Name: preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation
Uploaded: 2018-03-09T13:00:00.000+00:00
Duration: 6 min 23 s
Description: Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

这项研究得到了基础科学研究项目 (项目编号: 2016R1A2B4013467) 的支持, 通过韩国国家研究基金会 (NRF), 由科学部、信息和通信技术 & 未来规划2016提供资金, 并得到韩国基础科学的支持。研究所研究计划 (项目编号: C36707)。

1. DMMTA 的合成V
<ol><li>
化学制剂和摩尔比混合 DMAV, Na2S, 和 H2, 所以4 注意: DMAV: Na2S: H2所以4 = 1:1. 6:1. 6<ol>
<li>将 5.24 g 的 DMAV溶解在40毫升去离子和 N2中 (在50毫升离心管中清洗至少30分钟) 水。</li>
		<li>通过溶解14.41 克 na2S·9H2O 在50毫升去离子和 N2-被清洗的水在250毫升瓶中, 准备 na2S 试剂。</li>
		<li>准备 H2, 以便4试剂通过在40毫升离心管中添加3.3 毫升的浓硫酸 (96%) 至2毫升去离子和 N50清除水。 注意:DMA 的最终摩尔比率V: Na2S: H2所以4 = 1:1. 6:1. 6 13,4,8,15。</li>
		<li>将已准备好的40毫升 DMAV解决方案 (步骤 1.1.1) 添加到250毫升烧瓶 (步骤 1.1.2) 中包含的 Na2S 解决方案的50毫升。冲洗包含 DMAV的10毫升清洗水的管, 并将其添加到250毫升烧瓶中。</li>
		<li>用装有玻璃管的三孔橡胶塞子关闭烧瓶。分别使用用于 N2气体流入、流出和 H2的玻璃管, 因此4解决方案入口。关闭烧瓶后立即, 允许 N2气体流入烧瓶中。 注: 应保持气体压力, 以在反应溶液表面流动而不飞溅。</li>
		<li>将耐酸油管连接到 H2的玻璃管, 使4溶液入口与50毫升注射器一起添加准备好的40毫升的 h2, 因此4解决方案 (步骤 1.1.3)。添加40毫升的 H2, 所以4解决方案, 慢慢地和逐步地。 警告:加入硫酸后, 会产生白烟;使用通风良好的油烟罩。</li>
		<li>定期添加硫酸时, 监测反应混合物在烧瓶中的颜色变化。保持间隔为 4-5 毫升的 H2, 因此4;混合物应该是一个白色的多云溶液。 注: 由于浓硫酸的快速添加, 瞬时黄沉淀可能出现。</li>
		<li>确保反应解决方案自1.1.4 开始以来一直保持1小时。</li>
	</ol>
</li>
	<li>
用液-液萃取法提取 DMMTAV
	<ol>
<li>1小时后, 将反应溶液倒入含有大约200毫升乙醚的部位漏斗中。</li>
		<li>摇动漏斗 5-10 分钟, 释放气体几次通过切换旋塞阀。 注意: 合成的 DMMTAV将被转移到二乙基醚 (0.713 g·mL-1) 的上层。 警告:乙醚气体可能有害;使用通风良好的油烟罩。</li>
		<li>在烧杯中收集反应溶液, 并分别收集含有 DMMTAV的二乙基醚。将反应溶液放回同一部位漏斗中, 加入约200毫升的新鲜二乙基醚 reshaking。重复步骤 1.2.2-1.2.3 三次。</li>
		<li>再将所收集的二乙基醚从步骤1.2.3 倒入同一部位漏斗中, 并添加约100毫升 N2-清除去离子水。摇动 5-10 分钟, 并丢弃 N2-清除去离子水和几毫升二乙基醚为纯净目的。在玻璃培养皿中收集剩余的二乙基醚 (最小内径为160毫米, 最小高度为50毫米)。</li>
		<li>将玻璃培养皿转移到 N2大气手套盒中, 以防止物种转化。 警告:确保溶剂不被拉入真空泵通过出口的通行证箱。</li>
		<li>干燥直到 dimethylmonothioarsinate 的白色沉淀 (结晶 DMMTAV) 在玻璃培养皿上形成。 注意: 协议可以在这里暂停。</li>
	</ol>
</li>
	<li>
对合成 DMMTAV和存储的验证
	<ol>
<li>采取结晶 DMMTAV的白色沉淀, 并测量并记录其总重量。 警告:使用通风良好的油烟罩或手套箱, 防止吸入硫化氢气体。</li>
		<li>将结晶 DMMTAV分解为50毫升的 N2-清除去离子水, 并通过 0.2-µm 注射器过滤器过滤黄色沉淀。</li>
		<li>假定已使用的 DMAv的总计转换为 DMMTAv,即≈9,649 mg As·L-1。稀释 DMMTAV库存解决方案≈1毫克·L-1和≈40µg·L-1分别使用电喷雾电离质量 Spectromtery (ESI) 和 HPLC-电感耦合-ms 进行验证分析。</li>
		<li>使用ESI (MS11)16和在m/z 155 中的片段在正离子模式下或m/z 153 中的负离子模式 (表 1) 中, 分析 DMMTAV的m/z 。 注意: 请参阅m/z的引用值 (表 1)。</li>
		<li>用高效液相色谱-ICP-MS11,16,17 , 用适当的淋洗条件分析股票溶液中 DMMTAV的色谱, 并确认主要峰值在文学. 注意: 应使用步骤1.3.5 中的分析结果计算合成 DMMTAV的纯度。</li>
		<li>用 ICP-质谱分析酸消化后的总砷浓度11 , 并用稀释因子和纯度计算合成 DMMTAv库存溶液中的真实 DMMTAv浓度, 如下面的公式所示: 分析合计为浓度 (µg·L-1) ·稀释因素·纯度 (%) = True DMMTAv集中在 DMMTAv库存解决方案 (µg·L-1)</li>
		<li>存储 DMMTAV库存解决方案在4°c 在黑暗中进一步定量形态分析18。</li>
	</ol>
</li>
</ol>2. DMDTA 的合成V
<ol><li>
化学制剂和摩尔比混合 DMAV , Na2S, 和 H2, 所以4 注意: DMAV: Na2S: H2所以4 = 1:7. 5:7. 5<ol>
<li>在50毫升离心管中, 将1.38 克的 DMAV溶解在40毫升去离子水中。</li>
		<li>通过在50毫升去离子水中溶解18.01 克 na2S·9H2O, 将钠2的试剂制备为一瓶。</li>
		<li>准备 H2, 使4试剂添加4毫升浓硫酸 (96%) 到40毫升的去离子水中, 其中含有50毫升离心管。 注意:DMA 的最终摩尔比率V: Na2S: H2所以4 = 1:7. 5:7. 513,4,8,15。</li>
		<li>将已准备好的40毫升 DMAV解决方案 (步骤 2.1.1) 添加到250毫升烧瓶 (步骤 2.1.2) 中包含的 Na2S 解决方案的50毫升。用10毫升去离子水冲洗含有 DMAV的管, 并将其添加到250毫升烧瓶中。</li>
		<li>将准备好的40毫升 H2, 使4解决方案 (步骤 2.1.3), 慢慢地和逐步地, 进入烧瓶。</li>
		<li>定期添加硫酸时, 监测反应混合物在烧瓶中的颜色变化。保持间隔为 4-5 毫升的 H2, 因此4;混合物应为白色/黄色多云溶液。 注: 由于浓硫酸的快速添加, 瞬时黄沉淀可能会出现。 警告:加入硫酸后, 会产生白烟;使用通风良好的油烟罩。</li>
		<li>在瓶中保持反应液过夜不覆盖。 注意: 协议可以在这里暂停。</li>
	</ol>
</li>
	<li>
使用固相萃取 (SPE) 方法提取的 DMDTAV
	<ol>
<li>在隔夜反应之后, 用 C18 注射器型硅基 SPE 过滤反应溶液, 以便在树脂上捕获合成的 DMDTAV 。 警告:使用通风良好的油烟罩。</li>
		<li>用10毫米醋酸铵 (pH 6.3) 将0.77 克醋酸铵溶解在去离子水的1升中。通过 C18 注射器 (步骤 2.2.1) 洗脱足够的10毫米醋酸铵, 提取吸附 DMDTA V.在玻璃培养皿中收集过滤过的醋酸铵 (最小内径为160毫米, 最小高度为50毫米)。</li>
		<li>将玻璃培养皿转移到 N2大气手套盒中, 以防止物种转化。 警告:确保溶剂不被拉入真空泵通过出口的通行证箱。</li>
		<li>干燥直到 dimethyldithioarsinate 的白色沉淀形成 (结晶 DMDTAV) 在玻璃培养皿上。 注意: 协议可以在这里暂停。</li>
	</ol>
</li>
	<li>
对合成 DMDTAV和存储的验证
	<ol>
<li>取结晶 DMDTAV的白色沉淀, 并测量其总重量, 记录测量结果。 警告:使用通风良好的油烟罩或手套箱, 防止吸入硫化氢气体。</li>
		<li>在50毫升的 N2中溶解结晶 DMDTAV -在50毫升离心管中清除去离子水, 并通过 0.2-µm 注射器过滤器过滤任何沉淀。</li>
		<li>假定已使用的 DMAV的总计转换为 DMDTAv,即≈2,539 mg As·L-1。稀释 DMDTAV库存解决方案≈1毫克·L-1和≈40µg·L-1分别使用 ESI-ms 和高效液相色谱-ms 进行验证分析。</li>
		<li>使用ESI (MS11)16和在m/z 171 中的片段在正离子模式下或m/z 169 中的负离子模式 (表 1) 中, 分析 DMDTAV的m/z 。 注意: 请参阅m/z的引用值 (表 1)。</li>
		<li>用高效液相色谱-ICP-MS11,16,17 ,用适当的淋洗条件分析 DMDTA 中的V的色谱, 并确认在保留时间内发现的峰值文学。 注意: 应使用步骤2.3.5 中的分析结果计算合成 DMDTAV的纯度。</li>
		<li>用 ICP-质谱分析酸消化后的总砷浓度11 , 并用稀释因子和纯度计算合成 DMDTAv库存溶液中的真 DMDTAv浓度如下方程式: 分析合计为浓度 (µg·L-1) ·稀释因素·纯度 (%) = True DMDTAv在库存解决方案中 DMDTAv的浓度 (µg·L-1)</li>
		<li>存储 DMDTAV库存解决方案在4°c 在黑暗中进一步定量形态分析18。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Suzuki, K. T., 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=Dimethylthioarsenicals+as+arsenic+metabolites+and+their+chemical+preparation.">Dimethylthioarsenicals as arsenic metabolites and their chemical preparation.</a> Chem. Res. Toxicol. 17, 914-921 (2004).</li><li>Kuroda, 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=Microbial+metabolite+of+dimethylarsinic+acid+is+highly+toxic+and+genotoxic.">Microbial metabolite of dimethylarsinic acid is highly toxic and genotoxic.</a> Toxicol. Appl. Pharmacol. 198, 345-353 (2004).</li><li>Naranmandura, H., Iwata, K., Suzuki, K. T., Ogra, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+metabolism+of+four+different+dimethylated+arsenicals+in+hamsters.">Distribution and metabolism of four different dimethylated arsenicals in hamsters.</a> Toxicol. Appl. Pharmacol. 245, 67-75 (2010).</li><li>Naranmandura, 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=Comparative+toxicity+of+arsenic+metabolites+in+human+bladder+cancer+EJ-1+cells.">Comparative toxicity of arsenic metabolites in human bladder cancer EJ-1 cells.</a> Chem. Res. Toxicol. 24, 1586-1596 (2011).</li><li>Wallschlager, D., London, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+methylated+arsenic-sulfur+compounds+in+groundwater.">Determination of methylated arsenic-sulfur compounds in groundwater.</a> Environ. Sci. Technol. 42, 228-234 (2008).</li><li>Zhang, J., Kim, H., Townsend, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+assessing+thioarsenic+formation+potential+in+sulfidic+landfill+environments.">Methodology for assessing thioarsenic formation potential in sulfidic landfill environments.</a> Chemosphere. 107, 311-318 (2014).</li><li>Shimoda, Y., 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=Proposal+for+novel+metabolic+pathway+of+highly+toxic+dimethylated+arsenics+accompanied+by+enzymatic+sulfuration,+desulfuration+and+oxidation.">Proposal for novel metabolic pathway of highly toxic dimethylated arsenics accompanied by enzymatic sulfuration, desulfuration and oxidation.</a> Trace Elem. Med. Biol. 30, 129-136 (2015).</li><li>Naranmandura, H., Suzuki, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+dimethylthioarsenicals+in+red+blood+cells.">Formation of dimethylthioarsenicals in red blood cells.</a> Toxicol. Appl. Pharmacol. 227, 390-399 (2008).</li><li>Leffers, L., Ebert, F., Taleshi, S. M., Francesconi, A. K., Schwerdtle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+toxicological+characterization+of+two+arsenosugars+and+their+metabolites.">In vitro toxicological characterization of two arsenosugars and their metabolites.</a> Mol. Nutr. Food Res. 57, 1270-1282 (2013).</li><li>Wang, Q. Q., Thomas, J. D., Naranmandura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+of+being+thiomethylated:+Formation,+Fate+and+Effects+of+methylated+thioarsenicals.">Important of being thiomethylated: Formation, Fate and Effects of methylated thioarsenicals.</a> Chem. Res. Toxicol. 25, 281-289 (2015).</li><li>Kim, Y. T., Lee, H., Yoon, H. O., Woo, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+dimethylated+thioarsenicals+and+the+formation+of+highly+toxic+dimethylmonothioarsinic+acid+in+environment.">Kinetics of dimethylated thioarsenicals and the formation of highly toxic dimethylmonothioarsinic acid in environment.</a> Environ. Sci. Technol. 50, 11637-11645 (2016).</li><li>Cullen, W. R., 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=Methylated+and+thiolated+arsenic+species+for+environmental+and+health+research+-+A+review+on+synthesis+and+characterization.">Methylated and thiolated arsenic species for environmental and health research - A review on synthesis and characterization.</a> J. Environ. Sci. 49, 7-27 (2016).</li><li>Fricke, M., 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=Chromatographic+separation+and+identification+of+products+form+the+reaction+of+dimethylarsinic+acid+with+hydrogen+sulfide.">Chromatographic separation and identification of products form the reaction of dimethylarsinic acid with hydrogen sulfide.</a> Chem. Res. Toxicol. 18, 1821-1829 (2005).</li><li>Fricke, M., Zeller, M., Cullen, W., Witkowski, M., Creed, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethylthioarsinic+anhydride:+a+standard+for+arsenic+speciation.">Dimethylthioarsinic anhydride: a standard for arsenic speciation.</a> Anal. Chim. Acta. 583, 78-83 (2007).</li><li>Suzuki, K. T., Iwata, K., Naranmandura, H., Suzuki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+differences+between+twon+dimethylthioarsenicals+in+rats.">Metabolic differences between twon dimethylthioarsenicals in rats.</a> Toxicol. Appl. Pharmacol. 218, 166-173 (2007).</li><li>Jeong, S., 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=Development+of+a+simultaneous+analytical+method+to+determine+arsenic+speciation+using+HPLC-ICP-MS:+Arsenate,+arsenite,+monomethylarsonic+acid,+dimethylarsinic+acid,+dimethyldithioarsinic+acid,+and+dimethylmonothioarsinic+acid.">Development of a simultaneous analytical method to determine arsenic speciation using HPLC-ICP-MS: Arsenate, arsenite, monomethylarsonic acid, dimethylarsinic acid, dimethyldithioarsinic acid, and dimethylmonothioarsinic acid.</a> Microchem. J. 134, 295-300 (2017).</li><li>Li, Y., Low, C. -. K., Scott, A. J., Amal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic+speciation+in+municipal+landfill+leachate.">Arsenic speciation in municipal landfill leachate.</a> Chemosphere. 79, 794-801 (2010).</li><li>Conklin, D. S., Fricke, W. M., Creed, A. P., Creed, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+pH+effects+on+the+formation+of+methylated+thio-arsenicals,+and+the+effects+of+pH+and+temperature+on+their+stability.">Investigation of the pH effects on the formation of methylated thio-arsenicals, and the effects of pH and temperature on their stability.</a> J. Anal. At. Spectrom. 23, 711-716 (2008).</li><li>Hansen, H. R., Raab, A., Jaspara, M., Milne, F. B., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfur-containing+arsenical+mistaken+for+dimethylarsinous+acid+[DMA(III)]+and+identified+as+a+natural+metabolite+in+urine:+major+implications+for+studies+on+arsenic+metabolism+and+toxicity.">Sulfur-containing arsenical mistaken for dimethylarsinous acid [DMA(III)] and identified as a natural metabolite in urine: major implications for studies on arsenic metabolism and toxicity.</a> Chem. Res. Toxicol. 17, 1086-1091 (2004).</li><li>Mandal, B. K., Suzuki, K. T., Anzai, K., Yamaguchi, K., Sei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SEC-HPLC-ICP-MS+hyphenated+technique+for+identification+of+sulfur-containing+arsenic+metabolites+in+biological+samples.">A SEC-HPLC-ICP-MS hyphenated technique for identification of sulfur-containing arsenic metabolites in biological samples.</a> J. Chromatogr. B. 874, 64-76 (2008).</li><li>Bartel, M., Ebert, F., Leffers, L., Karst, U., Schwerdtle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+characterization+of+the+inorganic+and+organic+arsenic+metabolite+thio-DMAV+in+cultured+human+lung+cells.">Toxicological characterization of the inorganic and organic arsenic metabolite thio-DMAV in cultured human lung cells.</a> J. Toxicol. 2011, (2011).</li><li>An, J., 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=Formation+of+dimethyldithioarsinic+acid+in+a+simulated+landfill+leachate+in+relation+to+hydrosulfide+concentration.">Formation of dimethyldithioarsinic acid in a simulated landfill leachate in relation to hydrosulfide concentration.</a> Environ. Geochem. Health. 38, 255-263 (2016).</li><li>Chen, B., 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=Arsenic+speciation+in+the+blood+of+arsenite-treated+F344+rats.">Arsenic speciation in the blood of arsenite-treated F344 rats.</a> Chem. Res. Toxicol. 26, 952-962 (2013).</li><li>Alava, P., 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=HPLC-ICP-MS+method+development+to+monitor+arsenic+speciation+changes+by+human+gut+microbiota.">HPLC-ICP-MS method development to monitor arsenic speciation changes by human gut microbiota.</a> Biomed. Chromatogr. 26, 524-533 (2012).</li><li>Kurosawa, 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=A+novel+metabolic+activation+associated+with+glutathione+in+dimethylmonoarsinic+acid+(DMMTAV)-induced+toxicity+obtained+from+in+vitro+reaction+of+DMMTAV+with+glutathione.">A novel metabolic activation associated with glutathione in dimethylmonoarsinic acid (DMMTAV)-induced toxicity obtained from in vitro reaction of DMMTAV with glutathione.</a> J. trace Elem. Med. Biol. 33, 87-94 (2016).</li></ol>

作者没有什么可透露的。

已开发的协议澄清了以前研究的关键步骤1,3,4,8,15省略或缩写, 这可能导致困难或失败期间DMMTAv和 DMDTAv合成。由于 DMMTAV是氧化敏感的1,5, 使用 N2清除的去离子水 (步骤 1.1.1-1.1.3) 来制备合成试剂, 以防...

由于 dimethylarsinic 酸 (DMAV) 已被证明表现出急性毒性和遗传毒性由于正在接受甲基化和 thiolation 在摄取1,2, 砷 thiolation 的代谢途径有深入研究了体外和体内3,4以及环境介质 (例如,垃圾渗滤液)5,6。以前的研究在活细胞中发现了 DMAV的减少和 thiolated 类似物, 例如、 dimethylarsinous 酸 (DMAIII)、dimethylmonothioarsinic 酸 (DMMTAV) 和 dimethyldithioarsinic 酸 (DMDTAV)7,8,9, 与 dimethylated thioarsenicals 如 DMMTAV显示更大的毒性比其他已知的无机或有机砷10。高毒性 thioarsenicals 的丰度具有严重的环境影响, 因为它们在高度 sulfidic 条件下可能对人类和环境构成风险11。然而, DMMTAv和 DMDTAv (横贯) 形成的机制及其在环境媒体中的命运仍然需要进一步研究。因此, 需要对 thioarsenicals 进行定量分析, 以提高对 DMMTAv和 DMDTAv的环境影响的理解。
虽然标准化学品是定量分析的关键要求, 但由于物种转化为其他物种的风险很高, DMMTAv和 DMDTAv的标准很难通过复制以前的研究获得。原始社会合成过程12。此外, 所提到的方法有局限性, 可能会导致在合成标准化学品和进行定量分析的实际困难。DMMTAv和 DMDTAv通常是通过 dmav 2的解决方案混合 dmav、Na2和 H2, 所以4在某摩尔比率1或冒泡 H1314的气体中进行的。冒泡方法使用 H2S 气体的直接供应, 以硫磺代替氧气, 这是剧毒和难以控制的经验不足的用户。相反, 上述混合方法1, 广泛用于定性分析 DMMTAv和 DMDTAV在环境研究5,6,12, 功能 thiolation使用 H2生成的 DMAV由混合 Na2s 和 H2, 因此4和生产 DMMTAv和 DMDTAv, 允许更容易的化学计量控制生产目标化学品, 与直接使用 H2的气体。
参考混合方法过程1,3,4,8,15在本研究中提到的一些关键实验步骤的局限性, 这可能导致实验失败。例如, 具体溶剂的详细情况 (即,去离子水) 的制备和合成砷的提取和结晶是过于缩写或没有充分详细说明。有关程序性步骤的这种分散和有限的信息可能导致 thioarsenicals 的不一致形成和不可靠的量化分析。因此, 本文所开发的修改后的协议描述了 DMMTAv和 DMDTAv库存解决方案的合成, 并进行了定量的物种分离分析。

<table><tbody><tr><td>Cacodylic acid</td><td>Sigma-Aldrich</td><td>20835-10G-F</td><td></td></tr><tr><td>Sodium sulfide nonahydrate</td><td>Sigma-Aldrich</td><td>S2006-500G</td><td></td></tr><tr><td>Sulfuric acid 96%</td><td>J.T.Baker</td><td>0000011478</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A7262-500G</td><td></td></tr><tr><td>Formic acid 98%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>066-00461</td><td></td></tr><tr><td>Diethyl ether (Extra Pure)</td><td>Junsei Chemical</td><td>33475-0380</td><td></td></tr><tr><td>Adapter cap for 60 mL Bond Elut catridges</td><td>Agilent Technologies</td><td>12131004</td><td>Syringe type of SPE</td></tr><tr><td>Bond Elut C18 cartridge</td><td>Agilent Technologies</td><td>14256031</td><td>Syringe type of SPE</td></tr><tr><td>HyPURITY C-18</td><td>Thermo Scientific</td><td>22105-254630</td><td>5 um, 125 x 4.6 mm</td></tr><tr><td>Glovebox</td><td>Chungae-chun, Rep. of Korea</td><td></td><td>Customized&amp;nbsp;</td></tr><tr><td>Agilent 1260 Infinity Bio-inert LC</td><td>Agilent Technologies</td><td>DEAB600252, DEACH00245</td><td></td></tr><tr><td>Agilent Technologies 7700 Series ICP-MS</td><td>Agilent Technologies</td><td>JP12031510</td><td></td></tr><tr><td>Finnigan LCQ Deca XP MAX Mass Spectrometer System</td><td>Thermo Electron Corporation</td><td>LDM10627</td><td></td></tr></tbody></table>

preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation

Dimethylated thioarsenicals, 如 dimethylmonothioarsinic 酸 (DMMTAV) 和 dimethyldithioarsinic 酸 (DMDTAv), 是由 dimethylarsinic 酸 (DMAV) thiolation 的新陈代谢途径产生的, 最近在环境和人体器官中发现的。DMMTAv和 DMDTAv可以量化, 以确定 dimethylated thioarsenicals 的生态效应及其在环境介质中的稳定性。这些化合物的合成方法是原始社会的, 使得复制以前的研究具有挑战性。此外, 缺乏关于储存技术的信息, 包括没有物种转化的化合物的储存。此外, 由于只有有限的合成方法信息, 在合成标准化学品和进行定量分析方面可能存在实验困难。本文提出的协议为 dimethylated thioarsenicals、DMMTAv和 DMDTAv提供了一个实际改进的合成方法, 并将有助于利用高性能液体进行物种分离分析的量化。用电感耦合等离子体质谱 (HPLC-ICP) 相结合的色谱法。通过对化学试剂的制备、过滤方法和贮存的研究, 对该过程的实验步骤进行了改进。

由于 DMAIII合成方法19错误地编写了 DMMTAV , 因此对合成的 DMMTAv和 DMDTAv的验证是合成和提取和确定理想标准的关键步骤。化学材料。合成化学物质可以通过 DMMTAV (兆瓦 154 g·mol-1) 和 DMDTAV (兆瓦 170 g·mol-1) 质量-电荷比 (m/z) 的峰值来验证, 使用的是电喷雾电离质量的正负离子模式。通过?...

Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

使用 DMAv为环境应用 DMMTAv和 DMDTAv的准备: 合成、纯化和确认

本文介绍了 dimethylmonothioarsinic 酸 (DMMTAv) 和 dimethyldithioarsinic 酸 (DMDTAv) 合成、诱导 dimethylarsinic 酸 (dmav) thiolation 通过混合 DMAv 的改进实验协议。, Na2S, 和 H2, 所以4。修改后的协议提供了实验指导, 从而克服了在定量分析过程中可能导致实验失败的综合步骤的局限性。

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

preparation-dmmtav-dmdtav-using-dmav-for-environmental-applications

Universidad San Sebastian

Research

JoVE Journal

Environment

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

5.5K 次观看.  Korea Basic Science Institute, Seoul Center. 本文介绍了 dimethylmonothioarsinic 酸 (DMMTAv) 和 dimethyldithioarsinic 酸 (DMDTAv) 合成、诱导 dimethylarsinic 酸 (dmav) thiolation 通过混合 DMAv 的改进实验协议。, Na2S, 和 H2, 所以4。修改后的协议提供了实验指导, 从而克服了在定量分析过程中可能导致实验失败的综合步骤的局限性。

视频: Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

科研

环境科学

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

在体外的tRNA与阿米巴 DNA和tRNA甲基Dnmt2（Ehmeth）酶甲基化含量

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

免疫与感染

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

新生儿缺氧的生化测量

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

医学

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.

开发和测试针对物种的定量PCR检测环境DNA应用

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

使用硫酸二甲酯探测RNA结构 体 外 和细胞测序突变分析

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

生物化学

<meta charset="utf8"></head><body>使用数字液滴 PCR 进行病毒基因组定量的标准化方案

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/&#181;L), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/&#181;L and 10,000 copies/&#181;L, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

<meta charset="utf8"></head><body>作者聚焦:优化数字微滴 PCR 方法以实现准确的腺相关病毒基因组定量

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of ...

使用 DMA^v为环境应用 DMMTA^v和 DMDTA^v的准备: 合成、纯化和确认

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揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

在体外的tRNA与阿米巴 DNA和tRNA甲基Dnmt2（Ehmeth）酶甲基化含量

新生儿缺氧的生化测量

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析'

开发和测试针对物种的定量PCR检测环境DNA应用

使用硫酸二甲酯探测RNA结构体外和细胞测序突变分析

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

使用 DMAv为环境应用 DMMTAv和 DMDTAv的准备: 合成、纯化和确认

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使用硫酸二甲酯探测RNA结构 体 外 和细胞测序突变分析

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

使用 DMA^v为环境应用 DMMTA^v和 DMDTA^v的准备: 合成、纯化和确认

使用硫酸二甲酯探测RNA结构体外和细胞测序突变分析