**1. Method Development**

Panasiuk et al. [11] developed an ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for a range of target mycotoxins including deoxynivalenol (DON), 3- and 15-acetyl-DON, DON-3-glucoside, nivalenol (NIV), and fusarenone-X. Sample preparation for the method included solid–liquid extraction, dispersive solid-phase extraction (QuEChERS), solid-phase extraction with hydrophiliclipophilic balance column, and several immunoaffinity columns; the highest efficacy being achieved with the last. However, of the six immunoaffinity columns tested, none showed cross-reactivity to all of the mycotoxins, therefore no single immunoaffinity separation can be advised. The optimized method using a Mycosep 225 Trich column clean-up was validated with a large number of feedstuff samples including wheat, maize, and animal feeds. A similar LC-MS/MS-based procedure is reported by Nakhjavan et al. [12] for multi-mycotoxin analyses. The method employing immunoaffinity clean-up, solid-phase extraction, or QuEChERS sample preparation was optimized for simultaneous quantitation of aflatoxins (aflatoxins B1, B2, G1 and G2, AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), NIV, diacetoxyscirpenol, fumonisins (fumonisins B1, B2 and B3, FB1, FB2, and FB3), T-2 toxin and HT-2 toxin in feed, and it allows limits of detection (LODs) ranging between 0.0003 and 0.05 µg/mL for the various mycotoxins tested.

Majdinasab et al. [13] reviewed colorimetric methods for industrial monitoring of mycotoxins in food and feed e.g., grains and cereals, grape juice, or red wine, and discuss the advantages and disadvantages for each method. Colorimetric strategies for various mycotoxins including T-2, DON, OTA, aflatoxins, ZEN, or FB1 (but not to FB2 or FB3) consist of enzyme-linked assays, lateral flow assays, microfluidic devices, and homogenous in-solution strategies that can utilize various (bio)receptors such as antibodies or aptamers.

The development of several immunoanalytical methods for mycotoxin detection is presented in the Special Issue. A competitive nanoparticle-based magnetic immunodetection assay for the detection and quantification of AFB1 with a LOD of 1.1 ng/mL is reported by Pietschmann et al. [14]. The method is based on magnetic separation of streptavidin-labeled magnetic particles, using an immobilized AFB1 antigen and biotinylated monoclonal AFB1 specific antibodies. The binding of antibodies to the immobilized antigen is competed by the free analyte (AFB1) in the solution (sample). Bound (i.e., uninhibited) antibodies on the solid surface are detected by frequency mixing magnetic detection. The LOD of the method is 1.1 ng/mL, comparable to a laboratory-based enzyme-linked immunosorbent assay (ELISA) method with a LOD of 0.29–0.39 ng/mL. The development of a portable instrument for ZEN by enzyme-linked fluorescent immunoassay (ELFIA) is reported by Gémes et al. [15], but as this instrument is a novel application for detection of mycotoxins as emerging water contaminants, it is discussed among the applications of routine monitoring (see Section 3. Applications in routine monitoring).

Several immunosensors on the basis of the same ZEN-specific polyclonal antibody are presented in the Special Issue for the detection of ZEN. An immobilized antibodybased competitive optical planar waveguide-based immunosensor by Nabok et al. [16] allowed a concentration-dependent detection of ZEN in the 0.01–1000 ng/mL concentration range. The optimized experimental benchtop planar waveguide setup is planned to be further developed into a portable hand-held biosensor including the signal processing electronics, suitable for in-field use. Using a similar sensor design but utilizing both immobilized antibody- (direct) and immobilized antigen-based (competitive) architectures, novel optical waveguide light mode spectroscopy (OWLS)-based immunosensors are reported by Székács et al. [17]. Covalent immobilization on the sensor surface was devised by epoxy-, amino-, and carboxyl-functionalization, and standard sigmoid curves in the optimized sensor formats allowed an outstanding LOD of 0.002 pg/mL for ZEN in the competitive immunosensor setup with a dynamic detection range of 0.01–1 pg/mL ZEN concentrations. The OWLS format represents five orders of magnitude improvement in LOD compared to the corresponding competitive ELISA, and the selectivity of the immunosensor for ZEN is outstanding on the basis of cross-reactivities determined for structurally related and unrelated compounds. The method was shown applicable in maize extract.

In addition to immunoanalytical (antibody-based) setups, the development of a labelfree aptamer-based fluorescent sensor is reported by Qian et al. [18] for the detection of OTA. The aptasensor utilizing a nucleotide recombination hybridization chain reaction amplification element allows high selectivity for OTA with a LOD of 2.0 pg/mL (4.9 pM). The elegant aptamer setup utilizes two hairpin nucleotide probes (H1 and H2). H1 contains a central loop portion capable of specific complex formation with OTA and two 6-nucleotide long terminal sequences complementary with each other. H2 is similar in structure, where the central loop is a G-quadruplex sequence capable to bind with N-methyl-mesoporphyrin IX and thus, forms a complex with enhanced fluorescent excitability. In the system, complex formation between OTA and H1 initiates repeated recombination-driven binding of numerous H2 probes, each incorporating N-methyl-mesoporphyrin IX molecules into the elongating H2 chain and resulting in amplification of the fluorescent signal. Other mycotoxins (ochratoxin B, AFB1) do not cross-react with the detection system and do not disturb the binding of OTA either. The detection method was demonstrated to be effective in wheat flour and red wine as commodity matrices.
