这项工作的部分资金由来自南得克萨斯科技管理（POCrr 2009.041），整合从国家研究资源中心（UL 1RR025767）医学和科学学院的赠款和牙科及颅面研究国家研究所（ 5R21DE017294）。

1。制备官能幻灯片<ol><li>载玻片放置在一个可移动的滑动机架，洗浸泡在罐子染色，含99％乙醇（病理分级）的两倍。用幻灯片使用纸巾（确保不产生纸尘），干用压缩氮气射流清洗。 </li></ol> 注意:不要使用金的湿巾擦拭幻灯片，因为它会产生细尘纸。 <ol start="2"><li>沉浸充满在室温下与浓硫酸和孵育过夜染色罐中含有幻灯片幻灯片架。 </li></ol> 注:为了避免与皮肤接触，使用耐化学腐蚀的手套和护目镜，同时与浓酸，有毒和腐蚀性化学品的工作。 <ol start="3"><li>超声幻灯片，用Milli-Q 30分钟，洗为30分钟（18MΩ）水与另一洗cetone为5分钟。这种治疗暴露在玻璃表面上的硅醇基。 </li><li>大衣，用2.5％的清洁幻灯片（体积/体积水）3 - 氨丙基（APTES）解决方案，通过幻灯片，APTES机架浸泡30分钟，15分钟洗涤3次在Milli-Q水各洗，烘烤于一炉的幻灯片在110°C为15分钟。烘烤，使交联，APTES，造成表面官能氨基。 </li></ol> 注意:请在一个塑料容器，因为优先的墙壁，玻璃容器，APTES存款，APTES的解决方案。 <ol start="5"><li>使用旋转涂布机，外套1％（WT / VOL甲苯）聚苯乙烯-马来酸酐共聚物（PS-MA的）（Sigma公司）的幻灯片，实现了疏水涂层6单层。 2.0毫升的PS-MA的加入到一个干净的玻璃安装在旋转涂布机和大衣在3000转每分钟为30秒的幻灯片。这些条件可能会有所不同的基础上旋转涂布参数，如涂层，使lution，基材，涂层厚度，由下列公式7管辖</li></ol><img alt="式（1）" src="/files/ftp_upload/3845/3845eq1.jpg" /> 其中 h是涂膜的厚度，e为蒸发率，η，C和ρ是涂层溶液的粘度，浓度和密度分别为，ω是角速度。 注:甲苯当通风柜的使用时间长，吸入是有害的建议。 <ol start="6"><li>幻灯片可以存储在长达一个月的幻灯片机架在干燥，无粉尘条件在2 - 8°C。 </li></ol> 2。酵母菌剂的制备和胶原封装<ol><li>准备C.过夜培养白念珠菌菌株SC5314，在酵母蛋白胨葡萄糖[的YPD; 10克/ L酵母提取物，20克/ L蛋白胨20 g / L的葡萄糖液体培养基，接种单菌落的C.人bicans为10 - 20毫升的YPD 8。在30°C过夜孵育轨道摇床（约150 - 200转）文化。收获1毫升白念珠菌从隔夜（在5-10分钟5000转离心）的YPD培养酵母细胞，并用两次10分钟1毫升无菌磷酸盐缓冲液（PBS; 10 mM磷酸盐缓冲液，氯化钾2.7毫米， 137毫米氯化钠，pH值7.4）（Sigma公司），每洗。收获离心洗细胞在10分钟1900转。洗过的细胞悬浮于1毫升重建缓冲液（0.2 N氢氧化钠溶液与2.2％（WT /卷）碳酸氢钠和4.8％（重量/体积）肝素钠，pH值7.2）。 </li></ol> 注:C.白色念珠菌是一种风险集团1/BSL1的微生物。永远记住使用工作良好的无菌/灭菌技术，这种微生物的生物危险材料的妥善处置，并按照制度程序。 <ol start="2"><li>计数细胞使用hemocyt烁效应在明场显微镜和调整细胞密度5×10 7细胞/ ml。 </li><li>进一步稀释十倍暂停另外10×的RPMI-1640 L-谷氨酰胺，并辅以与morpholinepropanesulfonic（MOPS）酸（pH值7.2）缓冲。 </li><li>封装混合细胞悬液与胶原蛋白（1.8毫克/毫升）的RPMI-1640（从鼠尾1型胶原蛋白的酵母细胞，BD公司，贝德福德，马），取得了最终浓度4×10 6细胞/毫升。在冰上保持胶原细胞悬液，以防止在打印前胶原凝胶。 </li></ol> 3。制备的钙 BChip <ol><li>清理和消毒，微阵列的所有表面，包括源盘站，洗涤和真空站，真空幻灯片盘片和印刷室，70％的异丙醇擦拭。 </li><li>放置和真空麦克风幻灯片所需的PS-MA的镀膜玻璃幻灯片roarrayer。 </li><li>涡的细胞悬浮在胶原大力吸成一个以及一个96孔板100μL混合悬浮印刷之前，。把这个源板在装载站。 </li><li>加湿器，切换到保持100％的相对湿度在整个印刷过程中的微阵列室。 </li><li>打印一个相隔间距1.2毫米的使用芯片检举（Omnigrid微Digilab公司，Holliston，马）由非接触式沉积采用锥形锥形190微米的小孔陶瓷提示点48行和16列数组NL 50细胞悬液（Digilab）。 </li><li>总理，冲洗和真空干燥的提示两次后，每一轮的印刷。 </li><li>后立即印刷，放置在一个气密，加湿室（杂交录像带，加利福尼亚州桑尼维尔，ArrayIt公司）的幻灯片和孵化在37°C为24小时，使生物膜的形成。 </li></ol> 4。药敏试验的Pr在的钙 BChip eformed生物膜对抗真菌药物<ol><li>从股票的解决方案或抗真菌药物粉末，准备工作液中的RPMI-1640培养基。典型的工作方案的最高浓度为1,024微克/毫升的氟康唑和两性霉素B 5 16微克/毫升。其他浓度可用于不同的代理。 </li><li>准备稀释2倍稀释的原液，涵盖范围估计在中间化合物的IC 50的 8个不同的化合物的浓度。 </li><li>经过24小时的生物膜增长，使用微阵列打印50 NL八个不同浓度的药物，随着阳性（不只是媒体的药物，即细胞）和阴性（钠次氯酸钠处理20分钟）控制，至少在六个复生物膜上。 </li></ol> 注意:保持100％的相对湿度，以防止斑点干燥而附加ING的药物。 <ol start="4"><li>加入药物后不久，在37℃孵育24小时，在加湿室药物的芯片。 </li><li>扣篮的CaBChip 3-5次，每次2分钟的时间在PBS洗药和染色孵化在37 CA BChip 0.5微米FUN1°C为30分9。 </li><li>洗净的，扣篮钙 BChip 1-3分钟3-5次，每一个扣篮，在PBS染色，干燥氮气流的的钙 BChip使用。阅读的荧光强度在532 nm波长的光电倍增管的增益380微阵列扫描仪（GenePix 4100A，Axon仪器，CA）的使用。 </li><li>设置阳性对照（无药物）和死亡（漂白处理）在100％和0％的生物膜点的荧光强度分别。 </li><li>使用下列公式确定在控制点的平均荧光强度（F）的相对减少的百分比抑制</li></ol><img alt="公式（2）" src="/files/ftp_upload/3845/3845eq2.jpg" /> 其中，F，升F 最大的 F o原始荧光强度药物治疗，没有药物管制和漂白处理点，分别。扫描仪设置进行了调整，以获得F 最大和 F O，分别为30000和4000俄罗斯足协。 <ol start="9"><li>使用GraphPad棱镜软件（拉霍亚加州）变坡山方程拟合计算50％抑制浓度或IC 50。 </li></ol> 5。代表结果 钙 BChip一个代表，包括一个48×16阵列C 的纳米生物膜白念珠菌， 如图2所示。明亮的视野显微镜显示的纳米生物膜整体建筑特色。扫描电子显微图像生物膜节目，真菌的菌丝内嵌入矩阵胶原纤维，这是约2微米，直径100纳米，分别。染的FUN1芯片扫描图像显示酵母和菌丝形式，这是真菌生物膜的特点。的的FUN1染生物膜的二维和三维共焦荧光图像可以看出，有空间异质性，穿插代谢活跃的细胞内细胞外基质（胶原组成的封装材料，也是最有可能的exopolymeric生产地区生物膜细胞），这是不代谢染料染色。据估计，生物膜的厚度约为50微米。 钙 BChip被用来估计的两种药物，氟康唑和两性霉素B药敏，结果如图3所示。按照行业标准96孔板检测上发表的报告，生物膜是抗氟康唑10和计算IC 50<html>两性霉素B是0.3微克/毫升11。 <img alt="图1" src="/files/ftp_upload/3845/3845fig1.jpg" /> 图1。 钙 BChip的制作流程图。 <img alt="图2" src="/files/ftp_upload/3845/3845fig2.jpg" /> 图2。使用机器人的微阵列高能开出钙 BChip印刷，图片和PS-MA的涂层幻灯片上载有768点。每个半球点是50 nL的体积，直径大约700微米，与之间的斑点1.2毫米的分离。也显示（顺时针），光学显微镜，生物芯片扫描仪，二维和三维荧光显微镜和扫描电子显微镜对钙 BChip的个人生物膜图像。在25000 x高倍率的SEM图显示了一个宽2微米的菌丝长丝。 图3"SRC ="/ files/ftp_upload/3845/3845fig3.jpg"/&gt; 图3。药敏试验和IC 50（一）氟康唑和（B）两性霉素B使用钙 BChip值的测定结果。两性霉素B治疗生物膜的荧光显微镜图像显示在（C）。结果是平均值±标准误差平均为两个独立的芯片，含有10复制每个条件。

<ol>
	<li>Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+United+States+hospitals:+a+three-year+analysis.">Nosocomial bloodstream infections in United States hospitals: a three-year analysis.</a> Clin. Infect. Dis. 29, 239-244 (1999).</li><li>Ramage, G., Bachmann, S., Patterson, T. F., Wickes, B. L., Lopez-Ribot, J. L. <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+multidrug+efflux+pumps+in+relation+to+fluconazole+resistance+in+Candida+albicans+biofilms.">Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms.</a> J. Antimicrob. Chemother. 49, 973-980 (2002).</li><li>Srinivasan, A., Uppuluri, P., Lopez-Ribot, J., Ramasubramanian, A. K. <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+High-Throughput+Candida+albicans+Biofilm+Chip.">Development of a High-Throughput Candida albicans Biofilm Chip.</a> PLoS ONE. 6, 19036-19036 (2011).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Pierce, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reproducible+96-well+plate-based+method+for+the+formation+of+fungal+biofilms+and+its+application+to+antifungal+susceptibility+testing.">A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.</a> Nat. Protoc. 3, 1494-1500 (2008).</li><li>Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cellular+microarray+for+high-throughput+toxicology+assays.">Three-dimensional cellular microarray for high-throughput toxicology assays.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 59-63 (2008).</li><li>Meyerhofer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+resist+films+produced+by+spinning.">Characteristics of resist films produced by spinning.</a> Journal of Applied Physics. 49, (1978).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob. Agents Chemother. 45, 2475-2479 (2001).</li><li>Chandra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+fungal+pathogen+Candida+albicans:+Development,+architecture,+and+drug+resistance.">Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance.</a> Journal of Bacteriology. 183, 5385-5394 (2001).</li><li>Jabra-Rizk, M. A., Falkler, W. A., Meiller, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+biofilms+and+drug+resistance.">Fungal biofilms and drug resistance.</a> Emerg. Infect. Dis. 10, 14-19 (2004).</li><li>Tobudic, S., Lassnigg, A., Kratzer, C., Graninger, W., Presterl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+of+amphotericin+B,+caspofungin+and+posaconazole+on+Candida+albicans+biofilms+in+intermediate+and+mature+development+phases.">Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases.</a> Mycoses. 53, 208-214 (2010).</li></ol>

L.洛佩斯·何塞·里博·阿南德K. Ramasubramanian在MicrobeHTS技术公司，这是发展抗真菌药物的自身权益。 MicrobeHTS技术，这些研究没有提供财政支持。

我们已经开发了一个基于细胞的高密度芯片，CA BChip， 白念珠菌生物膜的纳升量组成。芯片上印改性玻璃基板，允许强大的胶原蛋白凝胶点附件，同时提供必要非蔓延，半球形的三维凝胶疏水。一个单一的钙 BChip可以取代约八96孔板，和几个芯片可以打印，并在同一时间处理。该芯片采用纳米级的文化，可以方便，快捷的处理，适合自动化，最大限度地减少手工劳动和成本，?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 3 - 氨丙基（APTES） </td><td> Sigma-Aldrich公司</td><td> 440140 </td></tr><tr><td>聚苯乙烯 - 马来酸酐共聚物（PS-MA的） </td><td> Sigma-Aldrich公司</td><td> 426946 </td></tr><tr><td>玻璃显微镜幻灯片</td><td> Fisher Scientific则</td><td> 12-549-3 </td></tr><tr><td>鼠尾胶原蛋白I型</td><td> BD公司</td><td> 354236 </td></tr><tr><td>机器人微阵列</td><td> omn​​igrid微</td><td> MICROSYS4000/4100A </td></tr><tr><td>微阵列扫描仪</td><td> genepix个人4100A </td><td> GENEPIX4100A </td></tr><tr><td>杂交盒式</td><td> ArrayIt公司</td><td> AHCXD </td></tr><tr><td> FUN1 [2-chloRO-4-（2,3 - 二氢-3 - 甲基 - （苯并-1,3 - 噻唑-2 - 基） - 亚甲基）-1 - 苯基quinoliniumiodide] </td><td> Invitrogen公司</td><td>架F-7030 </td></tr><tr><td>氟康唑</td><td> sicor制药公司</td><td> J02AC01 </td></tr><tr><td>两性霉素B </td><td>西格玛</td><td> A2411 </td></tr><tr><td>的RPMI-1640 </td><td>敏达，公司</td><td> 50-020-PC </td></tr><tr><td>陶瓷提示190微米的小孔</td><td> digilab </td><td> 60020441-00 </td></tr><tr><td> GraphPad棱镜软件</td><td> GraphPad软件公司</td><td></td></tr><tr><td> genepix临V4.1的</td><td>分子器件</td><td></td></tr></tbody></table>

candida albicans biofilm chip (cabchip) for high-throughput antifungal drug screening

白念珠菌仍然是主要病原念珠菌，目前第四个最常见的在美国的医院院内血流感染。这些机会性感染造成损害个人越来越多越来越大的威胁，并进行不可接受的高死亡率。这是由于抗真菌药物的有限阿森纳的一部分，但也出现抵抗最常用的抗真菌药物。进一步复杂化的治疗方法是，多数表现念珠菌生物膜的形成与相关的事实，并在这些生物膜的细胞显示临床使用抗真菌药耐药水平的提高。在这里，我们描述的C.组成的高密度芯片的发展白念珠菌生物膜纳米，我们有一个名为钙 BChip 3。简言之，一个机器人微阵列用tØ打印C.酵母细胞到固体表面的白色念珠菌 。在印刷过程中，酵母细胞被包围在一个三维矩阵使用量低至50 NL，并固定在一个合适的涂层的玻璃基板。经过最初的印刷，幻灯片培养24小时，使生物膜的发展，在37°C。在此期间的景点成长为全面发展的"纳米生物膜"，显示典型的结构和表型特征与成熟的三白念珠菌生物膜（ 即形态的复杂性，三维结构和耐药性）4。总体而言， 钙 BChip是〜750相当于和空间不同生物膜的组成与打印多个芯片可以同时处理额外的好处。估计FUN1代谢的荧光强度染色用芯片扫描仪通过测量细胞活力。这种真菌芯片非常适合为u在真正的高通量筛选抗真菌药物的发现本身。与现行标准（ 即 96酶标板模型生物膜形成5）相比，真菌生物膜芯片的主要优点是自动化，小型化，节省试剂和分析时间的量和成本，以及消除劳动密集的步骤。我们相信，这种芯片将显着加快的抗真菌的药物发现过程。

Watch this Scientific Journal Video about Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening at JoVE.com

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

我们已经开发出一种高密度芯片平台的3D纳米生物膜的组成<em&gt; C。白念珠菌</em&gt;称为<em&gt;钙</em&gt; BChip。上测试药物的敏感性个人资料<em&gt;钙</em&gt; BChip是传统的96孔板模式相媲美，表明真菌的芯片非常适合真正的高通量筛选抗真菌药物。

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

candida-albicans-biofilm-chip-cabchip-for-high-throughput-antifungal

Research

JoVE Journal

Bioengineering

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

13.6K 次观看.  University of Texas at San Antonio. 我们已经开发出一种高密度芯片平台的3D纳米生物膜的组成 C。白念珠菌称为钙 BChip。上测试药物的敏感性个人资料钙 BChip是传统的96孔板模式相媲美，表明真菌的芯片非常适合真正的高通量筛选抗真菌药物。

视频: Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

基于细胞中钙含量TRP通道功能的高通量筛选，使用FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

科研

生物工程

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

开源微孔板兼容照明面板的应用

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...