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| 测定纳米银在河口水中对副溶血弧菌抑制效应的高效方法 |
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侯向祎1,2, 王笑旸3, 张圆圆2, 梁若寒4, 卢峰5, 杨倩倩6, 普小丹7, 张艳8, 曲克明9, 张旭志10
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1.上海海洋大学海洋科学与生态环境学院 上海 201306;2.中国水产科学研究院黄海水产研究所
山东 青岛 266071;3.中国海洋大学食品科学与工程系 山东 青岛 266003;4.中国水产科学研究院黄海水产研究所
山东 青岛 266072;5.中国水产科学研究院黄海水产研究所
山东 青岛 266073;6.中国水产科学研究院黄海水产研究所
山东 青岛 266074;7.中国水产科学研究院黄海水产研究所
山东 青岛 266075;8.中国水产科学研究院黄海水产研究所
山东 青岛 266076;9.中国水产科学研究院黄海水产研究所
山东 青岛 266077;10.中国水产科学研究院黄海水产研究所
山东 青岛 266078
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| 摘要: |
| 由于河口水具有复杂的物理和化学性质，准确、高效地测定纳米银在其中的抑菌效应是个国际性挑战。本研究基于电子微生物生长分析仪建立了一种自动化表型方法。副溶血弧菌(Vibrio parahaemolyticus)在河口水中暴露于纳米银之后，暴露混合液直接被加进预装有Luria-Bertani液体培养基的检测管中，置入电子微生物生长分析仪测定细菌的生长动力学曲线，根据细菌生长曲线判读纳米银对副溶血弧菌的最小抑菌浓度。采用电子微生物生长分析仪测定了纳米银在8个河口水样品中的抑菌效应，结果表明，所得最小抑菌浓度值都与采用经典平板计数法和微量肉汤稀释法所得结果吻合良好。较之于经典方法，所建新方法的优势在于无需去除暴露混合液中诸如悬浮颗粒之类的复杂共存物，因此操作更简便、劳动强度小，总周期缩短至少20 min，并有效降低了主观和客观操作误差风险，具有良好的精密度和重现性。此外，电子微生物生长分析仪法测得的最小抑菌浓度值通常不低于平板计数法和微量肉汤稀释法所得数值，表明该基于传感器识别结果的方法比基于视觉判别结果的方法具有更高的灵敏度。本研究为准确、高效测定纳米材料在诸如河口水之类复杂介质中的环境毒理效应提供了新手段。 |
| 关键词: 纳米银 生态毒性 河口水 副溶血弧菌 电子微生物生长分析仪 |
| DOI：10.19663/j.issn2095-9869.20240327001 |
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| 基金项目:青岛市市南区科技计划(2023-2-026-YY)和山东省重点研发计划(2024TSGC0259)共同资助 |
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| An efficient analytical method for determining the effects of silver nanoparticles on Vibrio parahaemolyticus in estuarine water |
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HOU Xiangyi1,2, WANG Xiaoyang3, ZHANG Yuanyuan2, LIANG Ruohan4, LU Feng5, YANG Qianqian6, PU Xiaodan7, ZHANG Yan8, QU Keming9, ZHANG Xuzhi10
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1.School of Marine Ecology and Environment, Shanghai Ocean University, Shanghai 201306, China;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;3.Department of Food Science and Engineering, Ocean University of China, Qingdao 266003, China;4.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266072, China;5.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266073, China;6.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266074, China;7.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266075, China;8.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266076, China;9.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266077, China;10.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266078, China
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| Abstract: |
| In virtue of their distinctive antimicrobial properties, silver nanoparticles (Ag NPs) are some of the most commonly used nanomaterials in the world, with applications in medical equipment, cosmetics, textiles, electronics, toys, and household appliances. As a result, they inevitably end up in rivers, lakes, estuaries, and coastal waters via wastewater, atmospheric deposition, and other pathways. Recent explorations have increased concerns regarding their adverse effects on the ecological health of estuarine environments. For risk assessment of nanomaterials in estuarine environments, microorganisms - especially bacteria - are ideal candidates as bioreporters. Reliable and effective methods for determining the effects of nanomaterials on microorganisms are of significance for assessing ecotoxicities. Growth curve-based methods are popular because they can fully reflect the toxicity of nanomaterials. Genotypic methods, which are based on DNA analysis, provide attractive alternatives. These phenotypic and genotypic methods have performed well in determining the effects of nanomaterials on microorganisms in simple laboratory media. However, when they are used in realistic matrices, such as estuarine water, which is complex in physical, chemical, and ecological characteristics, pretreatment steps for separation and purification are unavoidably applied prior to the determination steps. These pretreatment steps usually pose the risk of subjective and objective errors and poor efficiency. To date, more efficient and accurate analytical methods are still needed for assessing the ecotoxicity of nanomaterials.
Recently, our research group contributed to an alternative concept for online monitoring of microbial growth by developing a multichannel capacitively coupled contactless conductivity (C4) detector. C4 detection is a particular type of conductivity-based analytical method, where the electrodes are not in direct contact with the tested medium. The magnitude of the detected signal (C4 output) is proportional to the concentration and mobility of the ionic charge carriers within the medium. It not only shares the advantages of common electrochemical techniques, such as instrumental simplicity, affordability, rapid response, nontransparent requirement, and easy miniaturization, but is also free of polarization, passivation, and fouling risks. Based on a 32-channel C4 detector and special algorithms, we developed a 32-channel electronic microbial growth analyzer (EMGA). EMGA could determine repeatable bacterial growth curves with a high temporal resolution in both homogeneous simple laboratory mediums and heterogeneous matrices.
The EMGA method was used to evaluate the antibacterial effects of Ag NPs on Vibrio parahaemolyticus, compared with the use of the broth microdilution method (BMD） and plate counting methods. The minimum inhibitory concentration (MIC) of Ag NPs against V. parahaemolyticus in estuarine water samples determined by using the EMGA method is 24.0 mg/L, which is consistent with the results obtained by using the BMD and plate counting methods. The results obtained by using the EMGA method are in good agreement with the MIC values of the BMD and plate counting methods, with an essential agreement (EA) of 75% and minor error (mE) of 25%. No major error (ME) was found, indicating that the EMGA method for measuring the effects of Ag NPs on V. parahaemolyticus in estuarine water samples is reliable. In addition, the MIC values obtained by using the EMGA method are often higher than or equal to the results obtained by using the BMD and plate counting methods, due to the higher sensitivity of automated instruments compared to visual observation. Therefore, the antibacterial activity obtained by using the EMGA method is reliable than the results based on visual judgment.
This study established a phenotypic method for determining the antibacterial activity of Ag NPs against V. parahaemolyticus in estuarine water. This method requires only two manual steps rather than three as in classical methods such as BMD and plate counting. In addition, due to the elimination of complex coexistent substances, it effectively reduces the risk of subjective and objective operational errors. This automated method based on sensor recognition results has higher sensitivity compared with the BMD and plate counting methods. Thus, the newly proposed method has the advantages of simplicity, time-saving, low-labor intensive, greater precision, and good repeatability. In addition, the sensitivity of this automatic instrument-based method is higher compared with eye-based methods. This efficient method provides a new approach for assessing ecotoxicity of nanomaterials in realistic environmental matrices, such as estuarine water. |
| Key words: Silver nanoparticles Ecotoxicity Estuarine water Vibrio parahaemolyticus Electronic microbial growth analyzer |
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