Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 10⁸ L mol⁻¹, which is about 4 times lower than that of NCA2 (5.36 × 10⁸ L mol⁻¹). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL⁻¹, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL⁻¹). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC₅₀) of optimized ELISA was 0.017 ng mL⁻¹ with a limit of detection (LOD) of 0.003 ng mL⁻¹. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals. Full article

Rapid screening methods are currently recognized as a strategic tool for mycotoxin issues management. Specific guidelines for validation and verification of mycotoxin screening methods are set in the Commission Regulation (EU) No 2014/519. This regulation establishes that the “aim of the validation is to demonstrate the fitness-for-purpose of the screening method” and focuses the entire validation procedure on determining specific cut-off values ensuring a maximum rate of false negative results of 5%. In addition, the assessment of the rate of false suspect results is addressed. With regard to rapid test-kits, ‘fitness-for-purpose’ includes not only the criteria more commonly considered when discussing laboratory-based methods (specificity, accuracy, and precision), but also more “practical” parameters such as speed and ease of implementation in a new operational environment. The latter means demonstrating under local conditions that performance parameters, as established during the validation, can be achieved by first time users. This goal can be achieved through “method verification”. The aim of the present study was to verify the fitness-for-purpose of mycotoxin screening methods when applied by first time users. This was achieved in one laboratory facility via results of a training course with multiple technicians attending. The verification study was organized similarly to a collaborative exercise and involved two groups comprising of 10 technicians each that used the methods for the first time. Different screening methods were applied for deoxynivalenol (DON) in wheat, which was mainly Enzyme Linked Immunosorbent Assay (ELISA), lateral flow device (LFD), fluorescence polarization immunoassay (FPIA), and liquid chromatography-high resolution mass spectrometry (LC-HRMS). An additional verification was done for aflatoxin B₁ (AFB₁) in maize and wheat using LFD and LC-HRMS, respectively. The results of analyses were used to calculate intermediate precision (RSD_ip, covering the inter-analyst variability in preparing the analytical samples and the precision under repeatability conditions) cut-off values and false suspect rates. RSD_ip ranged from 6.5% to 30% for DON, and from 16% to 33% for AFB₁. The highest obtained variances were associated with the AFB₁ analyses due to working with much lower mass fractions. The rate of false suspect results were lower than 0.1% for all tested methods. All methods showed a fit-for-purpose method performance profile, which allowed a clear distinction of samples containing the analytes at the screening target concentration (STC) from negative control samples. Moreover, the first time users obtained method performances similar to those obtained for validation studies previously performed on the screening methods included in the training course. Full article

We developed and tested a prototype of an antibody microarray immunoassay for simultaneous quantitative detection of four typical mycotoxins (aflatoxin B₁, ochratoxin A, zearalenone, and fumonisin B₁) in corn samples. The test kit consisted of a nitrocellulose membrane layered with immobilized monoclonal antibodies against mycotoxins. During the assay, the mycotoxin-protein conjugates were biotinylated. The signal detection was enhanced by a combination of the biotin-streptavidin system and enhanced chemiluminescence (ECL). This improved the sensitivity of the assay. Under the optimized conditions, four calibration curves with goodness of fit (R² > 0.98) were plotted. The results showed that the detection limits for aflatoxin B₁, ochratoxin A, zearalenone, and fumonisin B₁ were 0.21, 0.19, 0.09, and 0.24 ng/mL, with detection ranges of 0.47–55.69, 0.48–127.11, 0.22–31.36, and 0.56–92.57 ng/mL, respectively. The limit of detection (LOD) of this antibody microarray for aflatoxin B₁, ochratoxin A, zearalenone, and fumonisin B₁ in corn was 5.25, 4.75, 2.25, and 6 μg/kg, respectively. The recovery rates from the spiked samples were between 79.2% and 113.4%, with coefficient of variation <10%. The results of the analysis of commercial samples for mycotoxins using this new assay and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) were comparable and in good agreement. This assay could also be modified for the simultaneous detection of other multiple mycotoxins, as well as low-weight analytes, hazardous to human health. Full article

A rapid and sensitive electrochemical biosensor based on magnetic nanoparticles and screen-printed electrodes (MNPs-SPEs sensor) was developed for the detection of ochratoxin A (OTA) in cereal and feed samples. Different types of magnetic nanoparticles-based ELISA (MNPs-ELISA) were optimized, and the signal detection, as well as sensitivity, was enhanced by the combined use of screen-printed electrodes (SPEs). Under the optimized conditions, the calibration curve of the MNPs-SPEs sensor was y = 0.3372x + 0.8324 (R² = 0.9805). The linear range of detection and the detection limit were 0.01–0.82 ng/mL and 0.007 ng/mL, respectively. In addition, 50% inhibition (IC50) was detectable at 0.10 ng/mL. The limit of detection (LOD) of this MNPs-SPEs sensor in cereal and feed samples was 0.28 μg/kg. The recovery rates in spiked samples were between 78.7% and 113.5%, and the relative standard deviations (RSDs) were 3.6–9.8%, with the coefficient of variation lower than 15%. Parallel analysis of commercial samples (corn, wheat, and feedstuff) showed a good correlation between MNPs-SPEs sensor and liquid chromatography tandem mass spectrometry (LC/MS-MS). This new method provides a rapid, highly sensitive, and less time-consuming method to determine levels of ochratoxin A in cereal and feedstuff samples. Full article

Cycopiazonic acid (CPA) is a neurotoxin that acts through inhibition of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). CPA blocks the calcium access channel of the enzyme. The inhibition may involve the binding of CPA with a divalent cation such as Mg²⁺. The potential for CPA to act as a chelator also has implications for methods to detect this toxin. Certain of the lanthanide metals undergo a dramatic increase in luminescence upon coordination with small molecules that can transfer excitation energy to the metal. This report is the first to describe the coordination of CPA with lanthanide metals, resulting in a substantial enhancement of their luminescence. The luminescence expressed was dependent upon the type of lanthanide, its concentration, and the environment (solvent, water content, pH). Based upon the phenomenon, a competitive assay was also developed wherein terbium (Tb³⁺) and a series of metal cations competed for binding with CPA. With increasing cation concentration, the luminescence of the CPA/Tb³⁺ complex was inhibited. The chlorides of ten metals were tested. Inhibition was best with Cu²⁺, followed by Co²⁺, Al³⁺, Fe³⁺, Mn²⁺, Au³⁺, Mg²⁺, and Ca²⁺. Two cations in oxidation state one (Na⁺, K⁺) did not inhibit the interaction significantly. The interaction of CPA with lanthanides provides a novel recognition assay for this toxin. It also provides a novel way to probe the binding of CPA to metals, giving insights into CPA’s mechanism of action. Full article

Mycotoxins, which are toxic, carcinogenic, and/or teratogenic, have posed a threat to food safety and public health. Sensitive and effective determination technologies for mycotoxin surveillance are required. Immunoassays have been regarded as useful supplements to chromatographic techniques. However, conventional antibodies involved in immunoassays are difficult to be expressed recombinantly and are susceptible to harsh environments. Nanobodies (or VHH antibodies) are antigen-binding sites of the heavy-chain antibodies produced from Camelidae. They are found to be expressed easily in prokaryotic or eukaryotic expression systems, more robust in extreme conditions, and facile to be used as surrogates for artificial antigens. These properties make them the promising and environmentally friendly immunoreagents in the next generation of immunoassays. This review briefly describes the latest developments in the area of nanobodies used in mycotoxin detection. Moreover, by integrating the introduction of the principle of nanobodies production and the critical assessment of their performance, this paper also proposes the prospect of nanobodies in the field of food safety in the foreseeable future. Full article

A visual immunoaffinity test column (IATC) assay was developed to detect fumonisins in cereal samples for spot tests without the need for special instruments. The developed IATC assay had equivalent recognition capability for fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), or fumonisin B₃ (FB₃), and exhibited no cross-reactivity with aflatoxin B₁, ochratoxin A, zearalenone, or the T-2 toxin. The sample pretreatment was accomplished more rapidly and with greater ease, the entire assay procedure was completed in approximately 10 min, including sample pretreatment and testing. The limits of detection (LODs) of the IATC assay to detect fumonisins in the maize, barley, oat, and millet samples were 20 μg kg⁻¹. The results of the spiked maize, barley, oat, and millet and real maize samples by the IATC assay agreed well with the results obtained by the commercial fumonisin enzyme-linked immunosorbent assay (ELISA) test kit and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. The developed IATC assay can serve as a useful screening tool for the rapid, qualitative, and semi-quantitative detection of the total content of fumonisins (sum of FB₁, FB₂, and FB₃) in cereal samples on-site. Full article

T-2 toxin/HT-2 toxin (T-2/HT-2) and ochratoxin A (OTA) are mycotoxins that can contaminate a variety of agricultural commodities. To protect consumers’ health, indicative limits for T-2/HT-2 and maximum limits for OTA have been set by the European Commission, requiring food business operators and controlling agencies to conduct routine checks for the presence of these harmful contaminants. Screening methods are increasingly used for monitoring purposes. Due to the demand for new and improved screening tools, two individual detection methods, T-2/HT-2 and OTA enzyme-linked immunosorbent assays (ELISAs), were developed in this study. The T-2/HT-2 ELISA was based on a T-2 monoclonal antibody with an IC₅₀ (50% inhibitory concentration) of 0.28 ng/mL and 125% cross-reactivity with HT-2. As regards the OTA ELISA, a new sensitive monoclonal antibody specific to OTA with an IC₅₀ of 0.13 ng/mL was produced. Both developed ELISA tests were then validated in agricultural commodities in accordance with the new performance criteria guidelines for the validation of screening methods for mycotoxins included in Commission Regulation (EU) No 519/2014. The T-2/HT-2 ELISA was demonstrated to be suitable for the detection of T-2/HT-2 in cereals and baby food at and above the screening target concentration (STC) of 12.5 μg/kg and 7.5 μg/kg, respectively. The OTA ELISA was shown to be applicable for the detection of OTA in cereals, coffee, cocoa and wine at and above the STC of 2 μg/kg, 2.5 μg/kg, 2.5 μg/kg and 0.4 ng/mL, respectively. The accuracy of both ELISAs was further confirmed by analysing proficiency test and reference samples. The developed methods can be used for sensitive and high-throughput screening for the presence of T-2/HT-2 and OTA in agricultural commodities. Full article

Recent years have witnessed an overwhelming integration of nanomaterials in the fabrication of biosensors. Nanomaterials have been incorporated with the objective to achieve better analytical figures of merit in terms of limit of detection, linear range, assays stability, low production cost, etc. Nanomaterials can act as immobilization support, signal amplifier, mediator and artificial enzyme label in the construction of aptasensors. We aim in this work to review the recent progress in mycotoxin analysis. This review emphasizes on the function of the different nanomaterials in aptasensors architecture. We subsequently relate their features to the analytical performance of the given aptasensor towards mycotoxins monitoring. In the same context, a critically analysis and level of success for each nano-aptasensing design will be discussed. Finally, current challenges in nano-aptasensing design for mycotoxin analysis will be highlighted. Full article

Mycotoxins are one of the main factors impacting food safety. Mycotoxin contamination has threatened the health of humans and animals. Conventional methods for the detection of mycotoxins are gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), or enzyme-linked immunosorbent assay (ELISA). However, all these methods are time-consuming, require large-scale instruments and skilled technicians, and consume large amounts of hazardous regents and solvents. Interestingly, a microchip requires less sample consumption and short analysis time, and can realize the integration, miniaturization, and high-throughput detection of the samples. Hence, the application of a microchip for the detection of mycotoxins can make up for the deficiency of the conventional detection methods. This review focuses on the application of a microchip to detect mycotoxins in foods. The toxicities of mycotoxins and the materials of the microchip are firstly summarized in turn. Then the application of a microchip that integrates various kinds of detection methods (optical, electrochemical, photo-electrochemical, and label-free detection) to detect mycotoxins is reviewed in detail. Finally, challenges and future research directions in the development of a microchip to detect mycotoxins are previewed. Full article

A rapid fluorescence polarization immunoassay (FPIA) was optimized and validated for the determination of ochratoxin A (OTA) in rye and rye crispbread. Samples were extracted with a mixture of acetonitrile/water (60:40, v/v) and purified by SPE-aminopropyl column clean-up before performing the FPIA. Overall mean recoveries were 86 and 95% for spiked rye and rye crispbread with relative standard deviations lower than 6%. Limits of detection (LOD) of the optimized FPIA was 0.6 μg/kg for rye and rye crispbread, respectively. Good correlations (r > 0.977) were observed between OTA contents in contaminated samples obtained by FPIA and high-performance liquid chromatography (HPLC) with immunoaffinity cleanup used as reference method. Furthermore, single laboratory validation and small-scale collaborative trials were carried out for the determination of OTA in rye according to Regulation 519/2014/EU laying down procedures for the validation of screening methods. The precision profile of the method, cut-off level and rate of false suspect results confirm the satisfactory analytical performances of assay as a screening method. These findings show that the optimized FPIA is suitable for high-throughput screening, and permits reliable quantitative determination of OTA in rye and rye crispbread at levels that fall below the EU regulatory limits. Full article

Biosensing platforms based on peptide recognition provide a cost-effective and stable alternative to antibody-based capture and discrimination of ochratoxin-A (OTA) vs. ochratoxin-B (OTB) in monitoring bioassays. Attempts to engineer peptides with improved recognition efficacy require thorough structural and thermodynamic characterization of the binding-competent conformations. Classical molecular dynamics (MD) approaches alone do not provide a thorough assessment of a peptide’s recognition efficacy. In this study, in-solution binding properties of four different peptides, a hexamer (SNLHPK), an octamer (CSIVEDGK), NFO4 (VYMNRKYYKCCK), and a 13-mer (GPAGIDGPAGIRC), which were previously generated for OTA-specific recognition, were evaluated using an advanced MD simulation approach involving accelerated configurational search and predictive modeling. Peptide configurations relevant to ochratoxin binding were initially generated using biased exchange metadynamics and the dynamic properties associated with the in-solution peptide–ochratoxin binding were derived from Markov State Models. Among the various peptides, NFO4 shows superior in-solution OTA sensing and also shows superior selectivity for OTA vs. OTB due to the lower penalty associated with solvating its bound complex. Advanced MD approaches provide structural and energetic insights critical to the hapten-specific recognition to aid the engineering of peptides with better sensing efficacies. Full article

We developed an HT-2 toxin-specific simple ELISA format with a positive read-out. The assay is based on an anti-immune complex (IC) scFv antibody fragment, which is genetically fused with alkaline phosphatase (AP). The anti-IC antibody specifically recognizes the IC between a primary anti-HT-2 toxin Fab fragment and an HT-2 toxin molecule. In the IC ELISA format, the sample is added together with the scFv-AP antibody to the ELISA plate coated with the primary antibody. After 15 min of incubation and a washing step, the ELISA response is read. A competitive ELISA including only the primary antibody recognizes both HT-2 and T-2 toxins. The anti-IC antibody makes the assay specific for HT-2 toxin, and the IC ELISA is over 10 times more sensitive compared to the competitive assay. Three different naturally contaminated matrices: wheat, barley and oats, were used to evaluate the assay performance with real samples. The corresponding limits of detection were 0.3 ng/mL (13 µg/kg), 0.1 ng/mL (4 µg/kg) and 0.3 ng/mL (16 µg/kg), respectively. The IC ELISA can be used for screening HT-2 toxin specifically and in relevant concentration ranges from all three tested grain matrices. Full article

Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by several species of Aspergillus and Penicillium and frequently found as a natural contaminant in a wide range of food commodities. Novel and robust biorecognition agents for detecting this molecule are required. Aptamers are artificial nucleic acid ligands able to bind with high affinity and specificity to a given target molecule. In the last few years, three separate research groups have selected aptamers for ochratoxin A. While each of these three families of aptamers have been incorporated into various methods for detecting OTA, it is unclear if each aptamer candidate is better suited for a particular application. Here, we perform the first head-to-head comparison of solution-based binding parameters for these groups of aptamers. Based on our results, we provide recommendations for the appropriate choice of aptamer for incorporation into solution-based biorecognition assays and applications. Full article

Mycotoxin contamination threatens health and life of humans and animals throughout the food supply chains. Many of the mycotoxins have been proven to be carcinogens, teratogens and mutagens. The reliable and sensitive sensing methods are requested to monitor mycotoxin contamination. Advanced sensors based on antibodies or aptamers boast the advantages of high sensitivity and rapidity, and have been used in the mycotoxin sensing. These sensors are miniaturized, thereby lowering costs, and are applicable to high-throughput modes. In this work, the latest developments in sensing strategies for mycotoxin determination were critically discussed. Optical and electrochemical sensing modes were compared. The sensing methods for single mycotoxin or multiple mycotoxins in food samples were reviewed, along with the challenges and the future of antibody or aptamer-based sensors. This work might promote academic studies and industrial applications for mycotoxin sensing. Full article

The contaminations of Fusarium mycotoxins in grains and related products, and the exposure in human body are considerable concerns in food safety and human health worldwide. The common Fusarium mycotoxins include fumonisins, T-2 toxin, deoxynivalenol and zearalenone. For this reason, simple, fast and sensitive analytical techniques are particularly important for the screening and determination of Fusarium mycotoxins. In this review, we outlined the related advances in biosensors, chemosensors and assays based on the classical and novel recognition elements such as antibodies, aptamers and molecularly imprinted polymers. Application to food/feed commodities, limit and time of detection were also discussed. Full article

Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. Full article

A sample preparation method was developed for the screening of deoxynivalenol (DON) in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA) was compared to the sensor-based techniques of surface plasmon resonance (SPR) and biolayer interferometry (BLI) in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889) was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 μg/kg, LOD wheat dust: 458 μg/kg). Full article

To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G₁ and G₂. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G₁ and G₂, and did not cross-react with aflatoxins B₁, B₂, or M₁. Its IC₅₀ values for aflatoxins G₁ and G₂ were 17.18 ng·mL⁻¹ and 19.75 ng·mL⁻¹, respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL⁻¹. To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G₁ and G₂ and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B₁-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi. Full article

A label-free aptamer-based assay for the highly sensitive and specific detection of Ochratoxin A (OTA) was developed using a cationic polymer and gold nanoparticles (AuNPs). The OTA aptamer was used as a recognition element for the colorimetric detection of OTA based on the aggregation of AuNPs by the cationic polymer. By spectroscopic quantitative analysis, the colorimetric assay could detect OTA down to 0.009 ng/mL with high selectivity in the presence of other interfering toxins. This study offers a new alternative in visual detection methods that is rapid and sensitive for OTA detection. Full article

A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was developed for rapid and sensitive detection of zearalenone (ZEN). The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = −0.4287x + 0.3132 (R² = 0.9904). The working range was 0.07–2.41 ng/mL. The detection limit was 0.04 ng/mL and IC₅₀ was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%–111.9% and 91.7%–114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R² = 0.9283). We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories. Full article

Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC₅₀s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC₅₀. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage. Full article

We demonstrate for the first time, the development of titanium dioxide nanoparticles (TiO₂) quenching based aptasensing platform for detection of target molecules. TiO₂ quench the fluorescence of FAM-labeled aptamer (fluorescein labeled aptamer) upon the non-covalent adsorption of fluorescent labeled aptamer on TiO₂ surface. When OTA interacts with the aptamer, it induced aptamer G-quadruplex complex formation, weakens the interaction between FAM-labeled aptamer and TiO₂, resulting in fluorescence recovery. As a proof of concept, an assay was employed for detection of Ochratoxin A (OTA). At optimized experimental condition, the obtained limit of detection (LOD) was 1.5 nM with a good linearity in the range 1.5 nM to 1.0 µM for OTA. The obtained results showed the high selectivity of assay towards OTA without interference to structurally similar analogue Ochratoxin B (OTB). The developed aptamer assay was evaluated for detection of OTA in beer sample and recoveries were recorded in the range from 94.30%–99.20%. Analytical figures of the merits of the developed aptasensing platform confirmed its applicability to real samples analysis. However, this is a generic aptasensing platform and can be extended for detection of other toxins or target analyte. Full article

A sensitive electrochemical molecularly-imprinted sensor was developed for the detection of aflatoxin B1 (AFB1), by electropolymerization of p-aminothiophenol-functionalized gold nanoparticles in the presence of AFB1 as a template molecule. The extraction of the template leads to the formation of cavities that are able to specifically recognize and bind AFB1 through π-π interactions between AFB1 molecules and aniline moities. The performance of the developed sensor for the detection of AFB1 was investigated by linear sweep voltammetry using a hexacyanoferrate/hexacyanoferrite solution as a redox probe, the electron transfer rate increasing when the concentration of AFB1 increases, due to a p-doping effect. The molecularly-imprinted sensor exhibits a broad linear range, between 3.2 fM and 3.2 µM, and a quantification limit of 3 fM. Compared to the non-imprinted sensor, the imprinting factor was found to be 10. Selectivity studies were also performed towards the binding of other aflatoxins and ochratoxin A, proving good selectivity. Full article

Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb) designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. The median inhibitory concentration (IC₅₀) of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC) demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples. Full article