VSR是由一个IIF居里夫人资助项目（缠绕地球，缩写:创伤诱导拟南芥电信号）。

1.饲养蚜虫注:工厂和蚜虫的EPG录制的选择取决于研究目标。对拟南芥的研究中，蚜甘蓝蚜为宜。 <ol><li>后排B.蚜虫甘蓝上甘蓝温室。保持用于在笼蚜虫饲养的植物，以避免污染其他植物。保持蚜虫饲养厂和实验工厂（在我们的例子甘蓝和拟南芥 ）在不同的房间，为了避免实验工厂的污染与蚜虫。 </li><li>转移蚜虫至新鲜植物约每2周，造成显著植物的损害，或到达人口过剩之前。转移10-20只蚜虫成虫到一个新的饲养厂发起新的殖民地。 </li><li>监察不必要的蚜虫，其他昆虫食草动物，蚜茧蜂，并定期烽饲养厂污染i的可能影响蚜虫群体的健康​​。 </li><li>他们对EPG录制最后蜕皮后收集的成人，无翅蚜长达一个星期。 </li><li>实验结束后，返回不习惯生长室的实验工厂，因为他们往往​​有已录制，这可能无意中污染其他植物中已经产生了一些后代。 </li></ol>为EPG录制2.昆虫接线<ol><li>使昆虫电极，获得黄铜连接器引脚（钉，直径1.2毫米），薄铜线（直径0.2毫米），非常薄的金线（直径约20微米），水基银胶，一个简单的小焊接螺栓焊液和树脂芯焊丝，用体视显微镜放大10倍，小剪刀或手术刀，二细镊子和一个泡沫塑料板或框。注:旋涡混合器可能是有用的。注意:一步一步协议，使电极被示于图2。 </li><li>对于蚜虫处理和胶应用中，得到:小和软水彩驼毛刷（尺寸2或更小）和昆虫针如那些用于昆虫的集合，虽然微细缝纫针或牙签可正常工作。第4步展示如何启动EPG录制。 </li><li>第1步。 注意:步骤1和2中显示了如何准备昆虫电极减去蚜虫。步骤3示出了如何将蚜虫连接到电极。蚜虫的真空固定布线时建议，但并不总是需要缓慢移动的物种（ 例如，甘蓝B.）。 <ol><li>接通焊接螺栓和熔体一些焊接线在其前端（ 图2A）。滋润铜管连接器引脚焊接一些流体（ 图2B）的头蘸入熔化焊接金属（ 图2C）。 </li><li>申请熔化焊料金属的外皮上的长1-2厘米一块薄铜线的一端（ 图2D）。然后把针和铜线一起对抗炎热的螺栓（ 图2E）和移动它们一起离开冷却和固化（ 图2F）。 </li></ol></li><li>步骤2。 <ol><li>彻底摇（或旋涡）用银胶小瓶几分钟直至光滑乳剂被示出。剪切（剪刀或手术刀）几件体视显微镜（ 图2G）的对象板的金线（约1.5厘米长）的。 </li><li>以黄铜引脚焊接铜丝（2.3节制造）和浸铜导线的自由端插入打开它（ 图2H）后，将聚集在瓶的盖子里面的小银胶水库。注意:只有一个小墨滴是必要的。 </li><li>移动铜线片金线的胶浸结束，同时抬起一端，以避免涂抹胶水到立体物体板。尝试COPPE重叠r和金线为几个毫米（ 图2I），分发沿着两条线的重叠的粘合剂。 </li><li>等到胶水已干足以使电线团结。检查干燥后的胶水接触，并添加一些新鲜的胶水用小针或其他一段铜线，如果加入了线的某些部分表现出无胶部分。 </li><li>后昆虫电极准备就绪，储存，例如插入一块泡沫塑料。 注意:金线的长度将决定蚜虫的行动自由:如果它太短（小于5mm），蚜虫可能会感到受限且通常不会表现;如果它太长（&gt; 2的厘米），蚜虫将自由移动。蚜虫倾向于移动到叶子​​的正面侧，如果允许。如果金线接触叶，信号会被短路。 </li></ol></li><li>步骤3。 <ol><li>蚜虫可以保持在适当位置通过光吸引装置，利用真空;在这种情况下，安装吨他吸了体视显微镜下的设备。将吸入口在该场的中心。 </li><li>彻底摇匀有银胶的，直到顺利形成乳液几分钟（或涡流）的小瓶。收集的小刷子蚜虫。 </li><li>切换抽吸装置上，并安装在所述吸入口（ 图2J）的蚜虫，与腹部的背对着实验者。用细刷，去除腹部（丰富的甘蓝蚜）任何表面的蜡。 </li><li>打开胶瓶及湿销与银胶非常小的液滴（ 图2K）。适用的银胶液滴到蚜虫的腹部（ 图2L-M）的背面。让这个小滴在几分钟完全干透，再大力摇晃胶水瓶中，加入银胶第二液滴上的第一个顶部。注意:当银胶是一种电导体，它不会引起显著损坏昆虫的表皮。 </li><li>关闭胶瓶后，将金线的自由端到潮湿的墨滴，并保持线依然同时让胶水完全干燥（ 图2N）。避免胶水涂抹到腿或触角和丢弃蚜虫，如果发生这种情况。 </li><li>关闭吸气固定装置，并小心地将昆虫（ 图20）。如果需要，可使用细刷以帮助从抽吸装置的蚜虫的提升。 注:接线B.甘蓝不需要真空，因为它们可以在一片精密实验室组织的，粗糙的表面，其中提供有足够的抓地力的蚜虫，以便它不会施加一滴湿胶到其腹部后解除是有线的。胶水干燥后可以解除与从细刷的帮助下组织的蚜虫。 </li><li>插入黄铜销与有线昆虫进入泡沫塑料和如果需要的话，继续接线所有其他昆虫要用于在EPG记录会话。 注意:这些协议为蚜虫接线工作很适合我们。用户可以找到他/她自己的方法进行布线蚜虫。 </li></ol></li><li>步骤4。 <ol><li>把在法拉第笼（ 图2P）植物上的非导电支撑物:使用培养皿或玻璃或塑料的板。 </li><li>将植物电极插入每盆的土壤。插入有线昆虫的黄铜销插入在EPG前置放大器（ 图2Q）的输入连接器。注意:土壤电极不对应于其他电生理技术中使用的接地电极。它有需要调整和补偿电极极化电压的偏移电压。 </li><li>在采集软件钢丝+的接口，具有100Hz的固定采样频率，输入文件名，指定记录时间，并写入文本指定实验的细节（治疗，工厂/樱雪<html>克拉物种等 ）在注释行2和3。 </li><li>降低昆虫到植物的合适的着陆区，通过点击采集软件（钢丝+）接口的启动按钮开始录制会议。 注1:可同时使用在一个EPG设置了最大的8个通道。一个EPG电极或每株几个EPG电极都可以使用。 注2:当研究的重点是蚜虫的行为，之前蚜虫厂访问开始记录，以避免错过第一家工厂的渗透活动。 </li><li>为了研究社会企业的电生理反应的刺激，等待至少10分钟后的蚜虫已进入韧皮部的阶段，以确保该蚜虫是在持续韧皮部摄取相，并且该信号基线是稳定的。只有这样，启动任何植物刺激实验。 </li></ol></li></ol>tp_upload / 52826 / 52826fig2.jpg"/&gt; 图2。使EPG电极与蚜虫或其他半翅目昆虫刺探电位（EPG）的记录。 <a href="https://www.jove.com/files/ftp_upload/52826/52826fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 面板AI，所需的步骤准备EPG电极减去蚜虫。首先，熔融在焊接螺栓（A）的末端的片焊接金属。然后，浸黄铜销的头部成一滴焊接流体（B）的，并与熔融金属接触它在焊接螺栓尖端（C）。后，立即该步骤中，铜导线的端部接触到焊接螺栓的尖端，以将其粘合到所述黄铜销（EF）的头部。用手术刀或刀片，切下一块金线（G）的。倾角铜线（接合在另一端的黄铜销）上的银胶（H）的自由端，并迅速加入金线，它（Ⅰ）前的银干涸。金线是一个极好的导体，并且可以被极化。实际上，在大多数的情况下，偏振是太小而不能检测到，并且如果是的话，它可以被用于与偏置电压（V）的补偿。 面板JO，以蚜虫（或其他昆虫半翅目）连接到电极所需的步骤。首先，小心地提起用细的水彩笔蚜虫，并将其放置在真空吸附装置（J）开幕。打开真空泵上，盖上一张纸施加抽吸空气阀孔。浸昆虫针入银胶（K）的前端，并把对蚜虫的腹部上方的小液滴胶，在立体显微镜下（LM）。内下〜20秒，银胶液滴上的蚜虫干燥之前，将金线昆虫电极插入的银胶的湿液滴的结束，并保持它在的地方1-3分钟，直到银胶有完全风干（N）。在这一点上，通过去除一张纸覆盖抽吸装置的空气阀孔，小心地取出蚜虫，从抽吸装置吸禁用:解除接线后蚜虫往往需要通过细刷一些帮助（O）。 面板 P示出了整个的EPG的概述设置法拉第笼的内部，和面板 q表示植物蚜虫组合的EPG的概述。参见上述用于该方法的更详细的说明部分2。 小写字母标签指的是人们需要做EPG电极项目: 一 :螺栓焊接; B:熔焊接金属;C:焊液; D:黄铜连接器引脚（钉）; E:铜线; F:Ø18μm的金线; G:抽吸装置; H:蚜虫; I:水性银胶; 记者:法拉第笼; K:植物电极; 升 :在EPG前置放大器的输入连接器（BNC）。

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WFT launched EPG Systems as a retirement activity, and is affiliated with it financially.

本文提供了制造电气贯穿图（EPG）记录了详细的方案。该EPG技术已经非常成熟，具有100-200活跃用户的全球，并已实施了不同主题的许多研究，例如:a）寄主植物的抗蚜虫等钢丝轴承昆虫13; B）植物病毒和病原体传播机构14; C）杀虫作用方式，（毒性和行为的变化）15; D）的EPG甚至是有益的证明蚜虫打架是有利的赢家，因为它增加了饲养效益16。 ?...

以产生长距离的电信号的能力是​​多细胞生物的一个有利的特征，可实现有效的响应外部刺激。这个特点已经独立进化在植物和动物，因此代表趋同进化的情况。鉴于电信号被耦合以在动物中重要的功能，例如刺激诱导在动物中的电信号的神经传递和肌肉收缩的分子基础，传输的机制和功能是深入研究的主题。相比之下，刺激诱导植物电信号传导很少受到关注的研究。虽然植物有没有神经或肌肉，似乎有足够的证据来假定刺激诱导的电信号在植物在其对环境的响应中发挥关键作用。 韧皮部，植物脉管系统的活组分，已被假定为主要的子施特拉特用于刺激诱 ​​导的电信号的传输，从刺激/损坏到非刺激/未损坏区域2。主细胞中的韧皮部是筛网元件（SES），比较简单的，细长的细胞。的SE的端部被连接到其他的搜索引擎，形成一个连续，低电阻，筛筒系统，铺展整个植物。然而，有很少的研究在这些高度特化的细胞的电特性。在这些以往的研究中，研究人员访问与社会企业或者玻璃微电极3或与被耦合到玻璃电极厂插入蚜虫的钢丝，之后stylectomy（切割）4。玻璃微电极是由玻璃毛细管被拉到在一端与热成小于1微米的细尖直径，然后充入一个KCl溶液制成。甲的Ag / AgCl或铂丝插入到氯化钾填充的玻璃电极，然后连接到放大器输入端，和一个所指电极插入到所感兴趣的细胞周围的浴，在完成的电路。这种设置记录胞外指涉电极和细胞内测量电极， 即，在电池5的膜电位之间的电位差。用这种方法，Umrath从植物细胞制成的第一细胞内记录，使用藻类Nitella 6,7。Nitella是一个相对简单的生物体具有大的细胞，并因此适合于细胞内的电生理学实验。与此相反，细胞内的玻璃电极成多细胞三维陆生植物的小细胞的插入是技术要求高，需要高度熟练的研究人员，以及复杂的可视化，显微操作，和抗振动设备。虽然玻璃电极都适合从表层细胞的植物，如根表皮细胞8录制，细胞内recordin从细胞GS深深植根于植物的组织，如社会企业，很可能会造成的损害引起的反应，混乱的结果。在1989年，弗罗姆和Eschrich报道了用另一种方法，称为"蚜方法&#39;，其中玻璃电极连接到蚜虫钢丝stylectomy 4之后。蚜虫方法是微创的，因为柔性通管丝不会引起组织或细胞的损伤玻璃电极做。蚜虫口针是大自然的植物渗透伟大发明，和蚜虫是相当比人类寻找社企更熟练。不幸的是，这种方法蚜虫也是技术专长和装备方面要求极高。此外，每个实现此技术实验的成功完全取决于蚜虫在馈送模式为 - 具有稳定地插入到SE的管心针，在stylectomy的时间。在回顾性思维，人们可以看到，这项技术的成功的几率可能是我通过向实验装置的仪器，它允许识别所述蚜虫探针是否在SE施加stylectomy时mproved。 1964年，麦克莱恩和金赛所描述的"电子监控系统"对蚜虫的取食行为的实时9,10研究。在这个系统中，蚜虫和管心针穿透的植物被集成到一个电路。后来，在1978年，Tjallingii设计系统的修改的版本，称为"电气贯穿图形&#39;（EPG）系统11,12。而原来的电子监控系统是电阻-起源电位敏感只，与EPG系统中，电动势（电动势）起源电位， 即，在植物中或在昆虫中产生，可以被记录在除了从所产生的电位电阻（R）中的昆虫。这是一个重要的改进，因为这两个信号分量，电动势和R，由蚜虫厂渗透过程中提供生物事件的相关信息。是什么使在EPG前置放大器敏感到R-部件是1GΩ，这是接近的植物/蚜虫抗性的平均值的其相对较低的输入电阻。一个小的偏置电压（ 图1，V）为约100毫伏施加到植物，然后跨过植物和昆虫划分在一侧，而在另一输入电阻。的电压及其变化的点处（ 图1A，B）中的昆虫和输入电阻之间测量。因此，R-分量表示偏移电压的植物蚜虫抗性调制，而电动势组件是植物电位在细探针顶端和潜力引起了昆虫的某一分数。该工厂的潜力 - 最相关的在这里 - 是由蚜虫口针刺破了植物细胞的主要膜电位。昆虫的潜力似乎主要两个探针运河内引起流体运动的流势， 即食品和唾液运河;没有内部神经或肌肉电位被记录在EPG。在实践中，细探针顶端用作电极尖端。所有植物细胞相对于内部带负电荷到正外的细胞。的电流（ 即，在水溶液带电离子的移动）从内侧流向外侧， 反之亦然很由于细胞膜的高电阻的限制。通常静息电位保持恒定。然而，当负离子搬出或正离子通过细胞膜移动中，膜电位降低， 即，它的去极化"。去极化发生万一细胞激发。当特定的离子通道在膜被打开或当膜被破坏和离子泄漏和流出的离子然后移动或缩小。所有细胞具有离子通道和泵在叔他质膜带来的膜电位到其静止水平通过恢复细胞内的各种离子的初始浓度。静息电位及其变化是电动势组件，并且因此，在EPG的技术是适用于测量它们。 <img alt="图1" src="/files/ftp_upload/52826/52826fig1.jpg" /> 图1. EPG电极 。在EPG电极是活蚜虫集成到电气贯穿图（EPG）的电路，其通管针插入到在稳定的进给模式的筛元件（SE）。如果探针-刺穿SE是在休息（图A），在电路中，记录由EPG中的电压，是稳定的，在静息电位（图C，垫）。如果SE是兴奋，其膜去极化（图B），该可视化在EPG中电压（图C，去极化）逐渐增加。作为SE的离子平衡返回休息， 也就是说，它repolarizes，记录了电子节目指南的电压逐渐下降到静止电位电平（图C，复极化）。在图C中，"A"和"B"指的是在图A和B，分别示出的方案。 V =可调偏置电压源。 RI =输入电阻。在平行于1GΩ外部电阻，放大器有一个内部（在运放）高1.5TΩ电阻（图A和B，为灰色）。由开关的遥控器上的EPG前置放大器可以从正常到EMF模式，其允许获得高度精确的电压值来进行更改。 <a href="https://www.jove.com/files/ftp_upload/52826/52826fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在接下来的部分，我们提供一个基本的协议执行EPG实验证明是有效的两种昆虫为重点和植物为重点的研究读者。

<table><tbody><tr><td>Brass connector pins</td><td>EPG Systems/hardw.shop</td><td></td><td>&amp;Phi; 1.2 mm</td></tr><tr><td>Thin copper wire</td><td>EPG Systems/hardw.shop</td><td></td><td>approx. &amp;Phi; 0.2 mm</td></tr><tr><td>Thin gold wire</td><td>EPG Systems</td><td></td><td>&amp;Phi; 18 &amp;micro;m</td></tr><tr><td>Soldering fluid</td><td>hardware shop</td><td></td><td>matching the soldering wire</td></tr><tr><td>Resin-cored soldering wire</td><td>hardware shop</td><td></td><td></td></tr><tr><td>Styrofoam</td><td>any</td><td></td><td></td></tr><tr><td>Water-based silver glue</td><td>EPG Systems</td><td></td><td>&lt;a href=&quot;http://www.epgsystems.eu/&quot;&gt;recipe in: www.epgsystems.eu&lt;/a&gt;</td></tr><tr><td>Paper wipes</td><td>Kimberly-Clark</td><td>5511</td><td></td></tr><tr><td>Soldering bolt</td><td>any</td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td>Hund Wetzlar</td><td></td><td>minimum magnification is 10X</td></tr><tr><td>Small scissors</td><td>Fine Science Tools</td><td>14088-10</td><td></td></tr><tr><td>Scalpel</td><td>Fine Science Tools</td><td>10050-00</td><td></td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11231-20</td><td></td></tr><tr><td>Vortex</td><td>A. Hartenstein</td><td>L46</td><td></td></tr><tr><td>Watercolor brushes</td><td>any</td><td></td><td>Number 1 or 2</td></tr><tr><td>Air suction device</td><td></td><td></td><td>see description in: www.epgsystems.eu</td></tr><tr><td>Insect pins</td><td>any</td><td></td><td>No. 1 or 2</td></tr><tr><td>Solid table</td><td></td><td></td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td>Hand made</td></tr><tr><td>Computer</td><td>Fujitsu Siemens</td><td></td><td></td></tr><tr><td>Data acquisition software</td><td>EPG Systems</td><td></td><td>Stylet+d</td></tr><tr><td>Giga-4 (-8) Complete System</td><td>EPG Systems</td><td></td><td></td></tr><tr><td>includes the following:</td><td></td><td></td><td></td></tr><tr><td>Main control box with USB output</td><td></td><td>Di155/Di710</td><td>12/14 bit, rate 100&amp;nbsp;Hz (softw. fixed)</td></tr><tr><td>EPG probes 4 (8)</td><td></td><td></td><td>50x DC pre-amplifier</td></tr><tr><td>Swivel clamps on rod</td><td></td><td></td><td></td></tr><tr><td>DC power adaptor</td><td></td><td></td><td>bipolar, 230/115 VAC to -/+8 VDC</td></tr><tr><td>Plant electrodes and cables</td><td></td><td></td><td></td></tr><tr><td>Additional test and ground cables&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

a new application of the electrical penetration graph (epg) for acquiring and measuring electrical signals in phloem sieve elements

细胞的电生理特性往往在体外研究，从他们的本地环境游离在他们之后。然而，遥远的细胞在生物体之间的电气传动的研究需要在体内 ，细胞嵌入在其天然环境无瑕疵的录音。电信号的传输距离伤员未受伤的地区在工厂已经长期以来引起了植物学家的利益。韧皮部，即传遍植物的植物脉管系统的生活的一部分，已被假定为在电传输在植物的主要组织。合适的电生理学方法的缺乏造成对的韧皮部细胞在体内的电特性的研究很多挑战。在这里，我们提出了一种新的方法来筛分子细胞内电（SES），采用活蚜虫，或其他韧皮部饲养昆虫半翅目，一体化的电气贯穿GRApH值（EPG）电路。的通用性，鲁棒性，以及该方法的准确度使得可以记录和详细研究的伤口诱导的电信号中的SE模式植物拟南芥 1的中央静脉。在这里，我们表明，EPG电极可以很容易地对社会企业在边际静脉内电生理记录来实现，以及研究社会企业与电信号响应几个外部刺激的能力。施加的SE的胞内电生理在EPG的方法可以被实现为多种植物物种的，在大量植物/昆虫组合，并为许多研究的目的。

在先前的研究中，我们实现了与表征期间毛虫攻击1中的中脉的社会企业产生的电信号的目的在EPG电极技术。中脉是优选的插入位点对常规的玻璃电极，以及用于玻璃探针电极，因为它是SE-致密的，相对坚固的，因而适合于所需的实施这些技术的固定。在这里，我们采取了与从更难以收集电信息来访问社会企业，特别是那些在叶片的边缘静脉的目的在EPG电极的通用性的优点。 图3</stro...

Watch this Scientific Journal Video about A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements at JoVE.com

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

在电气贯穿图（EPG）的韧皮部筛分子获取和测量电信号中的新应用

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

a-new-application-electrical-penetration-graph-epg-for-acquiring

Research

JoVE Journal

Environment

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

14.7K 次观看.  University of Würzburg. Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants. 

视频: A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Measurement of Bioelectric Current with a Vibrating Probe

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. For example, they play an important role in directing cell migration during wound healing. Here we describe the manufacture and use of ultrasensitive vibrating probes for measuring extracellular electric currents. The probe is an insulated, sharpened metal wire with a small platinum-black tip (30-35 &mu;m), which can detect ionic currents in the &mu;A/cm2 range in physiological saline. The probe is vibrated at about 200 Hz by a piezoelectric bender. In the presence of an ionic current, the probe detects a voltage difference between the extremes of its movement. A lock-in amplifier filters out extraneous noise by locking on to the probe's frequency of vibration. Data are recorded onto computer. The probe is calibrated at the start and end of experiments in appropriate saline, using a chamber which applies a current of exactly 1.5 &mu;A/cm2. We describe how to make the probes, set up the system and calibrate. We also demonstrate the technique of cornea measurement, and show some representative results from different specimens (cornea, skin, brain).

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

生物电电流测量振动探头

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. ...

科研

生物工程

Development of a 3D Graphene Electrode Dielectrophoretic Device

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The protocol details the procedures to construct a versatile, reusable, multiple layer, laminated dielectrophoresis chamber. Specifically, six layers of 50 &#181;m x&#160;0.7 cm x&#160;2 cm graphene paper and five layers of double sided tape were alternately stacked together, then clamped to a glass slide. Then a 700 &#956;m diameter micro-well was drilled through the laminated structure using a computer-controlled micro drilling machine. Insulating properties of the tape layer between adjacent graphene layers were assured by resistance tests. Silver conductive epoxy connected alternate layers of graphene paper and formed stable connections between the graphene paper and external copper wire electrodes. The finished device was then clamped and sealed to a glass slide. The electric field gradient was modeled within the multi-layer device. Dielectrophoretic behaviors of 6 &#956;m polystyrene beads were demonstrated in the 1 mm deep micro-well, with medium conductivities ranging from 0.0001 S/m to 1.3 S/m, and applied signal frequencies from 100 Hz to 10 MHz. Negative dielectrophoretic responses were observed in three dimensions over most of the conductivity-frequency space and cross-over frequency values are consistent with previously reported literature values. The device did not prevent AC electroosmosis and electrothermal flows, which occurred in the low and high frequency regions, respectively. The graphene paper utilized in this device is versatile and could subsequently function as a biosensor after dielectrophoretic characterizations are complete.

A microdevice with high throughput potential is used to demonstrate three-dimensional (3D) dielectrophoresis (DEP) with novel materials. Graphene nanoplatelet paper and double sided tape were alternately stacked; a 700 &#956;m micro-well was drilled transverse to the layers. DEP behavior of polystyrene beads was demonstrated in the micro-well.

三维石墨电极介电泳装置的研制

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The ...

工程学

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

通过活生物膜表征电子传输

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

化学

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

微电极阵列接口微流体研究神经元通信和轴向信号传播

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

神经科学

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

具有透化检测功能的连续流动微电穿孔系统的制造和操作

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

<meta charset="utf8"></head><body>评估电穿孔对基因工程加标 HEK 细胞的影响

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

<meta charset="utf8"></head><body>监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

<meta charset="utf8"></head><body>用于 A431 细胞电穿孔分析和递送预测的 TEEI 方法

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

<meta charset="utf8"></head><body>作者聚焦：通过微通道和纳米通道优化多孔衬底电穿孔，以增强监测和中间阶段表征

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

基于多孔底物的电穿孔与跨上皮电阻抗监测

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

在电气贯穿图（EPG）的韧皮部筛分子获取和测量电信号中的新应用

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具有透化检测功能的连续流动微电穿孔系统的制造和操作

监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

基于多孔底物的电穿孔与跨上皮电阻抗监测