The state-of-the-art immunochemical methods for the determination of mycotoxins are considered. Both instrumental (enzyme-linked immunosorbent assay, polarization fluoroimmunoassay, and sensor devices) and noninstrumental methods are presented. The principles of particular methods are considered, and the examples of the use of these methods for the determination of mycotoxins from various groups in food products and animal feeds are given; the main lines of development are discussed

Electrospun nanofibers (ENFs) are promising materials for rapid diagnostic tests like lateral flow assays and dipsticks because they offer an immense surface area while excluding minimal volume, a variety of functional surface groups, and can entrap functional additives within their interior. Here, we show that ENFs on sample pads are superior in comparison to standard polymer membranes for the optical detection of biogenic amines (BAs) in food using a dipstick format. Specifically, cellulose acetate (CA) fibers doped with 2 mg/mL of the chromogenic and fluorogenic amine-reactive chameleon dye Py-1 were electrospun into uniform anionic mats. Those extract cationic BAs from real samples and Py-1 transduces BA concentrations into a change of color, reflectance, and fluorescence. Dropping a BA sample onto the nanofiber mat converts the weakly fluorescent pyrylium dye Py-1 into a strongly red emitting pyridinium dye. For the first time, a simple UV lamp excites fluorescence and a digital camera acts as detector. The intensity ratio of the red to the blue channel of the digital image is dependent on the concentration of most relevant BAs indicating food spoilage from 10 to 250 μM. This matches the permitted limits for BAs in foods and no false positive signals arise from secondary and tertiary amines. BA detection in seafood samples was also demonstrated successfully. The nanofiber mat dipsticks were up to sixfold more sensitive than those using a polymer membrane with the same dye embedded. Hence, nanofiber-based tests are not only superior to polymer-based dipstick assays, but will also improve the performance of established tests related to food safety, medical diagnostics, and environmental testing. Graphical Absract ᅟ.

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An immunochemically based test for non-instrumental simultaneous detection of zearalenone (ZEA) and T-2 toxin (T2) in feed was developed. The method combines clean-up of sample extract, pre-concentration of analytes by immunoextraction and immunodetection through the enzymatic reaction of horseradish peroxidase (HRP). The test is housed inside a standard 1-mL solid-phase extraction column and consists of three layers: two test layers (one for ZEA and another for T2) with immobilised specific antibodies and one control layer with bound anti-HRP antibodies. Feed extract was passed through an additional column with clean-up layer, which was disconnected after extract application. Total assay time was about 15 min for six samples and detection time was 4 min after chromogenic substrate application. Under optimised conditions a cut-off level for ZEA and T2 of 100 microg/kg was established. Different feed types were analysed for ZEA and T2 contamination by the proposed method and results were confirmed by LC-MS/MS.

To develop sensor technologies based on genetically engineered recognition elements, recombinant antibodies characterised by high stability are a prerequisite. Here we describe the first successful isolation of recombinant alpaca single-domain antibody fragments with high affinity to the mycotoxin ochratoxin A (OTA). Variable domains (VHH) of heavy-chain antibodies binding to OTA were cloned from an immunised alpaca. Selected VHH clones clustered in four major groups, based on protein sequence similarity. Six representative VHH antibody fragments were produced in Escherichia coli and characterised for their sensitivity and specificity by indirect competitive enzyme-linked immunosorbent assays. OTA concentrations causing 50% inhibition (IC50) ranged from 12 ng/ml for antibody fragment OCH-62 to 442 ng/ml for antibody fragment OCH-40. The IC50 of OCH-62 for ochratoxin B, an OTA analogue, was 476 ng/ml. No significant cross-reactivity was observed with common food constituents with structural similarities with parts of OTA, such as L-phenylalanine and coumarin. The performance of OCH-62 for detection of OTA in food matrices was comparable to the performance in buffer (white wine: IC50=17 ng/ml; red wine: IC50=21 ng/ml; instant coffee: IC50=17 ng/ml). In a membrane-based flow-through immunoassay, used for fast visual screening of samples, OCH-62 showed a cut-off level of 10 ng/ml OTA. Thereby, OCH-62 ranked among the best recombinant antibody fragments described for mycotoxins and is an excellent candidate for the design of food contamination sensors based on nanotechnology.

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