本研究部分由国家自然科学基金资助 (31370813、3147670、31670831和 31600672)、国家转基因植物专项基金 (2016ZX08012-003、2016ZX08012-005)、新世纪优秀人才项目大学, 中国国家重点研究开发项目 (2016YFA0500601) 和中国博士后科学基金 (2016M591667)。

注意: 本协议假定已为所需的微通道和加载适配器提供形状的不锈钢模具 (3D 文件为 补充文件 1 和 2).该协议还假定已经进行了植物 DNA 分离. 
 1. 制作毛细管阵列现成的盒式磁带 <ol> <li> 清洗不锈钢模具。
	<ol> <li> 通过洗涤剂、乙醇和去离子水清洗不锈钢模具, 去除潜在的污染物。用氮气烘干模具. </li>
	</ol> </li> <li> 清除毛细管 <ol> 戴上安全护目镜、实验室和 #160; 制服和化学防护手套. <li>
		<li> 将毛细管放入烧杯中。用30毫升丙酮清洁毛细血管5分钟去除有机物, 然后用去离子水冲洗毛细血管. 
		注意: 在油烟罩中处理丙酮. </li>
		<li> 将10毫升 h 2 O 2 放入烧杯和 #160; 然后将 30 ml h 2 , 所以 4 加入到 h 2 O 2 中, 以防止过热。确保解决方案 (食人鱼溶液) 完全覆盖毛细血管至少30分钟. 
		注意: 在处理食人鱼溶液时要小心 (H 2 , 以便 4 /h 2 O 2 = 3: 1, v/五) 。如果食人鱼溶液溢出, 用大量的水迅速冲洗溶液, 并用纸巾擦拭表面. 
		注: 对毛细管进行彻底清洗是保证灯反应成功的关键之一。在清洗过程中必须摇动酸缸, 去除毛细管中的气泡, 必要时也可延长清洗时间. </li>
		<li> 用大量的水冲走食人鱼溶液。用乙醇和去离子水冲洗毛细血管5分钟。在干燥的烤箱中干燥毛细管. </li>
	</ol> </li> <li> 倒烷 (硅橡胶) <ol> 重出了在50毫升管中有1:1 的比值的硅橡胶底座和硫化试剂。通常, 混合5克的弹性体基地, 以5克的弹性体固化剂. </li>
		<li> 用玻璃棒将混合物彻底搅拌约5分钟。将该管与硅橡胶在真空钟罐中放置30分钟吸管. </li>
		<li> 将其慢慢倒入不锈钢模具的气缸中。允许硅烷在60和 #176 的干燥炉中固化3小时; C 注意: 在浇注时要小心避免气泡。在浇注后, 让它站在5分钟的自然去除的气泡出的硅橡胶. </li>
	</ol> </li> <li> 在硫化硅橡胶后将模具从 "硅橡胶" <ol> 中卸下后, 通过用气缸拉出模具来拆卸模具。用手术刀把模具的边缘切掉, 从气缸中取出. </li>
		<li> 用乙醇和去离子水清洗三次。用氮气烘干的支持. </li>
	</ol> </li> <li> 将该支持的下表面视为疏水性。在室温下, 用疏的外套浸泡1秒的硅橡胶支架. </li> 干燥10分钟
	<li> 将毛细管插入到 "支持" <ol> <li> 中, 将已清洗的4毫米毛细管插入到该支持的孔中, 并将0.5 毫米的毛细管留在该支持的顶面外。确保毛细血管的两端处于同一水平. </li>
	</ol> </li> <li> 将毛细管的外表面和该支持的顶面表面视为疏水性. 
	<ol> <li> 添加15和 #181; 我疏大衣上表面的支持。请注意, 涂层立即蔓延到所有的顶部表面 (包括表面的支持和外部表面的毛细血管外露部分) 的毛细管力. </li>
		<li> 空气干燥疏改性毛细管阵列. </li>
	</ol> </li> <li> 将底漆固定到毛细管阵列 <ol> 准备底漆溶液中。重出0.65 克壳聚糖 (分子式: (C 6 H 11 无 4 ) n ), 并将其分解为50毫升去离子水, 通过调节 pH 值为 4.5-5.5, 用乙酸实现壳聚糖浓度为1.3%。根据 表 1 准备底漆组件组合。请参见 辅助表 1 以引物 </li> <li> 添加1.6 和 #181; 一组底漆的混合物, 根据预先设计好的顺序填充毛细管阵列的一个相应毛细管. 
		注: 其余的两个空白毛细管被设置为负控制. </li>
		<li> 将阵列锚定在标准平底96井板的透明井中, 并在60和 #176 处干燥阵列; C 至少 2 h. </li>
	</ol> </li> </ol> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"> <tbody> <tr> <td> 灯具底漆固定混合组件 (初始浓度) </td> <td> 音量 (#956; L) </td> </tr> <tr> <td> ddH 2 O </td> <td> 17.0 </td> </tr> <tr> <td> 壳聚糖 (1.3%) </td> <td> 1.0 </td> </tr> <tr> FIP/毕普底漆 (20 和 #956; m) </td> <td> 2.0/2.0 </td> </tr> <tr> <td> LoopF/LoopB 底漆 (20 和 #956; m) </td> td>> 1.0/1.0 </td> </tr> <tr> <td> F3/B3 入门 (20 和 #956; M) </td> <td> 0.5/0.5 </td> </tr> <tr> <td> 总卷 </td> <td> 25.0 </td> </tr> </tbody> </table> 表 1: 灯的组成部分底漆固定试剂. 在表的左列中列出了灯具底漆固定组合件的组成, 并在右列中列出了每个组件的音量. 
<ol start="9"> <li> 按如下方式组装卡带。将加载适配器放到固定阵列的顶部。确保适配器的碟只覆盖所有十毛细血管暴露的部分 (参见 图 1 ). </li>
</ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56597/56597fig1.jpg"/> 图 1: 磁带制作和组装。( a ) 不锈钢模具和硅橡胶支持。模具由3部分组成: 汽缸、坝板、柱板。( b ) 支持制造和组装毛细管盒的电路原理图。整个过程包含5步骤: 1. 在浇注, 2. 脱模, 3. 毛细管插入和表面涂层, 4. 底漆固定和5。磁带锚定。1. 将硅橡胶倒入模具的气缸中;2. 推出大坝板, 从支持的模式中取出模具;3. 将支持的下表面涂上涂层, 然后将毛细管插入到该技术支持中, 最后涂上表面的支持和暴露的毛细血管。粗蓝线表示疏外套;4. 将底漆装入单独的毛细血管;5. 将毛细管阵列固定在单96孔板上, 并在其上安装一个样品加载适配器。详细信息已在协议步骤 1.1-1.9 中描述。<a href="//ecsource.jove.com/files/ftp_upload/56597/56597fig1large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 2. 在毛细管阵列中灯反应的性能 <ol> <li> 准备反应组合 <ol> <li> 准备灯的反应混合, 每 T 能 2 . </li>
		<li> 添加试剂到混合物根据所选择的顺序, 如在 表 2 , 和涡流的反应混合物 5 s 后添加素。轻轻地在在添加 Bst 聚合酶后20次垂直于试管. </li>
	</ol> </li> </ol> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"> <tbody> <tr> <td> 灯组件和 #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160;和 #160; (初始浓度) </td> <td> 卷 (和 #956; L) </td> </tr> <tr> <td> ddH MgSO </td> <td> </td> 11.6 </tr> <tr> <td> , 4 (100 mM) </td> <td> 2.0 </td> </tr> <tr> <td> dNTPs (25 mm) </td> <td> 1。4d > </tr> <tr> <td> 甜菜碱 (5 米) </td> <td> 4.0 </td> </tr> <tr> <td> 缓冲 (10x) </td> <td> 2.5 </td> </tr> <tr> <td> 素 (1.25 mM) </td> <td> 0.5 </td> </tr> <tr> <td> MnCl 2 (25 mM) </td> <td> 0.5 </td> </tr> <tr> <td>/em > 聚合酶 (8 U/和 #956; l) </td> <td> 1.5 </td> </tr> <tr> <td> 植物 DNA (10 ng/和 #956; l) </td> <td> 1.0 </td> </tr> <tr> 总卷 <td> </td> 25.0 <td> </td> </tr> </tbody> </table>表 2: 毛细管阵列灯的反应系统。 在左列中列出了毛细管阵列灯组件的反应系统的组件, 并在右列中列出了每个组件的音量. 
<ol start="2"> <li> 加载试剂并密封卡带 <ol> <li> 吸管20和 #181; 与标准的100和 #181 的灯反应混合物; 我的小费。将笔尖插入加载适配器的入口以锁定它。将反应混合物轻轻地注入适配器的碟中;反应混合物会迅速填满盘子, 然后通过毛细管力自动加载毛细管. </li>
		<li> 卸下带有锁定尖端的适配器, 并通过与 PCR 兼容的透明密封膜密封该井. 
		注: 只有在亲水性盘中接触反应混合物的毛细血管才能填充。因此, 请确保所有毛细管的顶部都具有相同的高度 (请参见 图 2 ). </li>
	</ol> </li> </ol> <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56597/56597fig2.jpg"/> 图 2: 使用加载适配器进行采样加载的图示. 图片显示使用蓝色解决方案的加载过程为例。将笔尖插入进风口, 然后缓慢地将样品注入适配器, 然后用锁定的笔尖卸下适配器。<a href="//ecsource.jove.com/files/ftp_upload/56597/56597fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
<ol start="3"> <li> 在63和 #176 的恒温箱中孵育毛细管阵列; C 用于1小时 </li> </ol>
 3. 结果读出和数据分析 <ol> <li> 获取荧光发射的图像. 
	<ol> <li> 在小型 hand-held uv LED 手电筒的顶面上修复 uv 滤镜, 以过滤可见光. </li>
		<li> 激发离解的素发出荧光与紫外线手电筒。通过数码相机或智能手机从毛细管阵列顶端捕获图像. </li>
		<li> 拍摄图像时, 确保摄像机在毛细血管的区域内尽可能地放大, 以获得高质量、清晰的图像. </li>
	</ol> </li> <li> 分析结果 <ol> <li> 通过图像分析软件打开图像, 然后选择和 #34; 图像和 #62; 模具和 #62; 灰度和 #62; 是和 #34; 和 #34; 图像和 #62; 模具和 #62; 16bit 和 #62; 是和 #34;选择和 #34; 文件和 #62; 保存 #62; 格式和 #62; TIFF 和 #34; 将图像转换为16位 tiff 格式. </li>
		<li> 提取荧光强度值 <ol> <li> 打开微阵列分析软件。将16位 TIFF 格式图像拖动到软件的界面中, 然后选择波长 #34; 532 nm 和 #34; 以及 #34、绿色和 #34 的颜色; 显示图像. </li>
			<li> 创建一个新的块来定位来自毛细血管的荧光信号。选择和 #34; 工具和 #62; 新的块和 #34; 并输入列和行数, #34; 1; 1 和 #34; 和 #34; 2; 5 和 #34; 在和 #39; 块和 #39; 和 #39; 功能和 #39; 接口分开. </li>
			<li> 右键单击并选择和 #34; 功能和 #34; 模型, 然后调整位置和直径, 以适应毛细血管的荧光区域。选择和 #34; 分析和 #34; 提取荧光强度的值. </li>
			<li> 选择和 #34; 文件和 #62; 保存设置为和 #34; 保存块文档, 选择和 #34; 文件和 #62; 保存结果和 #34; 保存荧光强度结果。计算信噪比, 以确定是否成功地在毛细管中执行灯. 
			注: 通过记录斑点的灰度值和信噪比 (棚), 得到了毛细管的信号, 定义为目标的前景信号的平均值与两个负的前向信号平均值的比值。控件.确定正信号的截止阈设置为信噪比和 #62; 2. </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Urdea, 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=Requirements+for+high+impact+diagnostics+in+the+developing+world.">Requirements for high impact diagnostics in the developing world.</a> Nature. 444, 73-79 (2006).</li><li>Yager, P., Domingo, G. J., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+diagnostics+for+global+health.">Point-of-care diagnostics for global health.</a> Annu Rev Biomed Eng. 10, 107-144 (2008).</li><li>Opota, O., Jaton, K., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+diagnosis+of+bloodstream+infection:+towards+molecular+diagnosis+directly+from+blood.">Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood.</a> Clin Microbiol Infec. 21 (4), 323-331 (2015).</li><li>Guo, 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=MPIC:+A+High-Throughput+Analytical+Method+for+Multiple+DNA+Targets.">MPIC: A High-Throughput Analytical Method for Multiple DNA Targets.</a> Anal Chem. 83 (5), 1579-1586 (2011).</li><li>Shao, N., 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=MACRO:+a+combined+microchip-PCR+and+microarray+system+for+high-throughput+monitoring+of+genetically+modified+organisms.">MACRO: a combined microchip-PCR and microarray system for high-throughput monitoring of genetically modified organisms.</a> Anal Chem. 86 (2), 1269-1276 (2014).</li><li>Kamle, S., Ali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+crops:+detection+strategies+and+biosafety+issues.">Genetically modified crops: detection strategies and biosafety issues.</a> Gene. 522 (2), 123-132 (2013).</li><li>Galvin, S., Dolan, A., Cahill, O., Daniels, S., Humphreys, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+monitoring+of+the+hospital+environment:+why+and+how?.">Microbial monitoring of the hospital environment: why and how?.</a> J Hosp Infect. 82 (3), 143-151 (2012).</li><li>Sciancalepore, A. G., 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=Microdroplet-based+multiplex+PCR+on+chip+to+detect+foodborne+bacteria+producing+biogenic+amines.">Microdroplet-based multiplex PCR on chip to detect foodborne bacteria producing biogenic amines.</a> Food Microbiol. 35 (1), 10-14 (2013).</li><li>Saxena, G., Bharagava, R. N., Kaithwas, G., Raj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+indicators,+pathogens+and+methods+for+their+monitoring+in+water+environment.">Microbial indicators, pathogens and methods for their monitoring in water environment.</a> J Water Health. 13 (2), 319-339 (2015).</li><li>Hopwood, A. 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=Integrated+Microfluidic+System+for+Rapid+Forensic+DNA+Analysis:+Sample+Collection+to+DNA+Profile.">Integrated Microfluidic System for Rapid Forensic DNA Analysis: Sample Collection to DNA Profile.</a> Anal Chem. 82 (16), 6991-6999 (2010).</li><li>Estes, M. D., 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=Optimization+of+multiplexed+PCR+on+an+integrated+microfluidic+forensic+platform+for+rapid+DNA+analysis.">Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis.</a> Analyst. 137 (23), 5510-5519 (2012).</li><li>Niemz, A., Ferguson, T. M., Boyle, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+nucleic+acid+testing+for+infectious+diseases.">Point-of-care nucleic acid testing for infectious diseases.</a> Trends Biotechnol. 29 (5), 240-250 (2011).</li><li>Peeling, R. W., Mabey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+tests+for+diagnosing+infections+in+the+developing+world.">Point-of-care tests for diagnosing infections in the developing world.</a> Clin Microbiol Infec. 16 (8), 1062-1069 (2010).</li><li>Perkins, M. D., Kessel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+Ebola+tells+us+about+outbreak+diagnostic+readiness.">What Ebola tells us about outbreak diagnostic readiness.</a> Nat Biotechnol. 33 (5), 464-469 (2015).</li><li>Li, 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=A+universal+multiplex+PCR+strategy+for+100-plex+amplification+using+a+hydrophobically+patterned+microarray.">A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray.</a> Lab Chip. 11 (21), 3609-3618 (2011).</li><li>Notomi, 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=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Res. 28 (12), (2000).</li><li>Mori, Y., Nagamine, K., Tomita, N., Notomi, T. <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+loop-mediated+isothermal+amplification+reaction+by+turbidity+derived+from+magnesium+pyrophosphate+formation.">Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.</a> Biochem Biophys Res Commun. 289 (1), 150-154 (2001).</li><li>Tomita, N., Mori, Y., Kanda, H., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nat Protoc. 3 (5), 877-882 (2008).</li><li>Iwamoto, T., Sonobe, T., Hayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+direct+detection+of+Mycobacterium+tuberculosis+complex,+M-avium,+and+M-intracellulare+in+sputum+samples.">Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M-avium, and M-intracellulare in sputum samples.</a> J Clin Microbiol. 41 (6), 2616-2622 (2003).</li><li>Goto, M., Honda, E., Ogura, A., Nomoto, A., Hanaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+detection+of+loop-mediated+isothermal+amplification+reaction+by+using+hydroxy+naphthol+blue.">Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue.</a> Biotechniques. 46 (3), 167-172 (2009).</li><li>Iseki, 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=Development+of+a+multiplex+loop-mediated+isothermal+amplification+(mLAMP)+method+for+the+simultaneous+detection+of+bovine+Babesia+parasites.">Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites.</a> J Microbiol Methods. 71 (3), 281-287 (2007).</li><li>Liang, C., 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=Multiplex+loop-mediated+isothermal+amplification+detection+by+sequence-based+barcodes+coupled+with+nicking+endonuclease-mediated+pyrosequencing.">Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing.</a> Anal Chem. 84 (8), 3758-3763 (2012).</li><li>Shao, Y., Zhu, S., Jin, C., Chen, F. <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+multiplex+loop-mediated+isothermal+amplification-RFLP+(mLAMP-RFLP)+to+detect+Salmonella+spp.+and+Shigella+spp.+in+milk.">Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk.</a> Int J Food Microbiol. 148 (2), 75-79 (2011).</li><li>Fang, X., Chen, H., Yu, S., Jiang, X., Kong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+viruses+accurately+by+a+multiplex+microfluidic+loop-mediated+isothermal+amplification+chip.">Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip.</a> Anal Chem. 83 (3), 690-695 (2011).</li><li>Stedtfeld, R. D., 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=Gene-Z:+a+device+for+point+of+care+genetic+testing+using+a+smartphone.">Gene-Z: a device for point of care genetic testing using a smartphone.</a> Lab Chip. 12 (8), 1454-1462 (2012).</li><li>Liu, D., Liang, G., Zhang, Q., Chen, B. <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+Mycobacterium+tuberculosis+using+a+capillary-array+microsystem+with+integrated+DNA+extraction,+loop-mediated+isothermal+amplification,+and+fluorescence+detection.">Detection of Mycobacterium tuberculosis using a capillary-array microsystem with integrated DNA extraction, loop-mediated isothermal amplification, and fluorescence detection.</a> Anal Chem. 85 (9), 4698-4704 (2013).</li><li>Zhang, 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=Point-of-Care+Multiplexed+Assays+of+Nucleic+Acids+Using+Microcapillary-based+Loop-Mediated+Isothermal+Amplification.">Point-of-Care Multiplexed Assays of Nucleic Acids Using Microcapillary-based Loop-Mediated Isothermal Amplification.</a> Anal Chem. 86 (14), 7057-7062 (2014).</li><li>Shao, N., 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=Visual+detection+of+multiple+genetically+modified+organisms+in+a+capillary+array.">Visual detection of multiple genetically modified organisms in a capillary array.</a> Lab Chip. 17 (3), 521-529 (2017).</li><li>Lizardi, P. 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=Mutation+detection+and+single-moledule+counting+using+isothermal+rolling-circle+amplification.">Mutation detection and single-moledule counting using isothermal rolling-circle amplification.</a> Nat Genet. , (1998).</li><li>Piepenburg, O., Williams, C. H., Stemple, D. L., Armes, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+detection+using+recombination+proteins.">DNA detection using recombination proteins.</a> Plos Biol. 4 (7), 1115-1121 (2006).</li><li>Opota, O., Jaton, K., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+diagnosis+of+bloodstream+infection:+towards+molecular+diagnosis+directly+from+blood.">Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood.</a> Clin Microbiol Infect. 21 (4), 323-331 (2015).</li></ol>

作者没有什么可透露的。

这里展示的平静平台, 它结合了灯技术与毛细管阵列, 使同时检测多个转基因相关的基因目标在一个单一的, 高效, 易于操作的测试。
为了在卡带中成功地执行复用灯的反应, 需要注意三关键点。首先, 达到相同的高度, 在毛细血管的上部和亲水性和疏水性模式的毛细管阵列是关键的同时加载试剂到所有的毛细血管。在初始插入到 "支持" 后, 毛细血管应由一个板对齐, 以确保它们?...

在广泛的领域, 如临床诊断1、2、3、转基因检测4, 对多种核酸同时检测的系统来说, 需要低成本、快速且易于使用. 5,6, 微生物监视7,8,9, 鉴证分析10,11, 特别是点测试 (POCTs), 其中资源通常被限制为12,13,14。
聚合酶链反应 (pcr), 包括其衍生物方法 real-time pcr 和多重 pcr, 是最广泛应用于这些领域的检测技术。但是, 这些方法通常只在一个测试15中检测一个目标, 并且需要电力和复杂的专业设备。
另一个有前途的技术检测核酸是环介导的等温放大 (灯), 这是第一次描述在 2000年16。灯是一种高效的 DNA 检测方法。理论上, 它可以放大1拷贝到 109副本的增在一个小时内, 所有执行在一个恒定的温度, (即, 在 60-65 ° c)。成功的放大将产生大量的不溶性副产品焦磷酸盐和导致混浊的变化17, 这可以直接观察肉眼。还可以通过添加金属离子或荧光染料 (如素18、核酸染料19和羟基萘酚蓝20) 来观察颜色的变化。由于光源具有灵敏度高、操作方便等优点, 在核酸检测中得到了广泛的应用。
目前, 多路灯检测主要有两种策略。一是通过在一管21,22,23中的多个灯引套进行多灯检测。然而, 由于不同引物间的内在干扰和竞争, 其多样性和放大效率将受到限制。此外, 在同一反应中, 很难识别出不同的灯具产品。另一个策略是基于物理隔离。不同的引物集合被隔绝了入各自的小型化隔间, 并且多个灯反应然后同时执行24,25。这些方法, 通常是基于微流控芯片, 提供了一个潜在的解决方案的高通量灯的反应。然而, 生产的芯片和复合预的底漆集是复杂的, 这可能会增加成本和减少重复性。
最近, 有几项研究描述了在毛细血管中执行灯反应以绕过复杂的微流控芯片制造, 并实现了低成本检测26,27。然而, 在高通量分析中, 这些毛细血管与 PCR 条管的微型版本类似, 因为样品和反应试剂 (包括不同的底漆) 必须单独准备并送到不同毛细血管内的反应单位。为了实现并行和复用分析, 需要额外的设备, 例如多通道注射器泵, 是平行装载样品或试剂。
为了克服目前核酸多重检测方法的局限性, 我们开发了一种将视灯技术与毛细管阵列相结合的小型化平台。该平台具有多目标、体积小巧、成本低廉、易于操作的28。在此, 我们描述了如何制造毛细管阵列和执行在阵列中的灯反应的细节。这里描述的协议已被标准化使用转基因生物 (转基因) 检测作为一个模型。重要的是, 这个协议也可以用于高通量检测其他核酸靶。

<table>
	<tbody>
		<tr>
			<td>UltraEverDry(super-hydrophobic coat)</td>
			<td>UltraTech</td>
			<td>4001</td>
			<td>supplier:Exiron chemistry(CHINA) CO.,LTD.</td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>8332557</td>
			<td></td>
		</tr>
		<tr>
			<td>Bst polymerase</td>
			<td>New England BioLabs</td>
			<td>M0275L</td>
			<td></td>
		</tr>
		<tr>
			<td>betain</td>
			<td>Sigma-Aldrich</td>
			<td>B0300-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>MKBP0495V</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>New England BioLabs</td>
			<td>B1003S</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Shanghai Sangon</td>
			<td>B804BA0022</td>
			<td></td>
		</tr>
		<tr>
			<td>chitosan</td>
			<td>Shanghai Sangon</td>
			<td>LJ0805S309J</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop 7.0 software</td>
			<td>Adobe Systems Inc., CA, USA</td>
			<td></td>
			<td>Image analysis</td>
		</tr>
		<tr>
			<td>GenePix Pro 6.1</td>
			<td>Molecular Devices, CA, USA</td>
			<td></td>
			<td>microarray analysis software</td>
		</tr>
		<tr>
			<td>AutoCAD</td>
			<td>Adobe Systems Inc.</td>
			<td></td>
			<td>3D construction software</td>
		</tr>
		<tr>
			<td>UV filter (ZWB2)</td>
			<td>YXSensing</td>
			<td></td>
			<td>supplier : taobao</td>
		</tr>
	</tbody>
</table>

visual detection of multiple nucleic acids in a capillary array

在疾病诊断、微生物监测、转基因生物体 (转基因) 检测中迫切需要多目标、短期和资源可承受的多种核酸检测方法, 并法医分析我们先前描述的平台称为平静 (Capillary Array-based Loop 介导的等温放大为Multiplex 视觉检测核酸)。在此, 我们描述了改进的制造和性能的过程, 这个平台。在这里, 我们应用一个小的, 现成的磁带组装的毛细管阵列的多重视觉检测核酸。毛细管阵列被预处理成疏水和亲水模式之前固定环介导的等温放大 (灯) 底漆集在毛细管。加载适配器组装后, 灯反应混合物被加载和孤立到每一个毛细管, 由于毛细管力由一个单一的移步骤。灯的反应在毛细血管中平行进行。结果是视觉读出的照明与 hand-held 紫外线手电筒。利用这个平台, 我们展示了对8种常见的元素和基因在转基因样品中具有高特异性和敏感性的监测。总之, 本文所描述的平台是为了方便检测多种核酸。我们相信它将广泛应用于需要高通量核酸分析的领域。

在该方法中, 在试样加载过程中防止不同毛细管间的交叉污染是非常重要的。为此, 引入了壳聚糖, 可以保留单个毛细血管中的底漆。为了测试它是否工作, 我们 pre-fixed ADH1 (玉米内源性参考基因) 在毛细管盒中设置的 "T" 和 "U" 的模式, 如 图 3a中所示。如预期的那样, 只有毛细血管中包含了显示阳性信号 ( 图 3b</...

Watch this Scientific Journal Video about Visual Detection of Multiple Nucleic Acids in a Capillary Array at JoVE.com

Visual Detection of Multiple Nucleic Acids in a Capillary Array

毛细管阵列中多核酸的视觉检测

该协议描述了一个小的, 现成的卡带的制作, 可用于视觉检测的多个核酸在一个单一的测试, 这是易于操作。在这种方法中, 用毛细管阵列对转基因生物的目标进行多重和高效的检测。

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

visual-detection-of-multiple-nucleic-acids-in-a-capillary-array

Research

JoVE Journal

Bioengineering

7.2K 次观看.  Shanghai Jiao Tong University. 该协议描述了一个小的, 现成的卡带的制作, 可用于视觉检测的多个核酸在一个单一的测试, 这是易于操作。在这种方法中, 用毛细管阵列对转基因生物的目标进行多重和高效的检测。

视频: Visual Detection of Multiple Nucleic Acids in a Capillary Array

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

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

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

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

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 ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

使用CRISPR并发剂的小鼠小肠组织中多基因敲除的方案

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

多平行板流室对剪切应力暴露的内皮细胞基因表达分析

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...