioelectric Recognition Assay (BERA) for the detection of aflatoxin M1 (AFM1). Membrane-engineering was achieved by electroinsertion of AFM1-homologous antibodies into the membrane of the cells. The sensor measured the electric response of cultured membrane-engineered fibroblast cells suspended in an alginate gel matrix due to the change of their membrane potential after the interaction of the analyte molecules with the antibodies. The BERA-based sensor was able to detect AFM1 rapidly (3min) at very low concentrations (5 pg/ml = 5 ppt), thus demonstrating a higher sensitivity than most currently available biosensor-based methods. In addition, the assay was quite selective against other aflatoxins, such as aflatoxin B1 and ochratoxin A. Furthermore, the assay system allowed for high throughput AFM1 analysis (160 individual tests/h), due to its multiple cell–electrode interface array. Although further research is required for the optimization of the assay, the elimination of possible matrix effects and its validation by assaying actual food samples, this novel biosensor offers new perspectives for ultra-rapid, ultra-sensitive and low-cost monitoring of mycotoxins in food commodities.

Multiplex PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in rice and fingermillet collected from southern India

The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In the present study, a multiplex PCR was standardised for the group-specific detection of fumonisin-producing and trichothecene-producing strains of Fusarium species. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions 1 and 2 of rDNA. Primers for group-specific detection were designed from the tri5 and tri6 genes involved in trichothecene biosynthesis and the fum1 and fum13 genes involved in fumonisin biosynthesis. Among the various genera and their strains tested, all the 85 confirmed Fusarium strains were positive for rDNA gene and the rest stayed negative. From among the Fusarium strains, 15 had amplification for trichothecene- and 20 for fumonisin-encoding genes. All PCR positive trichothecene chemotypes of Fusarium species tested were positive for chemical analysis but in the case of fumonisins, of the 20 PCR positive cultures, only 13 showed positive for chemical analysis by HPTLC. The assay described here provided a rapid and reliable detection of trichothecene- and fumonisin-producing Fusarium directly from natural food grains and the results were always comparable with a conventional HPTLC detection method. It can, therefore, be used by the food industry to monitor quality and safety.

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC(1, 2) methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain(3) and screen printed electrodes as sensing platform. Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health(4). The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity(5). The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution. At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution. A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform. Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5. The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution. At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution. A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.