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

https://doi.org/10.1016/j.bioelechem.2021.107743Get rights and content

Highlights

  • A 3D-printed biosensor for combined toxicity assessment of mycotoxins is presented.

  • Cells/carbon nanofiber/gelatin methacryloyl composite hydrogel was constructed.

  • The “3D honeycomb” printing infill pattern was chosen to optimize conductivity.

  • The LOD of DON, 3-ADON, and 15-ADON were 0.07, 0.10 and 0.06 μg/mL, respectively.

Abstract

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.

Introduction

Deoxynivalenol (DON) is a secondary metabolite mainly produced by Fusarium fungi, which typically invades all kinds of crops, including cereal crops, creates high levels of pollution and is detected with high frequency worldwide [1]. It cannot be eliminated during food processing. DON mainly affects the immune system and gastrointestinal tract, causing vomiting, gastroenteritis, and immune dysfunction. Research into the relationships between airborne transmission, degree of mold exposure and inflammatory airway disease has also attracted wide attention. According to reports, farm workers are at risk of exposure to DON when handling grain or silage[2], [3], [4]. Studies on the induction of MAPK-dependent transcription factors by DON in human cell lines indicate that the expression of ATF3 mRNA was the highest in A549 cells after DON exposure[5]. Therefore, the A549 cell line is of great significance for exploring the toxicity mechanism of DON.

Similar to DON, 3-ADON and 15-ADON are also detected with high frequency in cereals (87% and 73% 3-ADON and 15-ADON positive samples, respectively) [6]. DON may be produced with its two acetylated derivatives, and their presence can be seen in up to 10–20% of DON containing samples (EFSA, 2011). In vivo experiments have shown that 3-ADON is rapidly converted into DON in blood [7]. Data indicate that acetylated DON is more quickly absorbed in the intestine and thus has been considered more toxic [8]. Studies on mycotoxin toxicity often focus on the effects of individual toxins, but few studies comprehensively discuss the mechanism of action underlying toxic effects due to combined mycotoxins. With the known co-existence of multiple toxins, a method for evaluating combined toxicity with multiple toxins needs to be established.

Toxicity evaluation methods mostly rely on cell experiments in two-dimensional culture and animal experiments. Two-dimensional cell experiments present advantages such as low cost and a short evaluation cycle. The cells also exhibit a certain degree of homology with the body, but the two-dimensional cell culture environment differs from the human in vivo environment [9]. Toxicology experiments using animals yield results that can truly and comprehensively reflect the effect of drugs on the body, these experiments entail high cost and require long experimental periods among other disadvantages. Owing to high sensitivity and portability, cell-based biosensors have recently become attractive substitutes for traditional methods. In the construction of cell sensors, cells as receptors are fixed to the interface. The magnitude of a change in signal can be used for the qualitative and quantitative analysis of drug stimulation on cells [10], [11], [12]. Compared with traditional techniques, a three-dimensional (3D) cell culture platform can simulate the microenvironment of cells in vivo more efficiently, and monitor the response of cells to exogenous hazards more accurately [13], [14], [15], [16]. As a simple and miniaturized detection method, the 3D cell-based biosensor has attracted considerable attention in the toxicity evaluation of drugs and pollutants.

As a type of computer-aided technology, 3D bioprinting can produce engineered tissue in a mechanized, organized, and optimized manner. It can assemble tissue by accurately locating biomaterials and living cells layer by layer and has a high spatial control capability. 3D printing technology can be applied not only to the creation of basic arrays but in the development of complex constructs by setting different sample retention times or repeating specific printer G code commands, also without the need to produce special molds or masks for patterning. Moreover, 3D printing has led to the availability of 3D tumor array chips, which are now required for specific, on demand, drug screening for rapid throughput [17], [18]. This technology is used on the development and preparation of biosensors, with the advantage of mass production, reduction of artificial error and convenient operation. Gelatin methacrylate (GelMA) hydrogel is widely used in 3D printing as a kind of bioink. It comprises natural gelatin functionalized using methacrylic anhydride, which shows good processability and biocompatibility [16], [19], [20]. The hydrogel can be used as an auxiliary bonding material to form a stereoscopic structure for the construction of a 3D cell environment. When prepared with culture medium, the hydrogel can provide a cell growth environment that allows cells to link with one another [21], [22].

With the advantages of high sensitivity, fast response, ease of operation, and simplicity of analysis, EIS can be used to evaluate the electrochemical behavior of cytotoxicity and has been recognized as one of the most attractive label-free technologies. EIS has also been successfully used to monitor morphological changes during cell adhesion and apoptosis, as well as to determine drug cytotoxicity [23], [24], [25]. Traditional electrodes, such as gold electrodes and glassy carbon electrodes, easily become polluted and require polishing and regeneration. They are complex and time-consuming, limiting their application. Screen-printed carbon electrodes (SPCEs) are low-cost, have a wide potential range, and exhibit good chemistry [26]. However, printing carbon electrodes on paper also involve challenges, such as limited sensitivity and high electrochemical resistance. These problems are addressed using carbon-conductive nanomaterials, which are widely used in electronic devices. These nanomaterials possess distinct properties, such as rapid electron transfer, high thermal conductivity, high mechanical flexibility, and good biocompatibility [27], [28]. With distinct mechanical properties and superior electrical properties, carbon nanofiber (CN) is a modified material widely used in electrochemistry [29].

In the present study, a cell electrochemical sensor based on 3D bioprinting was proposed to evaluate the individual and combined toxicity of DON, 3-ADON, and 15-ADON. GelMA provides a 3D growth environment for cells, which can better simulate in vivo environment and assess toxicity more accurately. Variations in electrochemical reactions caused by mycotoxins stimulating A549 cells are presented. Cell viability, reactive oxygen species (ROS) and [Ca2+]i concentration were also measured; the EIS signal was found to correspond to the changes in ROS, [Ca2+]i concentration, and apoptotic and necrotic cell ratios. The results indicate that the method is simple, convenient, and shows potential for high-throughput online detection analysis. The proposed method provides a promising alternative for the cytotoxic evaluation of mycotoxins.

Section snippets

Materials and apparatus

Chloroauric acid (HAuCl4·4H2O) and toxin standards (DON, 3-ADON and 15-ADON) were purchased from Sigma-Aldrich Inc (St. Louis, MO, USA). The toxin standards were dissolved in dimethyl sulfoxide (DMSO) to make a 25 mg/mL stock solution and stored at 4 °C for further use. Carbon nanofibers (XFM60) measured 200–600 nm in diameter and 5–50 μm in length were supplied by Xian Feng Nanomaterials Technology Co., Ltd (Nanjing, China). Human lung adenocarcinoma epithelial cells (A549) were purchased from

Characterization of screen-printed electrode constructed by 3D printing

An 8-channel “honeycomb” electrochemical biosensor was developed to evaluate the activities of 3D culture cells. Scheme 1 describes the preparation of the electrochemical sensor based on bioprinting. To improve the electrochemical sensitivity of SPCEs, gold nanoparticles (AuNPs) were electroplated on the working electrode of the SPCEs. The scanning electron microscopy (SEM) images in Fig. 1D (b) show that, the AuNPs were uniformly distributed on the electrode surface. Carbon nanofibers were

Conclusion

Sensors for assessing cytotoxic responses are highly suitable for various sensing applications, including toxin detection and drug evaluation. Using cells as biosensors can simulate human physiological responses to a certain extent. Numerous toxicity sensors have been proposed. Most of the electrochemical cell sensors are constructed based on manual operation, which inevitably introduces human error. In this study, a simple and multi-sample electrochemical biosensor based on 3D printing was

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Natural Science Foundation of China (No. 31772069, No. 31801660), the fellowship China Postdoctoral Science Foundation (2020M671343), Natural Science Foundation of Jiangsu Province (No. BK20190584), the Fundamental Research Funds for the Central Universities (1022050205205500), National first-class discipline program of Food Science and Technology (JUFSTR20180303), Collaborative Innovation Center for Food Safety and Quality Control.

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