我们想感谢美国宇航局天体生物学科学和技术仪器发展（ASTID）计划的资金支持（授权号码103498和103692），大风洛克伍德的大学Birck Nantechnology的中心在美国普渡大学人与生物圈设备的引线键合，和亨俊园CAD图纸的流动池室。

1。聚（3,4 -乙撑二氧噻吩）:聚（4 -苯乙烯磺酸钠）（PEDOT:PSS）的电化学解的H +和CO 3 2 -离子的制备<ol><li>加入70毫克聚（4 -苯乙烯磺酸钠）（Na +的PSS - ），直到完全分散（大约10秒）〜10毫升去离子（DI）水和涡。 </li><li> 10.7微升3,4-ethlyenedioxythiophene二氧噻吩（EDOT）加入到该溶液中，在1.1和旋涡，直到溶液完全混合。 </li></ol> 2。制备聚（3,4 -乙撑二氧噻吩）:硫酸钙（PEDOT:的CaSO 4）的Ca 2 +离子的电化学解<ol><li>添加136毫克的硫酸钙（ 硫酸钙）10毫升去离子水和旋涡;解决方案不会完全散去，并出现乳白色。 </li><li> 10.7微升EDOT 2.1和旋涡的解决方案，直到完全混合。 </li></ol> 3。 PEDOT基于电化学导电聚合物<ol><li>用于生物分析系统公司（BASI）C3细胞支架（ 图3）和EC小量电位/恒电流电化学形成电化学电池。将EDOT:PSS电聚合溶液的电化学电池中，氮气泡20分钟，以除去溶解的氧。 </li><li>载入夹有铂的纱布在反电极的电化学电池的位置。然后夹在MAB面临的铂 - 纱布与工作电极在电化学电池在工作电极上的位置。调整人与生物圈深度这样只有被淹没的圆形电极PEDOT:PSS电化学的解决方案。避免溶液接触的平方的电接触垫。 </li><li>放置一个BASI饱和银/氯化银（Ag / AgCl电极）电极与参比电极的电化学电池的位置。确保参比电极是不是在工作和逆流之间R电极。 </li><li> PEDOT:PSS沉积:泡泡电化学电池为20分钟，并使用EC小量电位/恒电流运行单个循环伏安从0V - 1.1V，扫描速率为20毫伏/秒在±100μA规模。 </li><li>对于PEDOT: 硫酸钙沉积:泡20分钟的电化学电池中，并使用EC小量电位/恒电流运行在814 nA的30分钟计时。 </li></ol> 4。循环伏安法在K的PEDOT基于高分子偶联3铁（CN）6 <ol><li>执行步骤3.1-3.3以上。 </li><li>使用EC小量的电位/恒电流运行单一的循环伏安曲线与不同的扫描速率毫伏/秒（25，50，75，100，L25，150，175，200），在±10μA规模从-653 mV至853 mV的。 </li></ol> 5。表面功能化协议<ol><li>存款具体在步骤3中的离子导电聚合物共轭。 </li><li>应用离子选择性膜，在步骤6中。 </li></ol> 6。应用离子选择性膜<ol><li>中心的人与生物圈计划在真空微调夹头。 </li><li>存款100微升膜到人与生物圈计划和运行的中心。 </li><li>离子选择性膜旋涂，旋涂机在1,500 rpm的转速下持续30秒，5秒斜坡向上和向下。 </li><li>旋涂的人与生物圈真空30分钟，并在烘箱中于70℃下进行20分钟的烘烤芯片。 </li></ol> 7。 PEDOT-PSS的共轭导电性高分子与pH和碳酸盐（CO 3 2 - ）离子选择性膜的校准<ol><li>条件的人与生物圈计划在10微米小苏打（ 碳酸氢钠 ）和5毫米氯化钾（KCl）的藻类媒体过夜。 </li><li>将人与生物圈计划到微流细胞芯片持有人。 </li><li>注射5毫升试验溶液的初始pH值或浓度（ 例如，pH值为4或10μM，CO 3 2 - ）。删除小家伙从流动池的芯片座的劳动和就业统计局。 </li><li>将芯片座到流动池的流动池的电夹具。 </li><li> EC小量软件打开并进入开路电位（OP）模式。将时间设定为300分钟，电压范围为±1V，和截止频率为10 kHz，并记录该值每2秒。 </li><li>让稳定的人与生物圈计划，（看平线），然后继续与校准过程。 </li><li>一旦，在MAB是稳定的，与试品溶液冲洗流动池，以进行校准，并注入下一个浓度（pH为5或25μMCO 3 2 - ）。确保无气泡被允许进入流通池。重复步骤7.5和7.6，在pH为6，7，8，和9或CO 3 2 -浓度为50，75，100，250，500，750，和1000μM。 </li><li>最后的浓度已运行后，删除的人与生物圈计划和干用氮气空气。 </li><li>将新鲜的空调解决方案，直到下次使用的人与生物圈计划放回。 </li></ol>硫酸钙氯化钙导电聚合物共轭<ol><li>条件的人与生物圈计划在7毫升0.1 M的氯化钙和10μM纳米3过夜。 </li><li>遵循类似步骤7.2 - 7.10。在步骤8.3中，将与初始浓度为0.01 mM的CaCl 2的碳酸试液。重复测试溶液的浓度为0.05，0.1，0.5，1和10毫米。 </li></ol>

<ol>
	<li>Migdalski, J., Bas, B., Blaz, T., Golimowski, J., Lewenstam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Miniaturized+and+Integrated+Galvanic+Cell+for+the+Potentiometric+Measurement+of+Ions+in+Biological+Liquids.">A Miniaturized and Integrated Galvanic Cell for the Potentiometric Measurement of Ions in Biological Liquids.</a> J. Solid State Electrochem. 13, 149-155 (2009).</li><li>Buehler, M. G., Kounaves, S. P., Martin, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+Water-quality+Monitor+with+Ion-selective-electrodes.">Designing a Water-quality Monitor with Ion-selective-electrodes.</a> 1, 331-338 (2001).</li><li>Adamchuk, V. I., Lund, E. D., Sethuramasamyraja, B., Morgan, M. T., Doberman, A., Marx, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Soil+Chemical+Properties+on-the-go+using+Ion-selective-electrodes.">Direct Measurement of Soil Chemical Properties on-the-go using Ion-selective-electrodes.</a> Journal Computers and Electronics in Agriculture. 48 (3), 272-294 (2005).</li><li>Oel&#946;ner, W., Hermann, S., Kaden, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Sensors+and+Sensor+Module+for+Studying+Biological+Systems+in+Space+Vehicles.">Electrochemical Sensors and Sensor Module for Studying Biological Systems in Space Vehicles.</a> Aerospace Science and Technology. 1, 291-296 (1997).</li><li>Bobacka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conducting+Polymer-based+Solid-state+Ion-selective+Electrodes.">Conducting Polymer-based Solid-state Ion-selective Electrodes.</a> Electroanalysis. 18 (1), 7-18 (2006).</li><li>Buck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion Selective Electrodes in Analytical Chemistry. , (1980).</li><li>Nam, H., Cha, G. S., Yang, V. C., Ngo, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18.">Chapter 18.</a> Biosensors and their Applications. , (2000).</li><li>Anatova-Ivanova, S., Mattinen, U., Radu, A., Bobacka, J., Lewenstem, A., Migdalski, J., Danielewski, M., Diamond, D. <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+Miniature+All-solid-state+Potentiometric+Sensing+System.">Development of Miniature All-solid-state Potentiometric Sensing System.</a> Sensors and Actuators B. 146, 199-205 (2010).</li><li>Michalska, A., Galuszkiewicz, A., Ogonowska, M., Ocypa, M., Maksymiuk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDOT+Films:+Multifunctional+Membranes+for+Electrochemical+Ion+sensing.">PEDOT Films: Multifunctional Membranes for Electrochemical Ion sensing.</a> J. Solid State Electrochem. 8, 381-389 (2004).</li><li>Bard, A. J., Faulkner, L. R., ed, 2. n. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electrochemical Methods: Fundamentals and Applications. , (2000).</li><li>Claussen, J. C., Artiles, M. S., McLamore, E. S., Mohanty, S., Shi, J., Rickus, J., Fisher, T. S., Porterfield, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Glutamate+Biosensing+with+Naanocube+and+Nanosphere+Augmented+Single-walled+Carbon+Nanotube+Networks:+A+Comparative+Study.">Electrochemical Glutamate Biosensing with Naanocube and Nanosphere Augmented Single-walled Carbon Nanotube Networks: A Comparative Study.</a> J. Mater. Chem. 21, 11224-11231 (2011).</li><li>Bobacka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+Stability+of+All-solid-state+Ion-selective+Electrodes+using+Conducting+Polymers+as+Ion-to-electron+Transducers.">Potential Stability of All-solid-state Ion-selective Electrodes using Conducting Polymers as Ion-to-electron Transducers.</a> Anal. Chem. 71, 4932-4937 (1999).</li><li>Lee, J. H., Yoon, I. J., Yoo, C. L., Pyun, H. J., Cha, G. S., Nam, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiometric+Evaluation+of+Solvent+Polymeric+Carbonate-selective+Membranes+based+on+Molecular+Tweezer-type+Neutral+Carriers.">Potentiometric Evaluation of Solvent Polymeric Carbonate-selective Membranes based on Molecular Tweezer-type Neutral Carriers.</a> Anal. Chem. 72, 4694-4699 (2000).</li><li>Song, F., Ha, J., Park, B., Kwak, T. H., Kim, I. T., Nam, H., Cha, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-solid-state+Carbonate+Selective+Electrode+based+on+a+Molecular+Tweezer-type+Neutral+Carrier+with+Solvent-soluble+Conducting+Polymer+Solid+Contact.">All-solid-state Carbonate Selective Electrode based on a Molecular Tweezer-type Neutral Carrier with Solvent-soluble Conducting Polymer Solid Contact.</a> Talanta. 57, 263-270 (2002).</li></ol>

我们什么都没有透露。

MAB生物芯片由的ASSISEs顶上基于PEDOT-CP的共轭转导层上的Pt电极的组合，其中一个可测量的电信号传导的离子浓度还从ISM构造。甲稳定的电极电位是指由CP层和ISM层。这两个层也决定MAB和质量的测量的电信号（噪声，漂移）的工作寿命。 PEDOT作为传导层由于其离子和电子的属性（在它的氧化形式）是特别有吸引力的。 PEDOT具有高氧化还原电容的能力，以尽量减少导电电极极化效...

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the items</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments </td>
		 </tr>
		 <tr>
			<td>3,4-Ethylenedioxythiophene</td>
			<td>Sigma-Aldrich</td>
			<td>483028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly(sodium 4-styrenesulfonate)</td>
			<td>Sigma-Aldrich</td>
			<td>243051</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EC epsilon galvanostat/potentiostat</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>e2P</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Saturated Ag/AgCl reference electrode</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>MF-2052</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pt gauze</td>
			<td>Alfa Aesar</td>
			<td>10283</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P-8131</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium nitrate</td>
			<td>J.T. Baker</td>
			<td>3190-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate</td>
			<td>Mallinckrodt/ Macron</td>
			<td>7412-12</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S-7127</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium chloride</td>
			<td>J.T. Baker</td>
			<td>1311-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>237132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C3 cell stand</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>EF-1085</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flow-cell chip holder</td>
			<td></td>
			<td></td>
			<td>Custom, courtesy of NASA Ames</td>
		 </tr>
		 <tr>
			<td>Flow-cell electrical fixture</td>
			<td></td>
			<td></td>
			<td>Custom, courtesy of NASA Ames </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Specific reagents and equipment.</td>
		 </tr>
		</tbody>
 </table>

multi-analyte biochip (mab) based on all-solid-state ion-selective electrodes (assise) for physiological research

一个芯片上实验室（LOC）应用在环保，生物医药，农业，生物，航天科研需要的离子选择性电极（ISE），可以承受长时间储存在复杂的生物介质1-4。一种全固态离子选择性电极（ASSISE）是上述的应用特别有吸引力的。电极应具有以下良好的特性:施工方便，维护成本低，和（潜在的）的小型化，可以进行批量处理。甲微加工ASSISE的用于量化的Ca 2 +，H +，CO 3 2 -离子的构建。它由一个贵金属电极层（ 例如铂），转导层和一个离子选择膜（ISM）的层。的转导层的功能的转导的浓度依赖型的离子选择性膜的化学势成可测量的电信号。 Ť他一生的ASSISE被发现依赖于保持电位的导电性层/膜界面5-7。要延长工作寿命ASSISE，在界面层，从而保持稳定的潜力，我们利用银/氯化银（Ag / AgCl电极的导电聚合物（CP），聚（3,4 -亚乙基二氧噻吩）（PEDOT）7-9）作为振子层。我们构建了ASSISE在一个芯片上的实验室的格式，我们称之为多分析物的生物芯片（MAB）（ 图1）。 校准测试解决方案表明，人与生物圈计划可监测pH值（业务范围pH值4-9），CO 3 2 - （测量范围0.01毫米- 1毫米），和Ca 2 +（对数线性范围为0.01毫米至1毫米）。人与生物圈计划海藻介质，存储在近一个月后，pH值，提供了近能斯特斜率响应。的碳酸酯的生物芯片显示类似于常规的离子选择性电极的电位的配置文件。生理学一例出现的测量值来监视系统的模型，生物活性小球藻 。 人与生物圈计划传达尺寸，多功能的优势，复分析物的感应能力，使得它适用于许多密闭监控的情况下，地球上或空间。 生物芯片的设计与实验方法生物芯片是10 x 11毫米的尺寸和有9 ASSISEs指定为工作电极（WES）和Ag / AgCl参考电极（RES）。每个工作电极（WE）的直径为240微米，平均间距为1.4毫米从RE的，直径为480微米的。这些电极连接到电接触垫的尺寸为0.5毫米×0.5毫米。的示意图如图2所示。 循环伏安法（CV）和恒电流沉积方法用于electropolymerize使用生物分析的PEDOT膜人系统公司（BASI）C3细胞支架（ 图3）。 PEDOT薄膜的反离子量身定制，以适应待测离子的利益。甲PEDOT与聚（苯乙烯磺酸）抗衡离子（PEDOT / PSS）是利用H +和CO 3 2 - ，，而硫酸钠（加入到该溶液中的CaSO 4）的是，利用的Ca 2 +。 PEDOT涂层的电化学性能，我们的分析中使用CVS氧化还原活性的解决方案（ 即 2毫米铁氰化钾（K 3的Fe（CN）6））。简历简介基于，兰德尔斯Sevcik分析被用来确定的有效表面积10。旋涂在1,500 rpm〜2微米厚的离子选择性膜（ISMS）的人与生物圈计划工作电极​​（WES）用于铸造。 人与生物圈被包含在一个微流体的流动池室充满了150微升体积的海藻介质接触垫电连接到的BASI系统（ 图如图4）。 小球藻光合活性的监测环境光线和黑暗条件下。

<html>的循环伏安图（CV）的PEDOT:PSS和其对应的还原峰电流（I P）与扫描速度（V 1/2）的一个例子示于图5a和5b分别。 PEDOT: 硫酸钙在不同的扫描率和它的还原峰电流将不会显示。使用兰德尔斯-Sevcik分析如图10所示，有效的表面区域的固体接触的PEDOT:PSS和PEDOT:离子选择性膜的CaSO 4没有被发现，分别为4.4×10 -11厘米<su...

Watch this Scientific Journal Video about Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research at JoVE.com

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

全固态离子选择性电极（ASSISEs）的建造由导电聚合物（CP）传感器提供几个月的功能寿命在液体介质中。在这里，我们描述了在实验室上的一个芯片格式ASSISEs的制造和校准过程。证明ASSISE长时间储存​​后，在复杂的生物媒体一直保持着近能斯特斜率曲线。

多分析物生物芯片（MAB），基于全固态离子选择性电极（ASSISE）的生理研究

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

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Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

17.2K 次观看.  Purdue University. 全固态离子选择性电极（ASSISEs）的建造由导电聚合物（CP）传感器提供几个月的功能寿命在液体介质中。在这里，我们描述了在实验室上的一个芯片格式ASSISEs的制造和校准过程。证明ASSISE长时间储存​​后，在复杂的生物媒体一直保持着近能斯特斜率曲线。

视频: Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

科研

生物工程

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

基于海胆大白鲨A接地采样:仿生为设计一个协议

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

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 ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

纤维素碳纤维溶胶单向冷冻干燥制备 Microhoneycomb 石柱: 方法和外延

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

使用基于曲线的转换工具量化纤维胶原蛋白组织

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

为生物医学和生物工程研究建立章鱼生态系统

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the dynamic properties of the cytoskeleton. This protocol offers a technique for human primary fibroblast transfection, which can be difficult because of the specifics of primary cell cultivation conditions. Additionally, cytoskeleton dynamic property maintenance requires a low level of transfection to obtain a good signal-to-noise ratio without causing microtubule stabilization. It is important to take measures to protect the cells from light-induced stress and fluorescent dye fading. In the course of our work, we tested different transfection methods and protocols as well as different vectors to select the best combination of conditions suitable for human primary fibroblast studies. We analyzed the resulting time-lapse videos and calculated microtubule dynamics using ImageJ. The dynamics of microtubules' plus-ends in the different cell parts are not similar, so we divided the analysis into subgroups - the centrosome region, the lamella, and the tail of fibroblasts. Notably, this protocol can be used for in vitro analysis of cytoskeleton dynamics in patient samples, enabling the next step towards understanding the dynamics of the various disease development.

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington's disease patients.

基于人类原发性皮肤成纤维细胞的亨廷顿舞蹈症模型微管加端动力学可视化

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...