Magnetic aptamer copper nanoclusters fluorescent biosensor for the visual detection of zearalenone based on docking-aided rational tailoring.

To address the food safety issues caused by toxins, we established a fluorescent copper nanocluster biosensor based on magnetic aptamer for the visual and quantitative detection of ZEN. Specifically, we utilized the docking-aided rational tailoring (DART) strategy to analyze intermolecular force and interaction sites between zearalenone (ZEN) and the aptamer, and optimize the long-chain aptamer step by step to enhance the binding affinity by 3.4 times. The magnetic bead-modified aptamer underwent conformational changes when competing with complementary sequences to bind with ZEN. Then, the released complementary sequences will be amplified in template-free mode with the presence of the terminal deoxynucleotidyl transferase (TdT), and generating T-rich sequences as the core sequences for the luminescence of copper nanoclusters. The luminescence could be visualized and quantitatively detected through ultraviolet irradiation. The proposed label-free aptasensor exhibited high sensitivity and specificity, with a low limit of detection (LOD) of 0.1 ng/mL.

Aggregation-induced emission nanoparticles facilitating multicolor lateral flow immunoassay for rapid and simultaneous detection of aflatoxin B1 and zearalenone.

Herein we developed a multicolor lateral flow immunoassay (LFIA) test strip for rapid and simultaneous quantitative detection of aflatoxin B1 (AFB1) and zearalenone (ZEN). Three differently colored aggregation-induced emission nanoparticles (AIENPs) were designed as LFIA signal tags, with red and green AIENPs for targeting AFB1 and ZEN at the test line, and yellow AIENPs for indicating the validity of the test strip at the control (C) line. After surface functionalization with antibodies, the developed AIENP-based multicolor LFIA allows simultaneous and accurate quantification of AFB1 and ZEN using an independent C-line assisted ratiometric signal output strategy. The detection limits of AFB1 and ZEN were 6.12 and 26 pg/mL, respectively. The potential of this method for real-world applications was well demonstrated in corn and wheat. Overall, this multicolor LFIA shows great potential for field screening of multiple mycotoxins and can be extended to rapid and simultaneous monitoring of other small molecule targets.

Impact of predicted climate change environmental conditions on the growth of Fusarium asiaticum strains and mycotoxins production on a wheat-based matrix.

Fusarium asiaticum is a predominant fungal pathogen causing Fusarium Head Blight (FHB) in wheat and barley in China and is associated with approximately £201 million in annual losses due to grains contaminated with mycotoxins. F. asiaticum produces deoxynivalenol and zearalenone whose maximum limits in cereals and cereals-derived products have been established in different countries including the EU. Few studies are available on the ecophysiological behaviour of this fungal pathogen, but nothing is known about the impact of projected climate change scenarios on its growth and mycotoxin production. Therefore, this study aimed to examine the interacting effect of i) current and increased temperature (25 vs 30 °C), ii) drought stress variation (0.98 vs 0.95 water activity; aw) and iii) existing and predicted CO2 concentrations (400 vs 1000 ppm) on fungal growth and mycotoxin production (type B trichothecenes and zearalenone) by three F. asiaticum strains (CH024b, 82, 0982) on a wheat-based matrix after 10 days of incubation. The results showed that, when exposed to increased CO2 concentration (1000 ppm) there was a significant reduction of fungal growth compared to current concentration (400 ppm) both at 25 and 30 °C, especially at 0.95 aw. The multi-mycotoxin analysis performed by LC-MS/MS qTRAP showed a significant increase of deoxynivalenol and 15-acetyldeoxynivalenol production when the CH024b strain was exposed to elevated CO2 compared to current CO2 levels. Zearalenone production by the strain 0982 was significantly stimulated by mild water stress (0.95 aw) and increased CO2 concentration (1000 ppm) regardless of the temperature. Such results highlight that intraspecies variability exist among F. asiaticum strains with some mycotoxins likely to exceed current EU legislative limits under prospected climate change conditions.

Construction of a self-reporting molecularly-imprinted electrochemical sensor based on CuHCF modified by rGNR-rGO for the detection of zearalenone.

A self-reporting molecularly-imprinted electrochemical sensor is prepared for the detection of Zearalenone (ZEA). Firstly, the reduced graphene nanoribbons and reduced graphene oxide (rGNR-rGO) were simultaneously modified onto a glassy carbon electrode (GCE) to improve the sensor's sensitivity. After electrodepositing copper nanoparticles onto the rGNR-rGO/GCE, cyclic voltammetry scanning was performed in potassium ferrocyanide solution, and copper hexacyanoferrate (CuHCF) was deposited onto rGNR-rGO/GCE to further improve the sensor's sensitivity while giving it self-reporting capability. Then, molecularly-imprinted polymer films were prepared on the CuHCF/rGNR-rGO/GCE to ensure the selectivity of the sensor. It is found that the linear range of ZEA detection by the constructed sensor is 0.25-500 ng·mL -1, with a detection limit of 0.09 ng·mL -1. This sensor shows the merits of good selectivity, high sensitivity and accurate detection, providing a great possibility for the precise detection of low concentration ZEA in food.

Dual function of magnetic nanocomposites-based SERS lateral flow strip for simultaneous detection of aflatoxin B1 and zearalenone.

Aflatoxin B1 (AFB1) and zearalenone (ZEN) are two mycotoxins that often co-occur in corn. A surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-LFIA) that can simultaneously detect AFB1 and ZEN in corn samples was developed employing the core-interlayer-satellite magnetic nanocomposites (Fe3O4@PEI/AuMBA@AgMBA) as dual-functional SERS tags. Under the optimal conditions, the detection ranges of AFB1 and ZEN in corn samples were 0.1-10 µg/kg and 4-400 µg/kg, respectively. Moreover, the test results for two mycotoxins in contaminated corn samples employing the suggested SERS-LFIA was in line with those of the HPLC technique. In view of its satisfactory sensitivity, accuracy, precision and short testing time (20 min), the developed system has a promising application prospect in the on-site simultaneous detection of AFB1 and ZEN.

Colorimetric and chemiluminescent enzyme immunoassays based on the alkaline phosphatase-tagged single-chain variable fragment fusion tracer for detecting zearalenone in agro-products.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin and seriously threatens food safety, which requires rapid and sensitive detection methods for monitoring ZEN in agro-products. Herein, an alkaline phosphatase-tagged single-chain variable fragment fusion protein (ALP-scFv) was used as a bifunctional tracer to develop a colorimetric enzyme immunoassay (CEIA) and a chemiluminescent enzyme immunoassay (CLEIA) for ZEN. In addition, the interactions between scFv and ZEN were exploited by computer-assisted simulation, and four key amino acid sites were preliminarily identified. After optimization, the CEIA and CLEIA exhibited a limit of detection of 0.02 and 0.006 ng/mL, respectively. Furthermore, both methods showed favorable accuracy in recovery experiments and good selectivity in cross reactions. Moreover, the detection results of the actual samples from both methods correlated well with those from high-performance liquid chromatography. Overall, the ALP-scFv fusion tracer-based CEIA and CLEIA are demonstrated as reliable tools for ZEN detection in food.

Exposure assessment of children to dietary mycotoxins: A pilot study conducted in Ribeirão Preto, São Paulo, Brazil.

Exposure to mycotoxins through food is a major health concern, especially for youngsters. This study performed a preliminary investigation on children's exposure to dietary mycotoxins in Ribeirão Preto, Brazil. Sampling procedures were conducted between August and December 2022, to collect foods (N = 213) available for consumption in the households of children (N = 67), including preschoolers (aged 3-6 years, n = 21), schoolers (aged 7-10 years, n = 15), and adolescents (aged 11-17 years, n = 31) cared in the Vila Lobato Community Social Medical Center of Ribeirão Preto. Ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was used to determine concentrations of the mycotoxins in foods. Mycotoxins measured in all foods comprised aflatoxins (AFs), fumonisins (FBs), zearalenone (ZEN), T-2 toxin, deoxynivalenol (DON) and ochratoxin A (OTA). Higher incidence and levels were found for FBs, ZEN, and DON in several commonly consumed foods. Furthermore, 32.86 % foods had two to four quantifiable mycotoxins in various combinations. The mean estimated daily intake (EDI) values were lower than the tolerable daily intake (TDI) for AFs, FBs, and ZEN, but higher than the TDI (1.0 µg/kg bw/day) for DON, hence indicating a health risk for all children age groups. Preschoolers and adolescents were exposed to DON through wheat products (EDIs: 2.696 ± 7.372 and 1.484 ± 2.395 µg/kg body weight (bw)/day, respectively), while schoolers were exposed through wheat products (EDI: 1.595 ± 1.748 µg/kg bw/day) and rice (EDI: 1.391 ± 1.876 µg/kg bw/day). The results indicate that wheat-based foods and rice may be risky to children, implying the need for stringent measures to avoid DON contamination in these products.

Pore Size Adjustment Strategy for the Fabrication of Molecularly Imprinted Covalent Organic Framework Nanospheres at Room Temperature for Selective Extraction of Zearalenone in Cereal Samples.

Covalent organic frameworks (COFs) are attractive adsorbents for sample pretreatment due to their unique structure and properties. However, the selectivity of COFs for the extraction of hazardous compounds is still limited due to the lack of specific interactions between COFs and targets. Herein, we report a pore size adjustment strategy for room-temperature synthesis of molecularly imprinted COF (MICOF) for selective extraction of zearalenone (ZEN) in complex food samples. The three-dimensional building block tetra(4-aminophenyl) methane was used as a functional monomer, while dialdehyde monomers with different numbers of benzene ring were used to adjust the pore size of MICOF to match with the size of ZEN molecules. The prepared MICOF gave the largest adsorption capacity of 177.2 mg g-1 and the highest imprinting factor of 10.1 for ZEN so far. MICOF was used as the adsorbent for dispersed solid-phase extraction in combination with high-performance liquid chromatography for the determination of trace ZEN in cereals. The high selectivity of the developed method allows simple aqueous standard calibration for the matrix effect-free determination of ZEN in food samples. The limit of detection and the recoveries of the developed method were 0.21 µg kg-1 and 93.7-101.4%, respectively. The precision for the determination of ZEN was less than 3.8% (RSD, n = 6). The developed method is promising for the selective determination of ZEN in complex matrices.

Natural compounds mitigate mycotoxins-induced neurotoxicity by modulating oxidative tonus: in vitro and in vivo insights - a review.

This review explores the repercussions of mycotoxin contamination in food and feed, emphasising potential threats to agriculture, animal husbandry and public health. The primary objective is to make a comprehensive assessment of the neurotoxic consequences of mycotoxin exposure, an aspect less explored in current literature. Emphasis is placed on prominent mycotoxins, including aflatoxins, fumonisins, zearalenone (ZEA) and ochratoxins, known for inducing acute and chronic diseases such as liver damage, genetic mutation and cancer. To elucidate the effects, animal studies were conducted, revealing an association between mycotoxin exposure and neurological damage. This encompasses impairments in learning and memory, motor alterations, anxiety and depression. The underlying mechanisms involve oxidative stress, disrupting the balance between reactive oxygen species (ROS) and antioxidant capacity. This oxidative stress is linked to neuronal damage, brain inflammation, neurochemical imbalance, and subsequent behavioural changes. The review underscores the need for preventive measures against mycotoxin exposure. While complete avoidance is ideal, exploration into the potential use of antioxidants as a viable solution is discussed, given the widespread contamination of many food products. Specifically, the protective role of natural compounds, such as polyphenols, is highlighted, showcasing their efficacy in mitigating mycotoxicosis in the central nervous system (CNS), as evidenced by findings in various animal models. In summary, countering mycotoxin-induced neurotoxicity requires a multifaceted approach. The identified natural compounds show promise, but their practical use hinges on factors like bioavailability, toxicity and understanding their mechanisms of action. Extensive research is crucial, considering the diverse responses to different mycotoxins and neurological conditions. Successful implementation relies on factors such as the specific mycotoxin(s) involved and achievable effective concentrations. Further research and clinical trials are imperative to establish the safety and efficacy of these compounds in practical applications.

Programmable DNA tweezers-SDA for ultra-sensitive signal amplification fluorescence sensing strategy.

BACKGROUND: DNA tweezers, classified as DNA nanomachines, have gained prominence as multifunctional biosensors due to their advantages, including a straightforward structure, response mechanism, and high programmability. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. Some small molecules, such as mycotoxins, often require more sensitive detection due to their extremely high toxicity. Therefore, more effective signal amplification strategies are needed to further enhance the sensitivity of DNA tweezers in biosensing. RESULTS: We designed programmable DNA tweezers that detect small-molecule mycotoxins and miRNAs through simple sequence substitution. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. We introduced the Strand Displacement Amplification (SDA) technique to address this limitation, proposing a strategy of novel programmable DNA tweezers-SDA ultrasensitive signal amplification fluorescence sensing. We specifically investigate the effectiveness of this approach concerning signal amplification for two critical mycotoxins: aflatoxin B1 (AFB1) and zearalenone (ZEN). Results indicate that the detection ranges of AFB1 and ZEN via this strategy were 1-10,000 pg mL -1 and 10-100,000 pg mL -1, respectively, with corresponding detection limits of 0.933 pg mL -1 and 1.07 pg mL -1. Compared with the DNA tweezers direct detection method for mycotoxins, the newly constructed programmable DNA tweezers-SDA fluorescence sensing strategy achieved a remarkable 104-fold increase in the detection sensitivity for AFB1 and ZEN. SIGNIFICANCE: The constructed programmable DNA tweezers-SDA ultrasensitive signal-amplified fluorescence sensing strategy exhibits excellent detection performance for mycotoxins. The superb versatility of this strategy allows the developed method to be easily used for detecting other analytes by simply replacing the aptamer and cDNA, which has incredible potential in various fields such as food safety screening, clinical diagnostics, and environmental analysis.

Role of ferroptosis in food-borne mycotoxin-induced toxicities.

Contamination by toxic substances is a major global food safety issue, which poses a serious threat to human health. Mycotoxins are major class of food contaminants, mainly including aflatoxins (AFs), zearalenone (ZON), deoxynivalenol (DON), ochratoxin A (OTA), fumonisins (FBs) and patulin (PAT). Ferroptosis is a newly identified iron-dependent form of programmed or regulated cell death, which has been found to be involved in diverse pathological conditions. Recently, a growing body of evidence has shown that ferroptosis is implicated in the toxicities induced by certain types of food-borne mycotoxins, which provides novel mechanistic insights into mycotoxin-induced toxicities and paves the way for developing ferroptosis-based strategy to combat against toxicities of mycotoxins. In this review article, we summarize the key findings on the involvement of ferroptosis in mycotoxin-induced toxicities and propose issues that need to be addressed in future studies for better utilization of ferroptosis-based approach to manage the toxic effects of mycotoxin contamination.

Estrogen receptor α interaction of zearalenone and its phase I metabolite α-zearalenol in combination with soy isoflavones in hERα-HeLa-9903 cells.

Risk assessment primarily relies on toxicological data of individual substances, with limited information on combined effects. Recent in vitro experiments using Ishikawa cells, an endometrial carcinoma cell line expressing both estrogen receptor isoforms, demonstrated interactive effects of phyto- and mycoestrogens. The mycoestrogens, zearalenone (ZEN), and α-zearalenol (α-ZEL) exhibited significantly enhanced estrogenic responses in the presence of isoflavones (ISF), depending on substance ratios and concentrations. This study investigated the impact of phyto- and mycoestrogen combinations on estrogenic response following OECD guideline 455, utilizing hERα-HeLa-9903 cells. Test substances included mycoestrogens (ZEN and α-ZEL) and isoflavones (genistein (GEN), daidzein (DAI), and S-equol (EQ), a gut microbial metabolite of DAI). Mycoestrogens were tested in the range of 0.001 to 100 nM, while isoflavones were used at concentrations 1000 times higher based on relevant occurrence ratios. Results showed that ZEN and α-ZEL induced ERα-dependent luciferase expression in concentrations above 1 nM and 0.01 nM, respectively. However, ISF caused a superinduction of the luciferase signal above 1 µM. A superinduction is characterized by an unusually strong or heightened increase in the activity of the luciferase enzyme. This signal is not affected by the estrogen receptor antagonist 4-hydroxytamoxifen (4-OH-TAM), which was additionally used to verify whether the increase of signal is a true reflection of receptor activation. This superinduction was observed in all combinations of ZEN and α-ZEL with ISFs. Contrary to the luciferase activity findings, RT-qPCR experiments and a stability approach revealed lower real ERα activation by ISFs than measured in the ONE-Glo™ luciferase test system. In conclusion, the OECD protocol 455 appears unsuitable for testing ISFs due to their superinduction of luciferase and interactions with the test system, resulting in experimental artifacts. Further studies are necessary to explore structure-activity relationships within polyphenols and clarify the test system's applicability.

Sensitive fluorescence ELISA for the detection of zearalenone based on self-assembly DNA nanocomposites and copper nanoclusters.

Zearalenone (ZEN), produced by Fusarium species, is a potential risk to human health. Traditional enzyme-linked immunosorbent assay (ELISA) is restricted due to low sensitivity for the detection of ZEN. Herein, enzyme nanocomposites (ALP-SA-Bio-ssDNA, ASBD) were prepared with the self-assembly strategy based on streptavidin-labeled alkaline phosphatase (SA-ALP) and dual-biotinylated ssDNA (B2-ssDNA). The enzyme nanocomposites improved the loading amount of ALP and catalyzed more ascorbic acid 2-phosphate to generate ascorbic acid (AA). Subsequently, Cu2+ could be reduced to copper nanoclusters (CuNCs) having strong fluorescence signal by AA with poly T. Benefiting from the high enzyme load of nanocomposites and the strong signal of CuNCs, the fluorescence ELISA was successfully established for the detection of ZEN. The proposed method exhibited lower limit of detection (0.26 ng mL-1) than traditional ELISA (1.55 ng mL-1). The recovery rates ranged from 92.00% to 108.38% (coefficient of variation < 9.50%) for the detection of zearalenone in corn and wheat samples. In addition, the proposed method exhibited no cross reaction with four other mycotoxins. This proposed method could be used in trace detection for food safety.

A Probiotic Bacillus amyloliquefaciens D-1 Strain Is Responsible for Zearalenone Detoxifying in Coix Semen.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium spp., which commonly and severely contaminate food/feed. ZEN severely affects food/feed safety and reduces economic losses owing to its carcinogenicity, genotoxicity, reproductive toxicity, endocrine effects, and immunotoxicity. To explore efficient methods to detoxify ZEN, we identified and characterized an efficient ZEN-detoxifying microbiota from the culturable microbiome of Pseudostellaria heterophylla rhizosphere soil, designated Bacillus amyloliquefaciens D-1. Its highest ZEN degradation rate reached 96.13% under the optimal condition. And, D-1 can almost completely remove ZEN (90 µg·g-1) from coix semen in 24 h. Then, the D-1 strain can detoxify ZEN to ZEM, which is a new structural metabolite, through hydrolyzation and decarboxylation at the ester group in the lactone ring and amino acid esterification at C2 and C4 hydroxy. Notably, ZEM has reduced the impact on viability, and the damage of cell membrane and nucleus DNA and can significantly decrease the cell apoptosis in the HepG2 cell and TM4 cell. In addition, it was found that the D-1 strain has no adverse effect on the HepG2 and TM4 cells. Our findings can provide an efficient microbial resource and a reliable reference strategy for the biological detoxification of ZEN.

[Determination of seven mycotoxins in cereals by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry based on the self-built database].

A method for the determination of seven mycotoxins in rice and wheat by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) based on self-built database was established. Samples were extracted with 0.2% formic acid aqueous solution-acetonitrile (50∶50, v/v), dehydrated and salted according to the QuEChERS method (4 g of magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate, 0.5 g of citrate disodium salt), and separated on an HSS T3 column (100 mm×2.1 mm, 1.8 µm). UPLC-Q-TOF/MS with MSE screening was performed, and the positive and negative ions of the screened mycotoxins were calibrated and quantified using matrix-matched standard curves with time of flight multiple reaction monitoring (TOF-MRM). The results showed that aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), and ochratoxin A (OTA) exhibited moderate matrix effects in rice, while OTA and zearalenone (ZEN) exhibited moderate matrix effects in wheat. The seven mycotoxins showed good linearities in their respective concentration ranges, with correlation coefficients (r) of 0.9900-0.9998. The limits of detection (LODs) for rice and wheat were 0.50-400 and 0.50-200 µg/kg, respectively, and the limits of quantification (LOQs) for both cereals were 1.00-800 µg/kg. In rice, the average recoveries at three spiked levels of low, medium, and high were 88.1%-123.9%, with relative standard deviations (RSDs) of 0.2%-13.6%. In wheat, the average recoveries at three spiked levels of low, medium, and high were 102.0%-123.4%, with RSDs of 0.8%-14.8%. As a result, one batch of 46 batches of rice was screened out for AFB1 and AFB2, with a screening rate of 2.2%, of which the measured values were 10.8 µg/kg and 1.2 µg/kg, respectively. According to GB 2761-2017, the maximum allowable level of AFB1 in rice is 10 µg/kg; thus, the exceeding rate for AFB1 is 2.2%. Deoxynivalenol (DON) was screened out in 9 out of 24 batches of wheat (screening rate, 37.5%), while ZEN was screened out in 19 batches (screening rate, 79.2%). According to GB 2761-2017, the maximum allowable levels of DON and ZEN in wheat are 1000 and 60 µg/kg, respectively. The levels of DON and ZEN detected in the wheat samples did not exceed these limits. The proposed method uses MSE for qualitative screening to avoid the occurrence of false positives caused by interfering compounds with mass numbers and retention times similar to those of the analytes. TOF-MRM mode is then used to quantify the positively screened mycotoxins. The method is fast, accurate, sensitive, and suitable for the isolation and quantitative detection of mycotoxin residues in rice and wheat samples. The findings provide powerful technical support for mycotoxin contamination monitoring in rice and wheat and early risk-warning efforts.

Effect of Aspergillus flavus contamination on the fungal community succession, mycotoxin production and storage quality of maize kernels at various temperatures.

Aspergillus flavus, a notorious saprobe and opportunistic plant pathogen, alters mycotoxin contamination and biochemical components in maize kernels during processing and storage, thereby reducing the possibilities of maize end use and compromising food safety. This study explored changes in mycotoxin production, fungal community succession and biochemical components in maize kernels stored at 20, 25 and 30 °C, exposed to A. flavus. Results showed that aflatoxin B1 concentration increased over time, reaching 4.88 µg/kg at 20 °C, 167.23 µg/kg at 25 °C and 349.64 µg/kg at 30 °C after 15 days of storage, whereas the zearalenone production was characterized by an increase followed by a decrease. Correspondingly, the number of molds gradually increased and reached a stable stage after 10 days. High-throughput sequencing of the internal transcribed spacer (ITS) revealed that Eurotium dominated the fungal communities, with A. flavus reaching maximum abundance in maize kernels stored at 30 °C for 15 days. Correlation analysis indicated that the relative abundance of A. flavus was significantly negatively correlated with the content of zein and moisture (P < 0.05). Moreover, the wet milling process of maize effectively eliminated the concentration of aflatoxin B1 and zearalenone from the starch. Pasting temperature and setback value of starch decreased while peak viscosity, final viscosity and breakdown value increased with storage. These findings indicate that interactions between the epiphytic fungal community and A. flavus at elevated storage temperatures aggravate both maize quality deterioration and mycotoxin contamination. Furthermore, they have a discernible impact on the pasting properties of starch. This insight informs strategies to control fungal infections during maize processing and storage.

Fabrication of a disposable aptasensing chip for simultaneous label-free detection of four common coexisting mycotoxins.

BACKGROUND: Coexisting multiple mycotoxins in food poses severe health risks on humans due to the augmented toxicity. Current multiplex detection methods for mycotoxins have evolved from instrumental analyses to rapid methods based on the specific recognition of antibody/aptamer using different signal transducers. However, nearly all of the reported aptasensors for multiple mycotoxins detection require external labels and can only simultaneous detection of two mycotoxins due to the limitation of distinguishable labels. The tedious labeling process definitely increases the operation complexity and the detection cost. Therefore, rapid method for simultaneous label-free detection of multiple mycotoxins in cereals is urgently needed. RESULTS: A disposable aptasensing chip was designed for simultaneous label-free detection of fumonisin B1 (FB1), aflatoxin B1 (AFB1), zearalenone (ZEN), and ochratoxin A (OTA) in one sample. Specifically, ITO conductive glass was divided into a rectangle (35 × 25 mm) and then etched by laser to set aside the required four ITO working electrodes (6 mm in diameter) with respective conductive channels. Gold nanoparticles were electrodeposited on the working electrodes to provide abundant anchoring sites for thiolated aptamers immobilization. On this basis, a disposable aptasensing chip for simultaneous label-free detection of four common coexisting mycotoxins has been developed, which used electrochemical impedance spectroscopy as transducer to measure direct biorecognition of the aptamer and corresponding target. This aptasensing chip provided wide linear ranges of 5-1000, 10-250, 10-1250, 10-1500 ng/mL for FB1, AFB1, ZEN, OTA, respectively, with the respective detection limit of 2.47, 3.19, 5.38, 4.87 ng/mL (S/N = 3). SIGNIFICANCE AND NOVELTY: This aptasensing chip shows fantastic characteristics of great simplicity and portability, easy operation, and multiple mycotoxins recognition. They are easy to produce on a large scale at low cost and the design concept can be easily expanded to screen a large panel of coexisting targets. This work provides a new avenue for multi-target detection and represents a substantial advance toward food quality and safety monitoring or other fields.

Efficacy of Two Commercially Available Adsorbents to Reduce the Combined Toxic Effects of Dietary Aflatoxins, Fumonisins, and Zearalenone and Their Residues in the Tissues of Weaned Pigs.

Mycotoxins present a significant health concern within the animal-feed industry, with profound implications for the pig-farming sector. The objective of this study was to evaluate the efficacy of two commercial adsorbents, an organically modified clinoptilolite (OMC) and a multicomponent mycotoxin detoxifying agent (MMDA), to ameliorate the combined adverse effects of dietary aflatoxins (AFs: sum of AFB1, AFB2, AFG1, and AFG2), fumonisins (FBs), and zearalenone (ZEN) at levels of nearly 0.5, 1.0, and 1.0 mg/kg, on a cohort of cross-bred female pigs (N = 24). Pigs were randomly allocated into six experimental groups (control, mycotoxins (MTX) alone, MTX + OMC 1.5 kg/ton, MTX + OMC 3.0 kg/ton, MTX + MMDA 1.5 kg/ton, and MTX + MMDA 3.0 kg/ton), each consisting of four individuals, and subjected to a dietary regimen spanning 42 days. The administration of combined AFs, FBs, and ZEN reduced the body-weight gain and increased the relative weight of the liver, while there was no negative influence observed on the serum biochemistry of animals. The supplementation of OMC and MMDA ameliorated the toxic effects, as observed in organ histology, and provided a notable reduction in residual AFs, FBs, and ZEN levels in the liver and kidneys. Moreover, the OMC supplementation was able to reduce the initiation of liver carcinogenesis without any hepatotoxic side effects. These findings demonstrate that the use of OMC and MMDA effectively mitigated the adverse effects of dietary AFs, FBs, and ZEN in piglets. Further studies should explore the long-term protective effects of the studied adsorbent supplementation to optimize mycotoxin management strategies in pig-farming operations.

High Bioaffinity Controllable Assembly Nanocarrier UiO-66-NH2@Quantum Dot-Based Immunochromatographic Assay for Simultaneous Detection of Five Mycotoxins in Cereals and Feed.

Herein, the UiO-66-NH2@quantum dot (NU66@QD) was synthesized with excellent fluorescence intensity and biocompatibility, which was used to develop a multiple immunochromatographic assay (ICA) for the detection of aflatoxin B1 (AFB1), fumonisin B1 (FB1), deoxynivalenol (DON), T-2 toxins (T-2), and zearalenone (ZEN) in cereals and feed. Five monoclonal antibodies and NU66@QD were efficiently labeled by a one-step mixed method to form a multiple detection probe. The limits of detection of the proposed NU66@QD-ICA for AFB1/FB1/DON/T-2/ZEN were 0.04/0.28/0.25/0.09/0.08 µg/kg. The recoveries ranged from 82.83-117.44%, with the coefficient of variation from 2.88-11.80%. A parallel analysis in 35 naturally contaminated cereal and feed samples was confirmed by LC-MS/MS, and the results showed a good correlation (R2 > 0.9), indicating the practical reliability of the multiple NU66@QD-ICA. Overall, the introduction of the novel nanomaterial NU66@QD provides a highly sensitive and efficient multiplex detection strategy for the development of ICA.

Regulated and Emerging Mycotoxins in Bulk Raw Milk: What Is the Human Risk?

Mycotoxins are abiotic hazards whose contamination occurs at the pre- and post-harvest stages of the maize value chain, with animal exposure through contaminated feed leading to their excretion into milk. Currently, only aflatoxin M1 is regulated in milk products. Since feed materials and complete feed present a multi-mycotoxin composition and are the main mycotoxin source into milk, it is important to recognize the occurrence of multiple toxins and their co-occurrence in this highly consumed food product. The aim of this study was to determine the content of regulated and emerging mycotoxins in milk samples, which allowed for evaluating the occurrence and co-occurrence patterns of different mycotoxins known to contaminate feed materials and complete animal feed. Human exposure considering the occurrence patterns obtained was also estimated. Aflatoxins, fumonisins, zearalenone, and emerging mycotoxins were among the mycotoxins found to be present in the 100 samples analyzed. Concentrations ranged from 0.006 to 16.3 µg L-1, with no sample exceeding the AFM1 maximum level. Though several mycotoxins were detected, no exceeding values were observed considering the TDI or PMTDI. It can be concluded that the observed exposure does not pose a health risk to milk consumers, though it is important to recognize vulnerable age groups.