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| 高效测定纳米银在河口水中对副溶血弧菌抑制效应的方法 |
| An efficient analytical method for determining the effect of silver nanoparticles on Vibrio parahaemolyticus in estuarine water |
| 投稿时间：2024-03-27 修订日期：2024-04-30 |
| DOI： |
| 中文关键词: 纳米银 生态毒性 河口水 V. parahaemolyticus 电子微生物生长分析仪 |
| 英文关键词: Silver nanoparticles, Ecotoxicity, Estuarine water, V. parahaemolyticus, Electronic microbial growth analyzer |
| 基金项目:山东省自然科学基金重点项目（ZR2020KB021）山东省重点研发计划（2020CXGC010703） |
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| 中文摘要: |
| 纳米银（Ag NPs）对水环境中微生物的威胁日益引人关注。由于河口水之类的实际样品往往具有复杂的物理和化学性质，迄今为止国际上尚未有方法能够高效、准确地测定纳米材料在其中对微生物的抑制效应。基于电子微生物生长分析仪（EMGA），以副溶血弧菌（V. parahaemolyticus）作为模式微生物，本研究建立了一种自动化程度较高的表型方法。V. parahaemolyticus在模拟河口水中急性暴露于系列浓度的Ag NPs，然后将一定量暴露混合液直接移入预装有LB液体培养基的检测管中，置入EMGA自动化测定细菌的生长动力学曲线，进而根据细菌生长曲线判读该纳米材料对V. parahaemolyticus的最小抑菌浓度（MIC）。采用EMGA法测定Ag NPs在8个模拟河口水样品中的抑菌效应，所得MIC值都与采用经典平板计数法和微量肉汤稀释（BMD）法所得结果吻合良好。新优势在于无需将暴露混合液的细菌分离出来，因此操作更简便、劳动强度低，总检测 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（比对试验得到的MIC值完全相同）。同时，该基于传感器识别结果的自动化方法比BMD 法和平板计数法具有更高的灵敏度。本研究所建EMGA法为准确测定纳米材料在诸如河口水之类复杂环境介质中的抑菌效应提供了较为高效的手段。 |
| 英文摘要: |
| In virtue of their distinctive antimicrobial properties, silver nanoparticles (Ag NPs) are one 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 the risk assessment of nanomaterials in estuarine environments, microbes - especially bacteria - are ideal candidates as bioreporters. Therefore, determining the ecotoxicity of Ag NPs toward bacteria is not only straightforward but also integral to environmental risk assessment. Reliable and effective methods for determining the effect 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 effect of nanomaterials on microorganisms in simple laboratory media. However, when they are used to determine the effects of nanomaterials on microorganisms 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 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 developed 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, Over the range of 102 – 106 CFU/mL, the linear regression equation was Tmgr (min) = ?83.601log [V. parahaemolyticus (CFU/mL)] + 714.33 with a correlation coefficient r of 0.9981. This suggests that the EMGA method can not only determine the growth curve of V. parahaemolyticus in LB liquid culture medium, but also be used for quantitative analysis. The correlation and accuracy were as good as or even better than those obtained with the automated OD method.
EMGA method was used to evaluate the antibacterial effect of Ag NPs on V. parahaemolyticus, and compared with OD method, BMD method, and plate counting method. The MIC of Ag NPs measured by EMGA in physiological saline is 6.0mg/L, which is consistent with the results obtained by OD method, BMD method, and plate counting method. The EMGA method has good accuracy in determining the antibacterial effect of Ag NPs on V. parahaemolyticus in estuarine water samples. In addition, the results of EMGA method and the MIC values of BMD and plate counting method are in good agreement, The results of EMGA method are in good agreement with the MIC values of BMD and plate counting method, with an essential agreement (EA) of 75%, minor error (mE) is 25% and no major error (ME) was found, indicating that the EMGA method for measuring Ag NPs is effective for V Parahaemolyticus has good accuracy in its antibacterial effect. In addition, the MIC values measured by the EMGA method are often higher than or equal to the results obtained by the BMD method and the plate counting method, due to the higher sensitivity of automated instruments compared to visual observation. Therefore, the antibacterial effect determination conclusion of EMGA method has higher reliability than the results based on visual judgment.
This study established a phenotypic method for determining the antibacterial effect of Ag NPs in simulated estuarine water. This method only requires two manual steps, and compared to classical measurement methods such as BMD and plate counting, its advantage is that it does not require the separation of bacteria from the exposed mixture. In addition, due to the absence of separation steps, it effectively reduces the risk of subjective and objective operational errors. This automated method based on sensor recognition results has higher sensitivity than BMD method and flat plate counting method. Thus this new method had the advantages of simplicity, time saving, low- labor strength, good precision and good repeatability. In addition, the sensitivity of this automatic instrument-based method is higher than that of eye-based. This efficient method provided a new approach for assessing ecotoxicity of nanomaterials in realistic environmental matrices, e.g. estuarine water. |
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