Emerging biotools for assessment of mycotoxins in the past decade
Introduction
Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Their ubiquity in nature is due to the ease with which mycotoxin-producing mould species grow on a wide range of substrates under different conditions (e.g., moisture, oxygen and temperature). Nowadays, the main problem caused by mycotoxins is spoiling agricultural products, which depends on environmental and storage conditions. It is estimated that approximately 25% of the world’s crops are contaminated to some extent with mycotoxins [1], [2], [3]. Also a matter of concern is the presence of airborne mycotoxins in mouldy buildings, responsible for so-called “sick-building syndrome” [4]. Additionally, some mycotoxins (e.g., aflatoxins) have been designated biowarfare agents due to their potential carcinogenicity. Whatever the contamination route (ingestion of contaminated foods, inhalation of toxigenic spores or direct dermal contact), the common problem is the serious threat to human and animal health. The adverse health effects due to mycotoxins were first mentioned in the tenth century when a chronicler described the disease “St. Anthony’s fire”. Many people from different regions around Europe died as a consequence of the ingestion of mouldy rye, after epileptic attacks, vomits and signs of madness caused by the sensation of internal burning. The knowledge acquired about such an undesirable disease, nowadays known as ergotism, and other mycotoxin effects have made it possible to react quickly and appropriately when faced with new outbreaks of mycotoxicosis.
Due to the increasing number and the variety of chemical structures, biosynthetic origins, toxicological effects and fungal sources, mycotoxins are very hard to classify. Moreover, many of them have been studied little. Table 1 shows the main features of the main mycotoxins and mycotoxin groups [5], [6], [7], [8].
Experts in pollutant-risk assessment consider mycotoxins to be the most important chronic dietary risk factor, above synthetic contaminants, plant toxins, food additives or pesticide residues [9].
The following citation, appeared on the BBC News the 11th January 2006, is a good example of the current concern about mycotoxins: “The head of Europe’s biggest pasta mill has been arrested in the Italian city of Bari on food adulterating charges… He is alleged to have tried to sell durum wheat, used in the manufacture of pasta, which was contaminated with a naturally occurring mould or fungus called ochratoxin, created by storage in unsuitably hot or humid conditions… Police said ochratoxin was present in quantities likely to cause the growth of cancers in consumers of pasta made from this consignment of wheat”.
Although sometimes news tend to be alarmist (the carcinogenic properties of ochratoxin A (OTA) have not yet been proved), the observation of both acute and chronic effects in animals due to the presence of mycotoxins has made evident the need to develop reliable, easy, cost-effective and fast analytical methods for monitoring and controlling them. The chemical diversity of mycotoxins and the wide range of matrices in which they can be found pose great challenges to analytical chemists. Apart from precision and accuracy, the fact that mycotoxins are often found as traces leads to the need for very sensitive techniques, able to detect the toxins at ppb, or even ppt, levels. Furthermore, the presence of several mycotoxins in a same sample may produce synergistic effects, which strongly encourage researchers to look for analytical techniques that can perform multi-toxin measurements.
Analysis of mycotoxins is usually performed by traditional methods, such as high-performance chromatography (HPLC), thin-layer chromatography (TLC) and gas chromatography (GC), coupled to ultraviolet-visible (UV-Vis) or fluorescence spectroscopy, mass spectrometry (MS), tandem MS (MS/MS), sequential MS (MSn) or electrochemical detection (ECD) [10]. These methods involve expensive, time-consuming steps: solid-phase extraction (SPE) with organic solvents from complex matrices; sample clean-up to remove interferents; pre-concentration; and, sometimes, analyte derivatization. The use of immunoaffinity columns (IACs) has improved the sample extraction and sample purification steps, but protocols are still laborious and require trained personnel.
Enzyme-linked immunosorbent assays (ELISAs) have also been widely applied to mycotoxin detection. They allow parallel analysis of multiple samples, being a powerful tool for rapid screening. Although most ELISA protocols require no sample clean-up, other than filtration and dilution, they still involve several washing steps.
There is evidently a need to develop high-performing methods for mycotoxin analysis that can deal with current drawbacks. In recent years, new approaches have appeared (e.g., dipsticks, biosensors, and multi-platforms) as well as novel recognition molecules (e.g., molecularly-imprinted polymers (MIPs)). Despite their early stage of development, preliminary results are promising.
This article reviews emerging biotechnological tools for mycotoxin detection. This state-of-the-art update on mycotoxin analysis will contribute to strengthening these trends.
Section snippets
Enzyme sensors
The electroactive characteristics of some mycotoxins lead us to think about the possibility of using electrochemical techniques for their detection.
Molina et al. [11] showed the electro-oxidation of alternariol (AOH) and alternariol monomethyl ether (AME) at glassy carbon and platinum electrodes. However, the high potentials required compromised the specificity of the assay.
In order to solve this problem, Moressi et al. [12] exploited the affinity of mushroom tyrosinase for these two
DNA sensors
The literature in this field is scarce, but merits a separate section in this article due to its originality.
The ability of aflatoxin M1 (AFM1) to interfere with DNA hybridization has been exploited to develop a rapid screening method [15]. The authors observed that this mycotoxin affects the kinetics and the time of signal generation due to double-helix formation. Although an LOD of 0.5 nM was achieved, the assay was not selective. Nevertheless, this work points the way towards new research
Immunoassay-based tools
Immunoassays are characterized by inherently high selectivity derived from affinity interactions between antibodies and antigens. They also allow analysis of multiple samples in parallel, and that makes them useful as simple screening tools for routine surveillance programs.
However, ELISA methods are still time-consuming. With the aim of simplifying procedures and developing robust, portable analytical tools, the trend is to focus on immunodipsticks and immunosensors.
MIP-based sensors
MIPs are synthetic receptors with several inherent advantages over biochemical or biological recognition systems (e.g., low cost, robustness and stability for long storage periods). MIPs have high-affinity sites that can selectively recognize the analyte, based on its shape, size or functional group distribution. These characteristics have led to consider MIPs excellent alternatives for clean up and pre-concentration of samples containing mycotoxins (e.g., ZON [35], [36] or OTA [37], [38], [39]
Biosensor arrays
Biosensor arrays can save time by detecting multiple target analytes simultaneously. They are very useful for analyzing multi-component samples, since the presence of several mycotoxin types in the same solution may involve synergistic effects that affect the individual determination of each mycotoxin. Moreover, the ability to perform parallel analyses enables assessment and quantification of matrix effects, so results are more accurate. Most of the biosensor arrays for mycotoxin analysis
Conclusions
Due to the presence of mycotoxins in a wide range of foodstuffs and their harmful effects to human health, research groups have devoted much effort to finding suitable mycotoxin detection techniques that fulfill the requirements for optimal analytical performance. While traditional mycotoxin analysis has been performed using mainly chromatographic techniques, which provide high sensitivity and accuracy, emerging techniques appearing in the past decade especially focus on developing fast
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