我们衷心感谢DARPA赞助的微/纳米射流基础焦点（MF3）根据合同编号N66001-10-1-4003，中心的资金和从DARPA的批N660001-09-C-2082。

1。 ITP的物理 ITP形成一个像电荷的离子之间的尖锐的移动边界。该技术可与阴离子或阳离子的样品，但我们裁缝介绍阴离子性ITP和注意的原则同样适用于阳离子性ITP。我们选择LE和TE缓冲乐离子等有较高幅度的有效电泳迁移。有效的电泳迁移率，μ= U / E的 ，是电场，电子和离子的漂移速度 u之间的比例常数。我们建立一个弥漫接口之间的LE和TE和应用直接从高电场电导率乐区的低导TE区。该系统迅速建立在强电场梯度在ITP接口，由于非均匀电导率剖面。按照它的名称（从希腊语“ISOS”的意思是“平等”，“takhos”意味着“速度”）的同时，统一的VE，TE和LE的离子旅游locity，作为一个非均匀的电场和电流保护的结果（这是所谓的“ITP的条件”， 见图1）。 ITP接口的自锐性，扩散到TE区的经验，强大的恢复通量和返回的领先区（TE在LE区离子反之亦然）的LE离子。样品离子集中在这个接口，如果他们在TE区的有效流动性大于TE合作离子，如果他们在LE区的有效流动性是比LE共同离子（ 见图1）少。自锐化和重点ITP的性能，有助于这一技术的鲁棒性ITP的接口（例如，由于压力驱动流或几何形状的变化，如收缩，扩张，曲折）的干扰比较敏感。 在峰值模式ITP（ 见图2a和视频1-2），样品的离子浓度在任何时候都显着低于LE和TE离子的浓度，因此可以忽略不计，以促进当地电导率。样品离子的分布取决于相邻区域的自锐性接口（TE和LE）和样品的有效流动性的价值相对于这些区域14多个样品离子作为主要集中在这个狭小的ITP接口地区。叠嶂。接口和峰宽度，以及相关富集的因素，规模与 ​​电流成反比（见实验图2b）14。 样品离子初始样本足够高的浓度和足够的时间积累，达到阈值浓度值。完全电离的物种，这个值决定由Kohlrausch调节功能（KRF）15弱电解质，Alberty和Jovin功能决定的。高原16,17模式，在图3和视频3，样品离子的分离和净化局部均匀和恒定浓度区的有效流动确定的顺序描绘。很稀离子仍可能集中在高原区之间的峰值模式。在ITP，样品离子可以引入有限之间的TE和LE（ 见图3）注射或交替TE和/或LE（ 见图2）混合在一起。我们是指在TE区“半无限”注射液，可用于不断积累分析离子混合。连续采样积累增加在这两个高峰，高原模式检测的灵敏度。然而，有限的注射是较为常见的高原模式。这是可能的，因为半无限注射的初步分析物浓度高，可大大增加TE的导电性和较低的集中率。此外，全面净化没有可能在半无限注射（因为仍留插件在TE有限的浓度）。 2。设备清洗和准备对于在本议定书提出的分析，我们使用各向同性湿式蚀刻（约D形截面）玻璃微流控芯片与跨通道的设计（ 见图1）。以下的清洗和准备协议进行了优化，高硼硅和石英渠道，但也可用于玻璃/ PDMS芯片。实验之前执行此清洁程序，以确保运行运行的重复性和成功应用的需要动态涂层抑制电渗流（EOF）。此协议的遗漏可能导致在强色散ITP接口14。 <ol><li>为了净化渠道，填补10-20μL10％漂白的北，东，南水库，并申请在2分钟西水库的真空。如果使用标准的钳芯片球童，真空可以有效​​地应用于简单attachinĞ200μL，广角端（未筛选）枪头芯片水库和2毫米内径管的真空连接线。 </li><li>清空水库和冲洗2分钟用1 M氢氧化钠的通道（如步骤1）。这轻轻的蚀刻通道的墙壁，产生一个干净的硼硅表面，以帮助建立统一的表面特性。 </li><li>空水库和清洁与去离子水（DI），然后用清水冲洗通道LE〜2分钟。在此期间，表面特性和动态涂层平衡通道内。 </li></ol> 3。在峰值模式ITP的荧光聚焦<ol><li>准备1毫升LE 100毫米，200毫米TRIS盐酸，1％的PVP组成。 </li><li>准备1毫升TE 100毫米肝素钠组成和200毫米TRIS。 90μLTE的结合与10μL1 mM的Alexa Fluor 488（AF488）。 </li><li>与Le冲洗后第2部分中所述，空西水库和清理与DI几次或为了淡化任何勒留在水库。 20微升TE含有AF488填补这个水库。 </li><li>在东水库将正极和地面（负）​​在西藏的电极和申请2μA（恒流）。样本高峰将在一个恒定的速度从西藏东水库（见图1）和低电导率的TE填补了通道，这些水库之间的电压会增加迁移。 </li></ol> 4。从培养大肠杆菌核酸提取和纯化大肠杆菌 能够有选择性地集中离子物种ITP理想的生物样品制备技术。我们来自未经处理的细胞裂解物的核酸，通过选择有效调动幅度低于目标核酸，但高于合作离子PCR抑制剂（如阴离子洗涤剂，蛋白质，有机溶剂，即使在HI后负离子净化GH浓度）。阳离子PCR抑制剂（如碱金属和阳离子蛋白和洗涤剂）在相反的方向迁移，所以也留下。 ITP的提取样品水库和重点目标核酸，而离开较慢的PCR抑制物种的背后（ 见图4）。 <ol><li>获取的样品或文化E.密度大于10 8 CFU / mL的大肠杆菌细胞。 </li><li>转移到一个安全锁的离心管和沉淀，离心4000克6分钟1毫升细胞培养。 </li><li>重悬沉淀在80μL无RNase的水，并添加10μL甘氨酸10毫米，10毫米之二 - 三，2毫米EDTA，0.1％的Triton-X的，5毫克/毫升溶菌酶溶解剂组成。轻轻混匀，静置5分钟。在室温（裂解协议适应Bercovici 等。11）。 </li><li>加入10μL1 M氢氧化钠提高裂解液的pH值〜12.5。轻轻开动移液器上下，直到解决方案变得清晰，在这一点溶藻是完整的。 </li><li>结合10μL裂解液90μL的50 mM的甘氨酸和100毫米之二 - 三。该解决方案现在可以被用来作为TE。 </li><li>准备1毫升勒BIS-TRIS，500毫米250毫米盐酸，1％的PVP和1X的SYBR Green II组成。填写与LE微流控芯片在第3部分所述。 </li><li>以片分析中提取纯化核酸的ITP患者后，取代了PCR兼容勒东水库的内容，含有50MM之二 - 三，25毫米盐酸，0.1％PVP。申请V至1000之间的东，西井开始实验。这些水库之间的电流将减少。 </li><li>在实验结束后的样品洗脱进入LE水库。与此洗脱重合，此系统的电流随时间通常到达高原值（因为阻力现在TE的离子通道内均匀分布为主）。轻轻混匀水库由内容重复移液和提取定量RT-PCR进行分析5μL的体积。 </li></ol> 5。氨基酸分离与的阳离子高原模式ITP和非重点示踪剂（NFT）的可视化 ITP的可用于分离和集中到毗邻和检测之间的TE和LE高原小离子。这可以根据当地的导电性，紫外吸收，温度传感，折射率，理化性质，如检测和鉴定。在这里，我们展示一个非重点示踪法（NFT）的荧光，合作离子物种被添加到了LE。这种荧光物种不集中，但其浓度适应当地的电场，从而使可视化纯化的高原区（ 见图5）。 <ol><li>准备1毫升LE组成的100毫米乙醇胺，甘氨酸200毫米，1％PVP，阳离子荧光罗丹明6G 100微米。 </li> &lt;李&gt;准备1毫升TE TRIS 20毫米，40毫米甘氨酸组成。准备通过混合90μL的TE 10μL50 mM的精氨酸，赖氨酸和50毫米的样本。 </li><li>免除20μL在西方LE和北东水库的水库和样品。申请在南水库1分钟的真空。 </li><li>冲洗东DI和TE（无样品TE）的更换。 </li><li>申请500之间的东，西水库至五。 </li></ol> 6。代表结果我们在图2b和图4中的核酸提取实验显示isotachopherograms峰值模式实验。荧光记者（如AF488的SYBR Green II）峰值模式实验中，整体的荧光强度可以集成和比对校准曲线获得定量的浓度信息。12此外，核酸提取实验中，样品被允许以洗脱到T他乐水库和提取与分析移液器定量RT-PCR。10,12，我们发现在图5中的氨基酸分离高原模式isotachopherograms。荧光强度（LE或TE区强度相对），可用于区域识别，同时区的宽度，使定量。 <img alt="图1。" src="/files/ftp_upload/3890/3890fig1.jpg" /> 图1。等速电泳（ITP）是一种用于微流体应用离子敏感的检测和分离技术在电动。 ITP患者提供了分离，选择性聚焦和富集能力。此外，它是非常强大，因为其自锐性，物理干扰不敏感。样品离子选择性集中之间的领先（LE）和尾随电解质（TE）和通过微旅行恒定速度在LE区的领先离子的速度决定。样品离子集中被包围的LE和TE离子的有效流动，如果其有效电泳迁移。原理图描绘了一个模型ITP的实验样品重点不断之间的LE和TE区。 <img alt="图2。" src="/files/ftp_upload/3890/3890fig2.jpg" /> 图2。在“高峰模式”ITP的稀释样品离子的焦点。一）两种截然不同的稀（C 样品 &lt;&lt; C LE）的物种在繁忙的LE和TE区之间形成的界面模式的重点。仅LE和TE确定的电场，样品物种不显着贡献当前。样品离子与TE（半无限注射）混合，并集中在大致高斯峰在ITP接口。注重标准（不平等所示）适用于强电离物种的。 B）应用电流范围为重点的实验显示的Alexa Fluor 488（AF488）（也见至五峰值模式IDEO 1）。样品峰宽成反比，电流（由于电渗流可以忽略不计的对流弥散）14自锐的界面是耐压力驱动流（ 视频2）由于色散。 <img alt="图3。" src="/files/ftp_upload/3890/3890fig3.jpg" /> 图3：在足够高浓度的重点，在“高原模式”的样品离子和支配当地的导电性。我们通常在有限注射TE和LE之间引入样品离子。在这种方式中，样品离子分开，以便根据其有效的电泳迁移率（见视频3）。对于强烈电离物种，TE和高原区浓度确定由Kohlrausch调节功能（KRF，最初由LE区不变集）。稀离子继续集中在峰值模式之间的高原地带包围的有效流动。重点Çriterion（不平等所示）适用于强电离物种。 <img alt="图4。" src="/files/ftp_upload/3890/3890fig4.jpg" /> 图4。裂解物具有峰值模式ITP的编制和核酸提取。从大肠杆菌中提取核酸大肠杆菌细胞培养使用溶菌酶辅助碱溶藻和选择性电泳纯化， 通过 ITP的重点。与裂解液混合的TE（棕色）包含目标（绿色）核酸，蛋白质，和潜在的PCR抑制化学。适当的选择，使落后和领先的离子选择性靶核酸的重点，同时离开落后PCR抑制剂。总核酸ITP的高峰，往往需要在一个非理想的形状，显示在这里的插图形象。由于此法的示范，我们总核酸提取革兰氏阴性菌， 大肠杆菌 （单独用氢氧化钠溶液溶解），纯化从裂解NA使用ITP的，收集提取的遗传物质，并进行定量RT-PCR分析，以验证成功纯化的16S rRNA（红色），并从细菌培养的16S rDNA（绿色）。阴性对照周期为16S rRNA基因（蓝色）和16S rDNA（黄色）的阈值以上，每30个循环。我们进行了定量RT-PCR使用电力的SYBR Green的RNA-C T 1步套件从150纳米（5&#39;-CGGATTGGAGTCTGCAACTCG）和反向（5&#39;-CACAAAGTGGTAAG CGCCCTC的）引物，在推荐的热循环条件下，应用生物系统公司。 <img alt="图5。" src="/files/ftp_upload/3890/3890fig5.jpg" /> 图5。非重点示踪剂（NFT）的检测，未标记的氨基酸的分离和检测。 NFT的检​​测示意图）。有限的样品离子注入引入的东通道。混合阳离子荧光示踪，有效的流动性比TE离子（μ 示踪 &lt;μ 的TE）的LE。的荧光是说作为示踪underspeeding“。作为荧光团的LE electromigrates到样品高原和TE现在占领的区域，它经历了一个更高的电场，从而其浓度的增加。这种浓度的变化创建的isotachopherogram的步骤。 B）分离两种氨基酸，精氨酸和赖氨酸，使用罗丹明6G的荧光示踪underspeeding NFT的检​​测。在ITP轻微高峰之间的TE和精氨酸是常见的，还不是很清楚，这里不干扰检测或量化的高原。 补充电影1。 <a href="http://www.jove.com/files/ftp_upload/3890/3890movie1.mpg" target="_blank">点击这里查看补充的电影</a> 。 补充电影2。 <a href="http://www.jove.com/files/ftp_upload/3890/3890movie2.mpg" target="_blank">点击这里查看补充电影</a> 。 补充电影（3） <a href="http://www.jove.com/files/ftp_upload/3890/3890movie3.mpg" target="_blank">点击此处查看补充的电影</a> 。

<ol>
	<li>Jung, B., Bharadwaj, R., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+millionfold+sample+stacking+using+transient+isotachophoresis.">On-chip millionfold sample stacking using transient isotachophoresis.</a> Anal. Chem. 78 (7), 2319-2319 (2006).</li><li>Jung, B., Zhu, Y., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+100+am+fluorophores+using+a+high-sensitivity+on-chip+CE+system+and+transient+isotachophoresis.">Detection of 100 am fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis.</a> Anal. Chem. 79 (1), 345-345 (2007).</li><li>Everaerts, F. M., Beckers, J. L., Verheggen, T. P. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotachophoresis:+Theory,+instrumentation,+and+applications.">Isotachophoresis: Theory, instrumentation, and applications.</a> Elsevier Science &#38; Technology. , (1976).</li><li>Khurana, T. K., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconcentration,+separation,+and+indirect+detection+of+nonfluorescent+analytes+using+fluorescent+mobility+markers.">Preconcentration, separation, and indirect detection of nonfluorescent analytes using fluorescent mobility markers.</a> Anal. Chem. 80 (1), 279-279 (2008).</li><li>Bercovici, M., Kaigala, G. V., Backhouse, C. J., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+carrier+ampholytes+assay+for+portable,+label-free+detection+of+chemical+toxins+in+tap+water.">Fluorescent carrier ampholytes assay for portable, label-free detection of chemical toxins in tap water.</a> Anal. Chem. 82 (5), 1858-1858 (2010).</li><li>Bercovici, M., Kaigala, G. V., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+analyte+identification+using+isotachophoresis+and+a+fluorescent+carrier+ampholyte+assay.">Method for analyte identification using isotachophoresis and a fluorescent carrier ampholyte assay.</a> Anal. Chem. 82 (5), 2134-2134 (2010).</li><li>Kaigala, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+system+for+isotachophoresis+assays.">Miniaturized system for isotachophoresis assays.</a> Lab Chip. 10 (17), 2242-2242 (2010).</li><li>Chambers, R. D., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantification+of+isotachophoresis+zones+using+nonfocusing+fluorescent+tracers.">Imaging and quantification of isotachophoresis zones using nonfocusing fluorescent tracers.</a> Anal. Chem. 81 (8), 3022-3022 (2009).</li><li>Schoch, R. B., Ronaghi, M., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+selective+extraction,+isolation,+preconcentration,+and+quantitation+of+small+RNAs+from+cell+lysate+using+on-chip+isotachophoresis.">Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.</a> Lab Chip. 9 (15), 2145-2145 (2009).</li><li>Persat, A., Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+nucleic+acids+from+whole+blood+using+isotachophoresis.">Purification of nucleic acids from whole blood using isotachophoresis.</a> Anal. Chem. 81 (22), 9507-9507 (2009).</li><li>Bercovici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+urinary+tract+infections+using+isotachophoresis+and+molecular+beacons.">Rapid detection of urinary tract infections using isotachophoresis and molecular beacons.</a> Anal. Chem. 83 (11), 4110-4110 (2011).</li><li>Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+malaria-infected+erythrocytes+using+isotachophoresis.">Extraction of DNA from malaria-infected erythrocytes using isotachophoresis.</a> Anal. Chem. 83 (24), 9715-9715 (2011).</li><li>Persat, A., Chambers, R. D., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+principles+of+electrolyte+chemistry+for+microfluidic+electrokinetics.+Part+I:+Acid-base+equilibria+and+pH+buffers.">Basic principles of electrolyte chemistry for microfluidic electrokinetics. Part I: Acid-base equilibria and pH buffers.</a> Lab Chip. 9 (17), 2437-2437 (2009).</li><li>Garcia-Schwarz, G., Bercovici, M., Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+dispersion+in+isotachophoresis.">Sample dispersion in isotachophoresis.</a> J. Fluid Mech. 1 (1), 1-1 (2011).</li><li>Kohlrausch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+concentrations-verschiebungen+durch+electrolyse+im+inneren+von+l&#214;sungen+und+l&#214;sungsgemischen.">&#220;ber concentrations-verschiebungen durch electrolyse im inneren von l&#214;sungen und l&#214;sungsgemischen.</a> Ann. Phys. 298 (10), 209-209 (1897).</li><li>Alberty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+boundary+systems+formed+by+weak+electrolytes.+Theory+of+simple+systems+formed+by+weak+acids+and+bases.">Moving boundary systems formed by weak electrolytes. Theory of simple systems formed by weak acids and bases.</a> J. Am. Chem. Soc. 72 (6), 2361-2361 (1950).</li><li>Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphasic+zone+electrophoresis.+I.+Steady-state+moving-boundary+systems+formed+by+different+electrolyte+combinations.">Multiphasic zone electrophoresis. I. Steady-state moving-boundary systems formed by different electrolyte combinations.</a> Biochem. 12 (5), 871-871 (1973).</li><li>Persat, A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling+by+simultaneous+selective+isotachophoresis+and+hybridization+with+molecular+beacons.">MicroRNA profiling by simultaneous selective isotachophoresis and hybridization with molecular beacons.</a> Anal. Chem. , (2011).</li></ol>

我们什么都没有透露。

ITP的方法介绍，在这里能够快速，灵敏，可靠的检测和处理的离子分子。在使用ITP的主要挑战是LE和TE缓冲液的正确选择。在阴离子ITP的，我们通常选择为主导的阴离子氯化物，因为它是一个具有绝对的流动性非常高的强酸，因而具有非常可预测的性能。因此，选择适当的TE和反离子缓冲阴离子ITP的选择通常是减少。选择性重点实验，TE的选择是至关重要的污染离子的排除。 TE的有效流动性较低的应用中是不存在的地方污染物，导致更快的对焦速度。我们建议使用以下，相对乖巧TE的阴离子离子：制造执行系统，公开资讯观测站，肝素钠，甘氨酸。反离子的选择影响体系的pH值和TE的有效流动。共同反三（PKA 8.1）和二，三（PKA 6.4）。对于要求较低或较高的pH值的检测，我们建议吡啶（PKA 5.25）和乙醇胺（PKA 9.5），分别。 表1和表2，阴离子和阳离子ITP的分别，我们总结了几个有用的缓冲区的例子。我们以阴离子LE阳离子LE离子钠离子和盐酸，我们假设100毫米离子强度的LE缓冲区。 读者会注意到，在我们的协议缓冲液的离子强度（ 表1-2）总是大于或等于10毫米。虽然在ITP的物理学理论应用于离子强度低于10毫米，近1毫米级的天然污染物（如碳酸之间的水和大气中二氧化碳含量的反应），往往限制在低离子强度缓冲的实际使用。 我们注意到，信号转导机制并不仅限于在本协议中的检测。作为第5部分，在高原纯净区模式，简要讨论，可以通过在当地电导率的变化，紫外线absor检测bance，温度，或折射率。在我们自己的小组中，我们已经开发出一种方法，利用荧光载体两性电解质敏感的检测和鉴定未知的待测。5在峰值模式试验，特异性探针，可以用来标记重点分析物。举一个例子，我们使用分子信标-寡核苷酸探针-荧光他们的目标序列杂交后的检测具有高度特异性的靶DNA或RNA分子11,18。 虽然ITP是相对强劲的分散造成的压力驱动流和EOF，EOF的过度可导致ITP接口，平均EOF速度成为ITP的速度等于停滞。14这种强烈的EOF发生，尤其是在高pH值条件和低离子强度。出于这个原因，我们建议使用100毫米的订单，如果方便的缓冲区pH值8或更低和离子强度。根据我们的经验，PVP是最有效的抑制硼硅芯片的EOF的涂层。然而，由于其筛分性能，PVP的可能不适合的一些应用，如基因组DNA对焦。在实验中，我们观察到，PVP的加入可以大大减少基因组DNA的绝对移动（点不集中）。醇基涂料在这些情况下，如在表面活性剂（如海卫X-100）一起选择Sigmacote还可以有效地减少的EOF 10。 <table border="1"><tbody><tr><td></td><td colspan="4"> TE的阴离子（PKA -1） </td></tr><tr><td> 缓冲反离子（PKA +1） </td><td> MES（6.10） </td><td> 公开资讯观测站（7.20） </td><td> 肝素钠（7.50） </td><td> 甘氨酸（8.15） </td></tr><tr><td>乙醇胺（9.50） </td><td> 21.41 </td><td> 20.40 </td><td> 17.38 </td><td> 22.85 </td></tr><tr><td>三（8.08） </td><td> 21.00 </td><td> 19.23 &lt;/ TD&gt; <td> 15.84 </td><td> 17.56 </td></tr><tr><td> BIS-TRIS（6.40） </td><td> 18.22 </td><td> 10.41 </td><td> 7.30 </td><td> 5.23 </td></tr><tr><td>吡啶（5.18） </td><td> 1.01 </td><td> 3.72 </td><td> 2.42 </td><td> 1.48 </td></tr></tbody></table> 表1。有效的移动幅度调整中阴离子ITP纯待测区LE是100毫米盐酸和200毫米的缓冲反离子（×10-9米2 / V / S）。 <table border="1"><tbody><tr><td></td><td colspan="4"> TE的阳离子（PKA +1） </td></tr><tr><td> 缓冲反离子（PKA -1） </td><td> 乙醇胺（9.50） </td><td> 三（8.08） </td><td> BIS-TRIS（6.40） </td><td> 吡啶（5.18） </td></tr><tr><td> MES（6.10） </td><td> 35.57 </td><td> 21.96 </td><td> 16.39 </td><td> 10.45 </td></tr><tr><td>莫PS（7.20） </td><td> 35.47 </td><td> 20.92 </td><td> 9.76 </td><td> 3.93 </td></tr><tr><td>肝素钠（7.50） </td><td> 35.35 </td><td> 19.97 </td><td> 7.77 </td><td> 2.88 </td></tr><tr><td>甘氨酸（8.15） </td><td> 34.77 </td><td> 16.72 </td><td> 4.50 </td><td> 1.44 </td></tr></tbody></table> 表2。有效的移动幅度调整阳离子ITP纯分析物区（×10-9米2 / V / S）的LE是钠100毫米和200毫米的缓冲反离子。

<table border="1"><tbody><tr><td colspan="4"> 试剂 </td></tr><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>蒸馏水</td><td> GIBCO公司</td><td> 10977 </td><td>核糖核酸酶/ DNA酶免费</td></tr><tr><td> Clorox公司超</td><td>高乐氏（Clorox） </td><td> 02489CT </td><td></td></tr><tr><td>氢氧化钠（NaOH） </td><td> mallinckrodt </td><td> 7708 </td><td></td></tr><tr><td>盐酸（HCl） </td><td>电解二氧化锰化学品</td><td> HX0603-4 </td><td></td></tr><tr><td> trizma基地（TRIS） </td><td> Sigma-Aldrich公司</td><td> T6066 </td><td></td></tr><tr><td>聚乙烯吡咯烷酮（PVP） </td><td> polysciences公司</td><td> 06067 </td><td>兆瓦百万</td></tr><tr><td>肝素钠</td><td> Sigma-Aldrich公司</td><td>的H-4034 </td><td></td></tr><tr><td>的Alexa Fluor 488羧酸</td><td> Invitrogen公司</td><td> A20000 </td><td></td></tr><tr><td>甘氨酸</td><td> Sigma-Aldrich公司</td><td>的T-9784 </td><td></td></tr><tr><td>双 - 三</td><td> Sigma-Aldrich公司</td><td> B4429 </td><td></td></tr><tr><td> EDTA的</td><td> GIBCO公司</td><td> AM9260G </td><td></td></tr><tr><td> TRITON-×100 </td><td> Sigma-Aldrich公司</td><td> X100 </td><td></td></tr><tr><td>溶菌酶</td><td> Sigma-Aldrich公司</td><td> L6876 </td><td></td></tr><tr><td>的SYBR Green II </td><td> Invitrogen公司</td><td> S7564 </td><td></td></tr><tr><td>乙醇胺</td><td> Sigma-Aldrich公司</td><td> 411000 </td><td></td></tr><tr><td>罗丹明6G </td><td> Acros Organics公司（Geel，比利时） </td><td> CAS号989-38-8 </td><td></td></tr><tr><td> L-氨基酸</td><td> Sigma-Aldrich公司</td><td> LAA21 </td><td></td></tr><tr><td> 电源的SYBR Green的RNA-C T 1步套件 </td><td>应用生物系统公司</td><td> 4389986 </td><td></td></tr><tr> &lt;TD&gt; PCR引物</td><td>集成DNA技术</td><td></td><td></td></tr><tr><td colspan="4"> 设备 </td></tr><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>高硼硅微芯片</td><td>卡尺生命科学公司</td><td> NS12A </td><td>提供带或不带塑料盒</td></tr><tr><td>真空泵</td><td>加斯特</td><td>的DOA-P104-AA </td><td></td></tr><tr><td>源</td><td>吉时利</td><td> 2410 </td><td>恒定电流和恒定电压操作模式</td></tr><tr><td>倒啶显微镜</td><td>奥林巴斯</td><td> IX70 </td><td>照明用汞灯（奥林巴斯）或LED（Thorlabs） </td></tr><tr><td>过滤立方体</td><td>欧米茄</td><td> XF115-2 </td><td>激发/发射：蓝/绿</td></tr><tr><td>安全锁定离心管</td><td> Eppendorf公司</td><td> 022363204 </td><td> 1.5毫升容量</td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5417C </td><td></td></tr></tbody></table> 表3。特定的试剂和设备。

on-chip isotachophoresis for separation of ions and purification of nucleic acids

电动技术是一个微型应用程序，因为其独特的能力，执行各种流体和电泳过程简单，无运动部件的紧凑型系统的主食。等速电泳（ITP）是一个简单的和非常强大的电动技术，可以实现基于离子迁移的百万倍的富集1,2和高效的分离和提取，例如，我们已经证明未标记的分离和敏感的检测ITP的应用。离子分子（如毒素，DNA，rRNA基因的miRNA），很少或根本没有样品制备4-8从复杂基质中的核酸，包括细胞培养，尿液和血液的提取和纯化。9-12 ITP的实现重点和分离使用外加电场和两个缓冲区内的流体通道系统。阴离子分析物，领先的电解液（LE）的缓冲区选择小号UCH，其阴离子具有更高的有效比尾随电解质（TE）的缓冲区（有效的流动性描述观察到的离子漂移速度，并考虑到离子的电离状态，在详细介绍由Persat 等人的阴离子电泳迁移13）。建立TE和LE之间的接口后，施加电场，LE的离子移动由TE离子占据该地区。样品离子中间有效的流动性比赛提前TE的离子，但不能超越的LE离子，使他们集中在LE-TE的接口（以下简称“ITP接口”）。此外，TE和LE的形式分别低和高导电性，这建立在ITP接口的一个陡峭的电场梯度地区。这一领域的的梯度preconcentrates样本的物种，因为他们注重。正确选择的重点目标物种的净化和其他非重点物种的TE和LE结果，并最终分离ð样本物种隔离。 在这里，我们审查的物理原理基础性ITP和讨论两个标准的操作模式：“高峰期”和“高原”的模式。在繁忙的模式，相对稀释样品离子集中在窄峰重叠在ITP接口。在高原模式，更丰富的样品离子达到稳态浓度，并分隔成高原毗连区的有效流动有序。峰和高原模式出现了相同的基本物理，但分化的初步分析物的浓度和/或样本积累分配的时间代表不同的制度。 我们首先详细描述模型峰值模式实验，然后表现出对大肠杆菌核酸提取的峰值模式分析大肠杆菌细胞的培养。我们的结论提出高原模式分析，我们使用非聚焦示踪剂（NFT）的物种，以可视化的分离和每形成氨基酸的定量分析。

Watch this Scientific Journal Video about On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids at JoVE.com

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

等速电泳（ITP）是一个强大的电动分离和富集技术的应用范围从毒素检测样品制备。我们回顾ITP的物理原理和两个具体的例子应用，应用这种技术的方法：小分子和细胞培养裂解核酸纯化分离和检测。

对芯片等速电泳离子的分离和纯化核酸

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

on-chip-isotachophoresis-for-separation-ions-purification-nucleic

Research

Education

JoVE Core

JoVE Journal

Physics

Bioengineering

Unit 1

Current and Resistance

SCIENTISTS IN ACTION

24.2K 次观看.  Stanford University. 等速电泳（ITP）是一个强大的电动分离和富集技术的应用范围从毒素检测样品制备。我们回顾ITP的物理原理和两个具体的例子应用，应用这种技术的方法：小分子和细胞培养裂解核酸纯化分离和检测。

视频: On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

lensfree芯片断层用人多角度的光照和像素超高分辨率的显微镜

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

片上内皮炎症表型

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

将伽马改性肽核酸自组装成有机溶剂混合物中复杂的纳米结构

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.