Restricted-Access Media Column Switching Online Solid-Phase Extraction UHPLC-MS/MS for the Determination of Seven Type B Trichothecenes in Whole-Grain Preprocessed Foods and Human Exposure Risk Assessment.

With increasing health awareness and the accelerating pace of life, whole-grain prepared foods have gained popularity due to their health benefits and convenience. However, the potential risk of type B trichothecene toxins has also increased, and these mycotoxins in such foods are rarely regulated. In this study, a quantitative method combining a single-valve dual-column automatic online solid-phase extraction system with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed for the first time using restricted-access media columns. This method can simultaneously determine trace residues of seven type B trichothecenes within 15 min. The method is convenient, sensitive (limit of detection and quantification of 0.05-0.6 µg/kg and 0.15-2 µg/kg, respectively), accurate (recovery rates of 90.3%-106.6%, relative standard deviation < 4.3%), and robust (>1000 times). The established method was applied to 160 prepared food samples of eight categories sold in China. At least one toxin was detected in 70% of the samples. Whole-wheat dumpling wrappers had the highest contamination rate (95%) and the highest total content of type B trichothecenes in a single sample (2077.3 µg/kg). Exposure risk assessment indicated that the contamination of whole-grain prepared foods has been underestimated. The total health risk index of whole-wheat dumpling wrappers, which are susceptible to deoxynivalenol, reached 136.41%, posing a significant threat to human health. Effective measures urgently need to be taken to control this risk.

3D "honeycomb" cell/carbon nanofiber/gelatin methacryloyl (GelMA) modified screen-printed electrode for electrochemical assessment of the combined toxicity of deoxynivalenol family mycotoxins.

A "honeycomb" electrochemical biosensor based on 3D printing was developed to noninvasively monitor the viability of 3D cells and evaluate the individual or combined toxicity of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), and 15-acetyldeoxynivalenol (15-ADON). Carbon nanofiber (CN)/gelatin methacryloyl (GelMA) conductive composite hydrogel with strong processability was printed on 8-channel screen-printed carbon electrodes (SPCEs) to maintain cell viability and form tight cell-to-cell contacts. A "3D honeycomb" printing infill pattern was selected in the construction of the biosensors to improve conductivity. Based on 3D printing technology, the electrochemical biosensor can prevent manual error and provide for high-throughput detection. Electrochemical impedance spectroscopy (EIS) was used to evaluate mycotoxin toxicity. The EIS response decreased with the concentration of DON, 3-ADON and 15-ADON in the range of 0.1-10, 0.05-100, and 0.1-10 µg/mL, respectively, with a limit of detection of 0.07, 0.10 and 0.06 µg/mL, respectively. Mycotoxin interactions were analyzed using the isobologram-combination index (CI) method. The electrochemical cytotoxicity evaluation result was confirmed by biological assays. Therefore, a novel method for evaluating the combined toxicity of mycotoxins is proposed, which exhibits potential for application to food safety and evaluation.

Development of a simple and convenient cell-based electrochemical biosensor for evaluating the individual and combined toxicity of DON, ZEN, and AFB1.

A simple and convenient cell-based electrochemical biosensor was developed to assess the individual and combined toxicity of deoxynivalenol (DON), zearalenone (ZEN), and Aflatoxin B1 (AFB1) on Hep G2 cells. The sensor was modified in succession with AuNPs (gold nanoparticles), cysteamine, and laminin. The cells interacting with laminin formed tight cell-to-electrode contacts, and collagen was used to maintain cell adhesion and viability. Electrochemical impedance spectroscopy (EIS) was developed to evaluate mycotoxin toxicity. Experimental results show that DON, ZEN, and AFB1 caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of DON, ZEN, and AFB1 in the range of 0.01-20, 0.1-50, and 0.1-3.5µg/mL, and IC50 obtained using the developed method was 48.5, 59.0, and 3.10µg/mL, respectively. A synergistic effect was observed between DON and ZEN, an additive effect was observed between DON and AFB1, and an antagonism effect was found in the binary mixtures of ZEN and AFB1 and ternary mixtures. These results were confirmed via CCK-8 assay. Utilizing SEM, we found that cells treated with mycotoxins caused significant changes in cell morphology, thus lessening cell adsorption and impedance reduction. Biological assay indicated that EIS patterns correlated with [Ca2+]i concentrations and apoptosis and necrotic cells ratios, thus effecting electrochemical signals. This method is simpler, more convenient, sensitive, and has a quicker response rate than most conventional cytotoxicity evaluation methods.

A Sensitive and simple macrophage-based electrochemical biosensor for evaluating lipopolysaccharide cytotoxicity of pathogenic bacteria.

In this study, a sensitive and simple electrochemical murine macrophage (Ana-1) cell sensor has been developed for early detection of lipopolysaccharides (LPS) to evaluate the toxicity of pathogenic bacteria. Magnetic glassy carbon electrode (MGCE), which possesses excellent reproducibility and regeneration qualities, was modified with a nanocomposite to improve electrochemical signals and enhance the sensitivity. The synthesized magnetic nanoparticles (MNPs) were internalized into murine macrophages, which completed the immobilization of macrophages onto the modified electrode for evaluating the cytotoxicity of LPS by electrochemical impedance spectroscopy (EIS). The MNPs facilitated reusability of the proposed sensor by allowing removal of the magnetic core from the electrode. Our results indicated that LPS caused a marked decrease in electrochemical impedance in a dose-dependent manner in range of 1-5µg/mL. By SEM, we found that microvilli on the plasma membrane became scarce and the membrane became smooth on cells incubated with LPS, which lessens the absorption of cells to reduce the impedance. And biological assay indicated that EIS patterns were correlated with the calcium concentration in cells, and suggested that [Ca(2+)]i production increased in cells incubated with LPS and its mobilization altered electrochemical signals. Compared with conventional methods, this electrochemical test is inexpensive, highly sensitive, and has a quick response, and thus provides a new avenue for evaluating the cytotoxicity of pathogens.

Development of a novel electrochemical sensor using pheochromocytoma cells and its assessment of acrylamide cytotoxicity.

We report on a sensitive, simple, label-free cell-based electrochemical sensor to monitor the toxic effect of acrylamide on the Pheochromocytoma cells. The surface of the electrode was modified with gold nanoparticles and electrochemically reduced graphene oxide. Cyclic voltammetry, impedance spectroscopy and differential pulse voltammetry were applied to characterize the modified electrode. Reduced graphene oxide was proved to increase electron-transfer rate between the cell and the surface of electrode, while gold nanoparticle retain cell bioactivity. The sensor exhibited good correlation to the logarithmic value of cell numbers ranging from 1.6×10(4) to 1.6×10(7) cells mL(-1), with R.S.D value of 1.68%. The value of differential pulse voltammetry (cell adsorption concentration of 1.6×10(7) cells mL(-1)) decreased with the concentration of acrylamide in range of 0.1-5 mM with the detection limit as 0.04 mM. Scanning electron microscope-based morphological and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis confirmed the results of the electrochemical study. This sensor was proved to be a useful tool for probing the toxicity of cells, and assisted in the development of a labeling-free, simple, rapid and immediate detection method.