Name: catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles
Uploaded: 2018-08-26T15:00:00.000+00:00
Duration: 9 min 46 s
Description: Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

这项工作得到了加利福尼亚大学、河畔分校和美国农业部食品与农业研究所的支持, 孵化项目 1009710 J.P.G.。该材料的基础是国家科学基金会根据1817363号赠款资助的 J.P.G. 的工作。

1. 种植芥的植物
<ol><li>在5厘米 x 5 厘米的一次性花盆中播种一个.把32这些花盆放入一个装满水的塑料托盘 (0.5 厘米深), 把塑料托盘和植物转移到植物生长室。<ol>
<li>设置生长室设置如下: 200 µmol/ms 光合活性辐射 (PAR), 24 1 °c 日和 21 @ 1 °c 夜, 60% 湿度, 14/10 h 天/夜光政权分别。</li>
	</ol>
</li>
	<li>在一周的发芽之后, 每壶都要细细地留下一株。注意保持每个花盆中大小相同的幼苗。</li>
	<li>每两天将自来水直接浇在塑料托盘上, 浇壶水。种植植物四周。南芥植物已准备好进一步使用。</li>
</ol>2. 民警的综合和特性
<ol><li>在50毫升圆锥管中, 将硝酸铈 (III.) 重1.08 克, 并将其溶解在2.5 毫升的分子生物学级水中。</li>
	<li>在50毫升圆锥管中, 将4.5 克的聚 (丙烯酸) 酸和溶解在5毫升分子生物级水中。</li>
	<li>使用数字涡旋混合器, 将这两个解决方案彻底地混合在 2000 rpm 15 分钟。</li>
	<li>将15毫升的氢氧化铵溶液 (7.2 米) 转移到50毫升玻璃烧杯中。</li>
	<li>搅拌在 500 rpm, 加入混合物从步骤2.3 滴到氢氧化铵溶液和搅拌在 500 rpm 在室温下24小时的通风罩。</li>
	<li>用一张纸盖上烧杯, 以避免夜间反应中的大量溶液损失。</li>
	<li>24小时后, 将产生的溶液转移到50毫升圆锥管, 并将其离心在 3900 x g 处, 以去除任何可能的碎片和大团聚体。</li>
	<li>将这22.5 毫升的上清液转化为三15毫升 10 kDa 过滤器, 用分子级水填充过滤器的其余部分, 使总稀释45毫升。</li>
	<li>用台式离心机在15毫升 10 kDa 过滤器中加入上清液, 用 3900 x g 的离心机在15分钟内, 从游离聚合物和其他试剂中纯化上清溶液. 重复此步骤至少六次。</li>
	<li>用 220-700 nm 的紫外-可见光分光光度计测量每个周期中淋洗的吸光度, 以确保最终的民警溶液中没有游离聚合物和其他试剂。</li>
	<li>将收集的民警解决方案放入5毫升注射器中, 并用20纳米孔径注射器过滤器进行过滤。在50毫升锥形管中收集经过过滤的民警解决方案。</li>
	<li>在塑料试管中采用稀释后的最终民警解决方案, 用 220-700 nm 的紫外可见光分光光度计测量其吸光度。刚果国民吸光度峰值为 271 nm。</li>
	<li>用啤酒-兰伯特定律计算其浓度: A = εCL。a 是给定样本峰值的吸光度, ɛ是国民 (cm-1米-1) 的摩尔吸收系数, L 是光路长度 (试管宽度, 1 厘米), C 是被测纳米粒子的摩尔浓度。</li>
	<li>使用粒子大小和泽塔电位分析仪测量合成的民警的水动力直径和泽塔电位 (图 1)。</li>
	<li>将最终的民警解决方案存储在冰箱 (4 摄氏度), 直到进一步使用。 注: 请参阅吴等。8有关民警特性的更多协议详细信息。</li>
</ol>3. 用 DiI 荧光染料标记民警
<ol><li>混合0.4 毫升5毫米 (58 毫克/升) 民警与3.6 毫升分子生物学级水在20毫升玻璃瓶和搅拌在 500 rpm。</li>
	<li>添加24µL 11 '-dioctadecyl 33, 3 ', 3 '-tetramethylindocarbocyanine 高氯酸染料溶液 (DiI, 2.5 毫克/毫升, 稀释在亚砜) 到176µL 的亚砜 (二甲基亚砜), 使 DiI 染料溶液。</li>
	<li>将 DiI 染料滴入到民警溶液中, 在室温下搅动 1000 rpm 1 分钟。</li>
	<li>将由此产生的混合物转化为15毫升 10 kDa 过滤器, 并用分子生物学级水填充管顶, 使总稀释15毫升。</li>
	<li>从亚砜和任何可能的游离 DiI 染料中纯化 DiI 标记的民警 (DiI) 溶液, 其台式离心与15毫升 10 kDa 过滤器在 3900 x g 处5分钟。<ol>
<li>重复步骤3.5 至少五次。</li>
	</ol>
</li>
	<li>通过20纳米孔径注射器过滤器过滤最终的 DiI-民警解决方案。</li>
	<li>用紫外-可见光分光光度法测量最终 DiI 的吸光度, 并根据啤酒-兰伯特定律计算其浓度 (图 2)。有关详细信息, 请参阅步骤2.13。</li>
	<li>将其存储在4摄氏度的冰箱中以供进一步使用。</li>
</ol>4. 植物叶片与民警的渗透
<ol><li>添加0.1 毫升的渗透缓冲 (100 毫米的附加费, 100 毫米氯化镁2, pH 值 7.5, 由 HCl 调整) 成0.9 毫升的0.5 毫米民警或 DiI-民警解决方案和涡旋。使用10毫米的污水附加入渗缓冲液作为负控制。</li>
	<li>将0.2 毫升的民警或 DiI 渗透液转移到1毫升无菌无针注射器。点击删除任何可能的气泡。</li>
	<li>在渗透纳米微粒之前, 从生长室中提取植株, 以避免在室温条件下可能的气孔闭合。</li>
	<li>在入渗前, 用叶绿素计测定拟南芥叶片的叶绿素含量。测量每片叶子的三个复制 (每个复制包括至少三测量)14。选择具有相似叶绿素含量的拟南芥叶, 进行浸润试验。</li>
	<li>用最近准备好的民警或 DiI--民警解决方案缓慢地将无针注射器的尖端向下渗透到叶子叶片的底部 (背面) 并压低柱塞 (图 3A)。</li>
	<li>用一个微妙的任务雨刷 (图 3C) 轻轻地擦掉残留在叶片表面上的多余溶液 (图 3B), 并给植物贴上标签。为每组叶子使用新的微妙的任务湿巾。</li>
	<li>在3小时内, 将 DiI 的植物放在长凳上进行叶适应和孵化. 注: 渗透到南芥植物, 然后准备进一步使用 (图 3D)。</li>
</ol>5. 共焦显微镜用叶样的制备
<ol><li>将豌豆大小的观察凝胶卷到大约1厘米半径 (图 4a), 然后将其摊开, 直到它在玻璃滑块上1毫米薄 (图 4B)。</li>
	<li>使用软木塞 (直径0.3 厘米) 在玻璃滑动的观察凝胶的中心切出圆形截面 (图 4C)。</li>
	<li>完全用 perfluorodecalin (PFD) 填充切口, 以便在叶组织中更深入和更好地共聚焦成像分辨率。</li>
	<li>使用软木塞 (直径0.2 厘米) 从适应的 DiI 收集叶盘--民警渗入了南芥植物 (图 4D)。</li>
	<li>在 PFD 填充井中安装叶盘;面对渗透 (背面) 的叶子的一侧。</li>
	<li>将方形盖玻片放在叶盘的顶部, 轻轻按下滑动盖玻片, 将其与观察凝胶的井封住, 确保没有气泡滞留 (图 4E)。</li>
</ol>6. 用共焦显微镜成像 DiI 叶组织中的民警
<ol><li>在反向激光扫描共聚焦显微镜中使用40X 物镜。</li>
	<li>在40X 物镜的顶部放置两到三滴 ddH2O。</li>
	<li>将准备好的 DiI 在倒置的40X 物镜上, 将已制备的叶片样品滑动放在上面。<ol>
<li>确保盖玻片一侧, 但不是玻璃滑动接触直接与 ddH2O 的镜头。</li>
	</ol>
</li>
	<li>在显微镜下, 用激光光或明亮的磁场找到一个感兴趣的区域。</li>
	<li>启动显微镜软件, 打开氩激光 (设定在 20%)。</li>
	<li>设置针孔, 以收集小于2µm 的光学切片, 线平均为4。</li>
	<li>图像的共聚焦显微镜设置的样品: 514 nm 激光激发 (30%);Z 堆叠截面厚度: 2 µm;PMT1: 550-615 nm (用于 DiI-民警成像);PMT2: 700-800 nm (用于叶绿体成像)。</li>
	<li>采取代表性的共焦图像的叶子样本从不同的人, 至少三生物复制。</li>
</ol>7. 用共焦显微镜对活体活性氧清除成像的实验观察
<ol><li>准备25µM 2 ', 7 '-dichlorodihydrofluorescein 双乙酸 (H2DCFDA, 一染料为表示一般 ROS) 和10µM dihydroethidium (DHE, 一种指示超氧化物阴离子) 染料的染料在工商业污水附加剂渗透缓冲 (pH 7.5) 在1.5 毫升离心管,分别。</li>
	<li>使用软木钻 (直径0.2 厘米) 从适应的民警中收集叶盘渗透到南芥植物中.<ol>
<li>使用钳尖尖, 使叶片上三到四孔, 加速染料加载过程。</li>
	</ol>
</li>
	<li>将叶盘转移到离心管, 分别用 H2DCFDA 和 DHE, 在黑暗下孵化30分钟。</li>
	<li>孵化后, 用 ddH2O 三次冲洗叶盘, 用观察凝胶将其装入玻璃滑梯 (见协议部分 5)。</li>
	<li>将幻灯片放在共焦显微镜上, 并手动聚焦到叶肉细胞的区域。有关详细信息, 请参阅协议6节。</li>
	<li>将叶盘暴露在紫外-A (405 nm) 激光器上, 3 分钟产生 ros, 并记录每叶圆盘的时间序列 ("xyt") 的 ros 信号强度变化。</li>
	<li>用共聚焦显微镜对叶盘进行图像设置: 40X 水目标;496 nm 激光激发;PMT1: 500-600 nm (用于 DHE 和 DCFDA 染料检测);PMT2: 700-800 nm (用于叶绿体检测)。使用仅渗透缓冲液渗透的植物作为阴性控制。</li>
</ol>8.从体外清除 H2O2
<ol><li>按照以前出版物3、8、15中的方法, 在体外进行合成民警的猫 (过氧化氢酶) 模拟活动
</li>
	<li>添加45.4 µL 的1x 附加费渗透缓冲 (10 毫米的附加费, 10 毫米氯化镁2, pH 7.5, 由 HCl 调整), 民警 (60 毫微米, 3 µL) 和 H2O2 (2 µM, 1 µL) 入井 (白色圆的底部96井板), 并且轻轻地混合它由吹打。</li>
	<li>加入 10-乙酰基 37-dihydroxyphenoxazine (工作浓度100µM, 0.5 µL) 和辣根过氧化物酶 (HRP; 工作浓度 0.2 U/毫升, 0.1 µL) 入井, 轻轻地混合它由吹打, 孵化它为30分钟 10-乙酰基 37-dihydroxyphenoxazine反应与 H2O2和被转换成试卤灵在存在的 HRP。<ol>
<li>在孵化过程中, 用铝箔包装盘子以避免光线。</li>
		<li>用反应缓冲器或水代替 H2O2, 准备一个负控制。</li>
		<li>除了库存解决方案, 在室温下准备所有其他解决方案。</li>
	</ol>
</li>
	<li>孵化后, 与一个板块阅读器, 监测吸光度在 560 nm 使用试卤灵, 以表明水平的 H2O2。设置时间制度在0、2、5、10、20和30分钟。</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyl+radical+scavenging+by+cerium+oxide+nanoparticles+improves+Arabidopsis+salinity+tolerance+by+enhancing+leaf+mesophyll+potassium+retention.">Hydroxyl radical scavenging by cerium oxide nanoparticles improves Arabidopsis salinity tolerance by enhancing leaf mesophyll potassium retention.</a> Environmental Science: Nano. 5 (7), 1567-1583 (2018).</li><li>Merad-Boudia, M., Nicole, A., Santiard-Baron, D., Saill&#233;, C., Ceballos-Picot, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+impairment+as+an+early+event+in+the+process+of+apoptosis+induced+by+glutathione+depletion+in+neuronal+cells:+Relevance+to+Parkinson's+disease.">Mitochondrial impairment as an early event in the process of apoptosis induced by glutathione depletion in neuronal cells: Relevance to Parkinson's disease.</a> Biochemical Pharmacology. 56 (5), 645-655 (1998).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+characterization+of+the+product+of+hydroethidine+and+intracellular+superoxide+by+HPLC+and+limitations+of+fluorescence.">Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence.</a> Proceedings of the National Academy of Sciences. 102 (16), 5727-5732 (2005).</li><li>Sun, C., Li, H., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+ceria-based+materials:+synthesis,+properties,+and+applications.">Nanostructured ceria-based materials: synthesis, properties, and applications.</a> Energy &amp; Environmental Science. 5 (9), 8475 (2012).</li><li>Hirano, M., Inagaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+monodispersed+cerium(iv)+oxide+particles+by+thermal+hydrolysis:+influence+of+the+presence+of+urea+and+Gd+doping+on+their+morphology+and+growth.">Preparation of monodispersed cerium(iv) oxide particles by thermal hydrolysis: influence of the presence of urea and Gd doping on their morphology and growth.</a> Journal of Materials Chemistry. 10 (2), 473-477 (2000).</li><li>Xi, D. M., Liu, W. S., Yang, G. D., Wu, C. A., Zheng, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed-specific+overexpression+of+antioxidant+genes+in+Arabidopsis+enhances+oxidative+stress+tolerance+during+germination+and+early+seedling+growth.">Seed-specific overexpression of antioxidant genes in Arabidopsis enhances oxidative stress tolerance during germination and early seedling growth.</a> Plant Biotechnology Journal. 8 (7), 796-806 (2010).</li><li>Wu, H., Santana, I., Dansie, J., Vivo Giraldo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+delivery+of+nanoparticles+into+plant+leaves.">In Vivo delivery of nanoparticles into plant leaves.</a> Current Protocols in Chemical Biology. 9 (4), 269-284 (2017).</li><li>Fukushima, K., Hasebe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaxial-abaxial+polarity:+The+developmental+basis+of+leaf+shape+diversity.">Adaxial-abaxial polarity: The developmental basis of leaf shape diversity.</a> Genesis. 52 (1), 1-18 (2014).</li><li>Monda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+stomatal+conductance+by+a+spontaneous+Arabidopsis+tetraploid,+Me-o,+results+from+increased+stomatal+size+and+greater+stomatal+aperture.">Enhanced stomatal conductance by a spontaneous Arabidopsis tetraploid, Me-o, results from increased stomatal size and greater stomatal aperture.</a> Plant physiology. 170 (3), 1435-1444 (2016).</li><li>Petrov, V., Hille, J., Mueller-Roeber, B., Gechev, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS-mediated+abiotic+stress-induced+programmed+cell+death+in+plants.">ROS-mediated abiotic stress-induced programmed cell death in plants.</a> Frontiers in Plant Science. 6, 1-16 (2015).</li><li>Chaves, M. M., Flexas, J., Pinheiro, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthesis+under+drought+and+salt+stress:+regulation+mechanisms+from+whole+plant+to+cell.">Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell.</a> Annals of Botany. 103 (4), 551-560 (2009).</li></ol>

作者没有什么可透露的。

在本协议中, 我们描述了在植物叶肉细胞内的纳米粒子的合成、表征、荧光染料标记和共焦成像, 以展示其体内活性氧清除活性。从硝酸铈的混合物和氢氧化铵中的临机溶液中合成了民警。民警的特点是吸收 spectrophotomery 和浓度确定使用啤酒 Lamberts 法。泽塔电位测量证实了民警的负电荷表面, 以增强对叶绿体8的传递。用荧光 DiI 染料对民警进行标记,在叶片叶肉细胞?...

氧化铈纳米粒子 (nanoceria) 广泛应用于生物, 从基础研究到生物工程, 由于其独特的催化活性氧 (ROS) 清除能力1,2,3。Nanoceria 有 ROS 清除能力由于大量表面氧气空缺交替在二氧化状态 (ce3 +和 Ce4 +) 4,5,6。Ce3 +悬垂键有效地清除 ROS, 而纳米晶格菌株通过氧化还原循环反应7促进这些缺陷部位的再生。Nanoceria 最近也被用于研究和工程植物功能8,9。非生物胁迫下的植物积累了 ROS, 对脂质、蛋白质和 DNA 造成氧化损伤10。在 nanoceria 植物中, 在体内活性氧的催化清除能在高光、高温和冷应力下改善植物光合作用8。nanoceria 对土壤的施用也增加了小麦的生物质量和籽粒产量11;nanoceria 处理的油菜 (甘蓝) 植株在盐胁迫下具有较高的植物生物量12。
Nanoceria 提供生物工程师和植物生物学家一个基于纳米技术的工具, 以了解非生物胁迫反应和提高植物非生物胁迫耐受性。Nanoceria 的体内活性氧清除能力是独立于植物物种, 并在植物组织的简便交付有潜力, 使广泛应用外的模型有机体。不像其他基于基因的方法, nanoceria 不需要产生植物线与抗氧化酶的过度表达, 以提高 ROS 清除能力13。nanoceria 对植物的叶片浸润是实验室研究的一种实用方法。
本议定书的总目标是描述 1) 带负电荷的聚 (丙烯酸) 酸 nanoceria (民警) 的合成和表征, 2) 在整个叶细胞内提供和跟踪全国民警, 3) 监测在体内。本协议合成了带负电荷的聚丙烯酸 (nanoceria) 酸 (民警), 并以其吸收谱、流体力学直径和泽塔电位为特征。我们描述了一种简单的叶片入渗方法, 以提供民警到植物叶组织。在体外显像的纳米颗粒在叶肉细胞中的分布, 用荧光染料 (DiI) 标记民警 (DiI), 并通过共焦荧光显微镜观察纳米粒子。最后, 我们解释如何通过共聚焦显微镜监测体内的ROS 清除。

<table><tbody><tr><td>Cerium (III) nitrate hexahydrate</td><td>Sigma-Aldrich</td><td>238538-100G</td><td></td></tr><tr><td>Molecular Biology Grade Water, Corning</td><td>VWR</td><td>45001-044&amp;nbsp;</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>VWR</td><td>14-959-49A</td><td></td></tr><tr><td>Poly (acrylic acid) 1,800 Mw</td><td>Sigma-Aldrich</td><td>323667-100G</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer Accessory, Insert Retainer</td><td>Fisher Scientific</td><td>02-215-391</td><td></td></tr><tr><td>Fisherbrand Digital Vortex Mixer Accessories: Foam Insert Set</td><td>Fisher Scientific</td><td>02-215-395</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma-Aldrich</td><td>05002-1L</td><td></td></tr><tr><td>PYREX Griffin Beakers, Graduated, Corning</td><td>VWR</td><td>13912-149&amp;nbsp;</td><td></td></tr><tr><td>RCT basic</td><td>IKA</td><td>3810001</td><td></td></tr><tr><td>Eppendorf Microcentrifuge 5424</td><td>VWR</td><td>80094-126</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>Millipore-Sigma</td><td>UFC901024</td><td></td></tr><tr><td>Allegra X-30 Series Benchtop Centrifuge</td><td>Beckman Coulter</td><td>B06314</td><td></td></tr><tr><td>UV-2600 Sptecrophotometer</td><td>Shimadzu</td><td>UV-2600 120V</td><td></td></tr><tr><td>Whatman Anotop 10 syringe filter</td><td>Sigma-Aldrich</td><td>WHA68091102</td><td></td></tr><tr><td>BD Disposable Syringes with Luer-Lok Tips</td><td>Fisher Scientific</td><td>14-829-45</td><td></td></tr><tr><td>Zetasizer Nano S</td><td>Malvern Panalytical</td><td>Zen 1600</td><td></td></tr><tr><td>1,1&amp;prime;-Dioctadecyl-3,3,3&amp;prime;,3&amp;prime;-tetramethylindocarbocyanine perchlorate</td><td>Sigma-Aldrich</td><td>42364-100MG</td><td></td></tr><tr><td>Dimethyl Sulfoxide, ACS</td><td>VWR</td><td>BDH1115-1LP</td><td></td></tr><tr><td>Sunshine Mix #1 LC1</td><td>Green Island Distributors, Inc</td><td>5212601.CFL080P</td><td></td></tr><tr><td>Adaptis 1000</td><td>Conviron</td><td>A1000</td><td></td></tr><tr><td>TES, &gt;99% (titration</td><td>Sigma-Aldrich</td><td>T1375-100G</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266-1KG</td><td></td></tr><tr><td>Air-Tite All-Plastic Norm-Ject Syringe</td><td>Fisher Scientific</td><td>14-817-25</td><td></td></tr><tr><td>Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers</td><td>Fisher Scientific</td><td>06-666A</td><td></td></tr><tr><td>Carolina Observation Gel</td><td>Carolina</td><td>132700</td><td></td></tr><tr><td>Corning microscope slides, frosted one side, one end</td><td>Sigma-Aldrich</td><td>CLS294875X25-72EA</td><td></td></tr><tr><td>Cork Borer Sets with Handles</td><td>Fisher Scientific</td><td>S50166A</td><td></td></tr><tr><td>Perfluorodecalin</td><td>Sigma-Aldrich</td><td>P9900-25G</td><td></td></tr><tr><td>Micro Cover Glasses, Square, No. 1</td><td>VWR</td><td>48366-045</td><td></td></tr><tr><td>Leica Laser Scanning Confocal Microscope TCS SP5</td><td>Leica Microsystems</td><td>TCS SP5</td><td></td></tr><tr><td>2&amp;prime;,7&amp;prime;-Dichlorofluorescin diacetate</td><td>Sigma-Aldrich</td><td>D6883-250MG</td><td></td></tr><tr><td>Dihydroethidium</td><td>Sigma-Aldrich</td><td>D7008-10MG</td><td></td></tr><tr><td>Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Eppendorf Uvette cuvettes</td><td>Sigma-Aldrich</td><td>Z605050-80EA</td><td></td></tr><tr><td>Chlorophyll meter&amp;nbsp;</td><td>Konica Minolta</td><td>SPAD-502</td><td></td></tr></tbody></table>

catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles

活性氧 (ROS) 的积累是植物非生物胁迫反应的标志。ROS 在植物中起着双重作用, 在低水平上充当信号分子, 在高水平上破坏分子。在被胁迫的植物中, ROS 的积聚会破坏代谢物、酶、脂类和 DNA, 从而减少植物生长和产量。氧化铈纳米粒子 (nanoceria) 在体内催化清除活性氧的能力为理解和生物工程植物非生物胁迫耐受提供了独特的工具。在这里, 我们提出了一个合成和表征聚 (丙烯酸) 酸涂层 nanoceria (民警) 的协议, 通过叶片渗透与植物界面的纳米微粒, 并监测其分布和 ROS 清除在体内使用共焦显微镜。目前用于操作植物中 ROS 积累的分子工具仅限于模型物种, 需要费力的转化方法。本协议的体内ROS 清除有可能适用于野生类型的植物, 宽叶和叶结构, 如拟南芥。

民警的合成和表征. 民警的合成、纯化和特点遵循了议定书第2节所述方法。图 1A显示了硝酸铈、临机局、硝酸铈和临机局的混合物和民警的溶液的着色。在合成了民警后, 从白色到浅黄色的颜色变化是可见的。用 10 kDa 过滤器纯化后, 民警的特征是紫外-可见光分光光度计。在 271 nm (图 1</st...

Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

在这里, 我们提出了一种合成和表征氧化铈纳米粒子 (nanoceria) 的协议, 用于在体内清除活性氧, nanoceria 在植物组织中进行共焦显微成像, 并在体内用共焦显微镜监测 nanoceria ROS 的清除。

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

catalytic-scavenging-plant-reactive-oxygen-species-vivo-anionic

Research

JoVE Journal

Bioengineering

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

8.8K 次观看.  University of California. 在这里, 我们提出了一种合成和表征氧化铈纳米粒子 (nanoceria) 的协议, 用于在体内清除活性氧, nanoceria 在植物组织中进行共焦显微成像, 并在体内用共焦显微镜监测 nanoceria ROS 的清除。

视频: Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

去除微量元素的氧化铜纳米粉体制备铀<em&gt;原位</em&gt;恢复泌水及其对细胞活力的影响

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

科研

环境科学

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

卷烟主流烟气中检测和清除气相自由基议定书

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

生物工程

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

铱氧化物纳米颗粒对硒化镉硫化镉@纳米棒光化学氧化增长

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

化学

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical photocatalytic structures. However, the influence of such chemical bonding on the optical and surface properties of the photocatalyst and thus its photocatalytic activity/reaction selectivity behavior has not been systematically studied. In this investigation, TiO2 has been supported on the surface of SiO2 by means of two different methods: (i) by the in situ formation of TiO2 in the presence of sand quartz via a sol-gel method employing tetrabutyl orthotitanium (TBOT); and (ii) by binding the commercial TiO2 powder to quartz on a surface silica gel layer formed from the reaction of quartz with tetraethylorthosilicate (TEOS). For comparison, TiO2 nanoparticles were also deposited on the surfaces of a more reactive SiO2 prepared by a hydrolysis-controlled sol-gel technique as well as through a sol-gel route from TiO2 and SiO2 precursors. The combination of TiO2 and SiO2, through interfacial Ti-O-Si bonds, was confirmed by FTIR spectroscopy and the photocatalytic activities of the obtained composites were tested for photocatalytic degradation of NO according to the ISO standard method (ISO 22197&#8722;1). The electron microscope images of the obtained materials showed that variable photocatalyst coverage of the support surface can successfully be achieved but the photocatalytic activity towards NO removal was found to be affected by the preparation method and the nitrate selectivity is adversely affected by Ti-O-Si bonding.

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The focus of the present work is to establish means to generate and quantify levels of Ti-O-Si linkages and to correlate these with the photocatalytic properties of the supported TiO2.

界面化学键合在TiO中的作用<sub&gt; 2</sub&gt; -SiO<sub&gt; 2</sub&gt;复合材料对其光催化NOx消除性能的影响

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

生物化学

Preparation of Polyoxometalate-based Photo-responsive Membranes for the Photo-activation of Manganese Oxide Catalysts

This paper presents a method to prepare charge-transfer chromophores using polyoxotungstate (PW12O403-), transition metal ions (Ce3+ or Co2+), and organic polymers, with the aim of photo-activating oxygen-evolving manganese oxide catalysts, which are important components in artificial photosynthesis. The cross-linking technique was applied to obtain a self-standing membrane with a high PW12O403- content. Incorporation and structure retention of PW12O403- within the polymer matrix were confirmed by FT-IR and micro-Raman spectroscopy, and optical characteristics were investigated by UV-Vis spectroscopy, which revealed successful construction of the metal-to-metal charge transfer (MMCT) unit. After deposition of MnOx oxygen evolving catalysts, photocurrent measurements under visible light irradiation verified the sequential charge transfer, Mn &#8594; MMCT unit &#8594; electrode, and&#160;the photocurrent intensity was consistent with the redox potential of the donor metal (Ce or Co). This method provides a new strategy for preparing integrated systems involving catalysts and photon-absorption parts for use with photo-functional materials.

Here, we present a protocol to prepare charge transfer chromophores based on a polyoxometalate/polymer composite membrane.

氧化锰催化剂光活化 Polyoxometalate 基光响应膜的制备

This paper presents a method to prepare charge-transfer chromophores using polyoxotungstate (PW12O403-), transition metal ions (Ce3+ or Co2+), and organic ...

A Continuous-flow Photocatalytic Reactor for the Precisely Controlled Deposition of Metallic Nanoparticles

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic nanoparticles is developed. Guidelines for the replication of the reactor and its operation are provided in detail. Three different composite systems (Pt/graphene, Pt/TiO2, and Au/TiO2) with monodisperse and uniformly distributed particles are produced by this reactor, and the photodeposition mechanism, as well as the synthesis optimization strategy, are discussed. The synthesis methods and their technical aspects are described comprehensively. The role of the ultraviolet (UV) dose (in each excitation pulse) on the photodeposition process is investigated and the optimum values for each composite system are provided.

For a continuous and scalable synthesis of noble-metal-based nanocomposites, a novel photocatalytic reactor is developed and its structure, operation principles, and product quality optimization strategies are described.

一种用于金属纳米粒子精确控制沉积的连续流动光催化反应器

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic ...

A Facile Synthetic Method to Obtain Bismuth Oxyiodide Microspheres Highly Functional for the Photocatalytic Processes of Water Depuration

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of materials is highly related to its photocatalytic performance, it is necessary to standardize the synthetic methods in order to obtain the most functional architectures and, thus, the highest photocatalytic efficiency. Here, we report a reliable route to obtain BiOI microspheres via the solvothermal process, using Bi(NO3)3 and potassium iodide (KI) as precursors, and ethylene glycol as a template. The synthesis is standardized in a 150 mL autoclave, at 126 &#176;C for 18 h. This results in 2-3 &#181;m-sized mesoporous microspheres, with a relevant specific surface area (61.3 m2/g). Shortening the reaction times in the synthesis results in amorphous structures, while higher temperatures lead to a slight increase in the porosity of the microspheres, with no effect in the photocatalytic performance. The materials are photo-active under UV-A/visible light irradiation for the degradation of the antibiotic ciprofloxacin in water. This method has demonstrated to be effective in interlaboratory tests, obtaining similar BiOI microspheres in Mexican and Chilean research groups.

This article describes a synthetic method to obtain bismuth oxyiodide microspheres, which are highly functional to perform the photocatalytic removal of organic pollutants, such as ciprofloxacin, in water under UV-A/visible light irradiation.

一种用于水降解光催化过程的高效双氧化二碘微球合成方法

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of ...

Metal Oxide Heterojunction for Photocatalytic Activities

There is a significant global demand for improvements in synthesis techniques and their optimal characteristics, especially for industrial-scale applications. Sol-gel-based solution combustion synthesis (SG-SCS) is a simple method to produce ordered porous materials. In this regard, Pearson&#39;s hard and soft acids and bases theory assists in selecting host-dopant reactivity to form a proper heterojunction.
The formation of a heterojunction also changes the essential properties of the materials, improving photocatalysis via charge transfer or synergistic activities. A calcination temperature of 500 &#176;C is ideal for this process based on the results of the stability assessment via a differential thermogravimetry ratio analysis (DTG).
The nanoscale dimensions of the nanoparticles (NPs) and nanocomposites (NCs) generated were validated using X-ray diffraction and high-resolution transmission electron microscopy (HRTEM). Furthermore, the scanning electron microscopy micrographs and BET analyses confirmed the porosity nature of the materials. HRTEM, X-ray photoelectron spectroscopy, and energy-dispersive X-ray investigations established the materials composition. The study found that NCs degraded the acid orange 8 (AO8) color more efficiently than bare ZnO.

The development of a heterojunction boosts the photocatalytic activities of solution combustion synthesis, which is a time-/energy-efficient process. Advanced analytical characterization techniques were used in this protocol to evaluate the materials&#39; characteristics, and nanocomposites demonstrated improved acid orange-8 dye degradation.

There is a significant global demand for improvements in synthesis techniques and their optimal characteristics, especially for industrial-scale ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

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去除微量元素的氧化铜纳米粉体制备铀