Name: a method for growing bio-memristors from slime mold
Uploaded: 2017-11-02T12:30:00.000+00:00
Duration: 7 min 46 s
Description: Watch this Scientific Journal Video about A Method for Growing Bio-memristors from Slime Mold at JoVE.com

这项研究是由普利茅斯大学的人文和表演艺术学院资助的。作者想承认化提供他们的导电 PLA 样品。

1. 使用打印机接口将打印床温度设置为85和 #176, 制作3D 打印插座 <ol> <li> 腔、盖和底座 <ol>, 用高冲击聚苯乙烯 (臀部) 加载3D 打印机; C挤出机到 230 #176;当温度达到时, 松开惰臂, 插入灯丝, 并向下推直到它开始挤出热端。然后, 固长丝惰臂, 取出挤出材料. </li>
		<li> 将3D 插座 STL 模型文件导入到3D 打印切片软件中, 通常可以通过导航到 "文件" 选项卡并选择导入/打开选项 ( 图 2 ) 来实现. </li>
		<li> 如果切片软件提供高和低质量的打印设置, 请选择 "高质量", 同时确保选择了正确的材料配置文件. 
		注意: 如果在一次运行中打印多个插座, 请确保该软件已设置为每次打印一个对象。如果跳过此步骤, 则可能会降低打印质量, 这可能会在将部件装配在一起时导致公差问题. </li>
		<li> 打印完成后, 等待打印床温度低于50和 #176; C 移除部件. </li>
		<li> 使用细丝刷, 轻轻地清除任何不完美的电极插座, 当配有电极时, 可能会造成障碍物. </li>
	</ol> </li> <li> 电极 <ol> <li> 更换清洁灯丝的臀部灯丝, 并通过打印头运行大量的材料. </li>
		<li> 用导电聚乳酸 (PLA) 灯丝来加载打印机, 其体积电阻率为0.75 和 #937;-cm 或更低. </li>
		<li> 将打印床温度设置为60和 #176; c 和挤出机到230和 #176; c (请参见步骤1.1.1 以指导). </li>
		<li> 当温度达到时, 通过打印头挤出几厘米长的灯丝。这一过程将有助于确保删除前几届会议的所有微粒. </li>
		<li> 使用3D 打印切片软件, 加载电极 STL 文件 ( 图 3 ). </li>
		<li> 在打印设置中, 指定以下内容: 图层高度 = 0.16 mm、外壳厚度 = 1.7 mm、底部/顶部厚度 = 0.74 mm、填充密度 = 100% ( 图 4 ). </li>
		<li> 如果在一次运行中打印多个电极, 请设置打印机一次打印一个. </li>
		<li> 一旦打印出来, 将电极放在打印床上, 直到冷却到室温。这可确保部件不变形和畸形. </li>
	</ol> </li> <li> 插座组件 <ol> <li> 将电极插入两个腔中的每一个。如果步骤1.1.5 已正确完成, 电极应进入商会没有太大的力量. </li>
		<li> 使用锋利的手术刀, 切割10毫米的聚氯乙烯 (PVC) 油管 (4 毫米内径和6毫米外径) 小心, 以确保每一个结束是切直和干净. </li>
		<li> 轻轻地缓解两个电极边缘上的 10 mm PVC 油管的每一端. </li>
		<li> 连接后, 两个腔夹插入底座. </li>
	</ol> </li> </ol> 2。容器准备和 P. 多头 接种 <ol> <li> 2% 琼脂培养基制备 <ol> <li> 将2克营养微生物琼脂粉放入250毫升玻璃瓶中. </li>
		<li> 添加100毫升去离子水和混合好. </li>
		<li> 压釜瓶为 12-15 分钟, 在121和 #176; C 或地方在沸水浴为 15-20 min. </li>
	</ol> </li> <li> 将琼脂基板设置为容器和 #39; s 室 <ol> <li> 使用水浴或微波炉将琼脂熔化. </li>
		<li> 用熔融琼脂填充2毫升吸管. </li>
		<li> 通过将吸管的笔尖悬停在内部底座的上方大约5毫米, 然后慢慢地将这些井填充到连接管孔的底部, 来填满每个插座和 #39;
		<li> 在填充完井后立即放置一个盖子, 并将容器放在一旁, 直到琼脂设置并达到室温. </li>
	</ol> </li> <li> P. 多头 接种 <ol> <li> 在这两个腔中的每个房间中放置一个燕麦片. </li>
		<li> 从一个饥饿的 (大约 12 h) 疟原虫的文化中去除一个2毫升的 pseudopods, 并将其放在两个腔中的一个。为了促进快速生长, 试着从生物体的最活跃的前部取原生质. </li>
	</ol>
	</li>
</ol>

<ol>
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作者没有什么可透露的。

本文提出了一种从 myxomycete P 多头中器生长的方法。生物体生长在3D 的印刷容器内, 旨在克服与实施生物器相关的一些限制。这些限制包括设置时间、样品生长时间和缺乏标准化的 sample-to-sample 生长条件和电观测。
我们的插座是第一次透露, 2015年在印刷宣传材料为半岛艺术当代音乐节 2016 (PACMF) 和各自的网站15。在这里, 我们的技术被用来开发一个混合的硬件 bioware 互动音乐系统, 能够产生音乐伴奏的现场音乐家。在参考14中, 我们报告了对我们的容器进行的广泛测试, 并将结果与以前的方法进行比较9,10。随着这些发展, 另一组研究人员随后探索创造生长环境来研究生物体的 thermistive 属性16, 但这些特性与记忆属性不同。但是, 在实现P. 多头器13、17的控制方法方面, 仅有两个其他尝试。在这些实验中, 油井是由一种凝胶状的生物相容性弹性体材料制成, 称为烷 (), 电极是用各种金属或聚 (34-吩):p oly (苯乙烯磺酸) (PEDOT: PSS) 制造的。虽然这些材料通常用于电子, 微, 和仿生工程, 它们是昂贵的, 需要一些专门知识使用。例如, PEDOT: PSS 需要自旋涂层和掺杂来提高其导电性。因此, 对于那些无法获得专家资源的人来说, 这些技术是遥不可及的。本文所介绍的容器使用的方法和材料易于访问和便宜。此外, 该设计为疟原虫的栖息地提供了一个好客的环境, 这与其他的P. 多头阻原型不同, 在这种情况下, 没有试图在任何时间段内使细胞存活。
到目前为止, 很难用以前的方法对培养皿中的生物体进行持续的 i-v 测量 (图 5, 左侧)。我们的方法显著地改善了这种情况 (图 6)。我们的容器的测试结果表明, 该设计减少了生长时间, 延长寿命, 标准化的成分反应, 并创造了一个受保护的微环境封装的有机体。此外, 该装置还提供了将生物体作为电气方案的组成部分的可行方法。
该方法缓解了与在电气系统中利用P. 多头器相关的一些问题。然而, 需要进一步研究和发展的限制。首先, 如果贮器受到温度的快速变化或长时间的高压, 则冷凝可以聚集在连接管的内表面。后者是由于机体的高电阻导致电能被转化为热能。如果有意义的话, 冷凝可以在连接管的一端的两个电极之间形成低阻通路。通过确保器不超载, 可以有效地管理此限制。其次, 使用所提出的方法产生的器的整体电阻可能不同于 component-to-component。这种现象可能是由于不限制原生质管外径的方法造成的。因此, 用户可能需要将校准过程纳入器的应用中。
由于这种方法, 我们现在可以开始研究在P. 多头中引起记忆观测的生物过程。这类过程很可能具有动态参数, 我们可以利用它来增加元素的使用。我们已经开始进行一些初步实验, 其中细胞外离子浓度改变, 以审查, 如果电压门控离子通道发挥作用的 memristance。
所提供的容器仅用于实现P. 多头器。但是, 这些设备有可能使用超过实现单个组件的用途。例如, 在参考12,18中, 原生质管被作为自和自我修复的生物导线进行了研究。在这两项调查中, 研究人员表示, 根据一项计划, 需要进一步开展工作, 建立原生质管的生长方法。本文提出的贮器提供了一种在两个或可能更多的点之间描述管子的生产方法。图 7显示了两张照片, 说明插座可用于长100毫米以上的健康管。在参考18中, 研究了原生质管的传递函数。这项调查的结果表明, 如果将生物体整合到一个电气系统中, 生长试管所需的琼脂可能会引起问题。这是由于基板的电容。这里提出的容器仍然需要琼脂保持湿度高。然而, 随着插座的设计的小变化, 它是可能创建一个可拆卸的管。一旦生长完成并被夹入一个电气系统, 这种设置可能会使管子与腔室断开。此外, 一旦管的健康开始恶化, 它可以重新连接到新的商会的食物和喘息, 直到它已经修复自己, 可以再次使用。图 8显示了与腔室断开连接的长管的照片。未来的研究需要调查原生质管的电气性能没有琼脂和当长的长度使用所提出的方法。

今天的计算机是使用三两个终端的基本无源电路元件: 电容器, 电阻, 电感器。被动元素只能够消散或储存能量, 而不是产生它。这些元素在 18th和 19th世纪中建立, 并通过麦克斯韦方程组进行连接。我们定义了这些三电路元件中的每一个, 它们之间的关系, 四电路变量, 即电流 (I), 电压 (V), 电荷 (Q) 和通量链 (φ)。电荷是电流的积分, 法拉第定律将电压定义为通量的时间积分。因此, 电容器是由电压和电荷之间的关系定义的, 电阻是由电压和电流之间的关系定义的, 电感是由流量和电流之间的关系定义的。一个世纪以来, 这些元素都是电子学的基石。然而, 它们只代表三可能的四关系对之间的电路变量, 离开磁链和电荷取消链接。在 1971年, 利昂蔡发表了一篇论文1 , 他假设, 有一个缺失的第四元素, 连接其余的两个变量, 他称之为阻。阻可以被描述为一个电阻器, 它能记住它的历史, 因此收缩 ' 记忆电阻 '。这一元素的功能, 通过改变其阻力, 根据大小的前施加的电压和它的持续时间。此外, 一旦电压不再适用, 阻保留其最后的电阻状态。不同于电容器, 电阻, 电感, 阻的行为是非线性的, 这是明显的, 在其 i-v 的轮廓, 其中一个捏滞环是形成在交流电压。这个循环采取的形式是一个李萨的数字包含两个垂直振荡的高和低阻力状态。在蔡氏形式化的 memristance 理论之前, 其他研究人员报告了某些频率的记忆阻力效应, 例如聚合物和金属氧化物等材料的实验, 以及在千分尺上开发电子器件。缩放2。然而, 在许多情况下, 这些影响被认为是不可取的。花了近四十年的时间, 蔡氏的形式化连接到一个物理设备和研究人员开始开发的方法, 利用记忆的效果。惠普实验室的一个团队成功地在 2008年3中制造了一个记忆设备, 这点燃了对元素的巨大兴趣。
计算机科学家对阻有浓厚的兴趣, 因为它被誉为第一个将处理和记忆能力结合在一个单元中的元素。它还显示类似于某些神经过程的行为, 如钉定时相关的可塑性 (STDP)4, 只命名一个。这种行为引起了构建类似大脑的计算技术的观点, 从而放弃了内存和中央处理器 (或 CPU)5之间的区别。与流行的开发器的方法不同 (例如, 使用2), 我们的目标是开发一个有机生物阻。此外, 我们感兴趣的是, 这个组件如何能够提供方法, 探索超越传统方法的工程计算设备的范例;例如, 在计算机音乐领域的创造性应用程序6。
Memristance 是研究人员最近在一系列生物系统中发现的一种效应。例如, 在芦荟植株7和人体皮肤8中观察到了记忆的特性, 但有两种。这些发现表明, 在生物基质上实现加工和存储设备是可能的。利用技术中的有机系统可以让我们探索诸如自组装、自我修复、低环境影响和电站等激动人心的概念。然而, 在我们可以调查这些机会之前, 需要解决几个挑战。许多具有记忆特性的生物系统有很大的限制, 限制了它们作为实际电子元件的生存能力。例如, 芦荟叶7需要光照, 寿命有限, 很难集成到电路中。此外, 还有一些其他的在体内记忆现象, 如人类汗液管道8, 目前在实验室和日常电子系统之外的开发系统中是行不通的。然而, 在所有的记忆现象中, 有一个潜在的候选者: P. 多头。
疟原虫的P. 多头是一个无定形的单细胞系统, 已被发现充当记忆组件9,10。这个有机体是一个理想的候选的研究混合硬件-湿电子出于多种原因。首先, 有机体是致病, 宏观, 并且不需要专家设备用途, 使疟原虫容易接近对工程师和 non-biologists。其次, 细胞是无定形的, 形成线状静脉网络, 并将在大多数基底上生长 (图 1)。这些特性使得细胞的形态学很容易被描述为符合传统的电气方案。还有研究表明, 疟原虫可以活四年以上的11, 它的静脉可以作为自修复传导通路12。几项实验室研究证实了生物体的记忆能力9,10,13现在时机成熟, 探索其潜能。
使用P的想法。多头器是相对较新的。因此, 没有确定的标准来测量和观察其电学特性。在相同的研究组和组之间的实验过程中缺乏一致性, 可能是因为发布的结果9,10之间存在不一致的原因。这种变异在样品生长条件和处理中很可能是最突出的。因此, 我们需要建立方法来生产和测试P. 多头器, 其中可能导致错误的因素得到更好的控制和监控。 此外, 我们需要创建实现P. 多头器的方法, 使其能够稳定且易于集成到电气系统中。
本文所提出的方法提供了一种将生物体作为元件并入电气原理图中的多头的器的实际应用的探索平台。这些技术很可能会吸引那些希望探索湿系统的真实世界使用的工程师。此外, 非 (例如, 开源电子原型爱好者) 可以对非常规计算的某些方面感兴趣, 但发现很难找到适合自己的原型。需要.一些潜在的应用可能包括实施利用器扣球行为的概率模型, 开发执行有状态 l 的方法逻辑操作, 并建模神经过程的信息存储和处理。

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a method for growing bio-memristors from slime mold

我们的研究旨在更好地了解生物体的电子特性, 以便设计出新的 bioelectronic 系统和基于生物学的计算体系结构。本论文的重点是利用单细胞粘液霉菌多头多头开发生物器 (或生物器) 和生物计算设备。阻是一种具有记忆的电阻器。它是第四基本的无源电路元件 (其他三是电阻、电容器和电感器), 为新型计算系统的设计铺平了道路;例如, 可能放弃存储和中央处理单元之间的区别的计算机。当在交流电压下应用时, 阻的电流与电压特性是一个夹紧的滞后回路。结果表明, P. 多头在交流电压下产生夹滞滞后回路, 并显示与阻的功能相媲美的自适应行为。本文介绍了利用P. 多头实现生物器的方法, 并介绍了一种用于培养生物体的容器的开发, 它便于将其作为电子电路组件进行部署。我们的方法已被证明可以减少生长时间, 增加元件寿命, 并标准化电观测。

为了产生代表性的结果, 我们建立了5样品使用的确切方法如上所述。对于控件, 还使用早期P. 多头阻调查910中描述的方法安排了5示例。在这里, 我们定位两个电极间隔约10毫米的距离在 60 mm 培养皿。每个电极由一个圆 (直径约20毫米) 镀锡铜丝 (16 站在0.2 毫米), 充满了2% 营养去离子琼脂 (〜2毫升)。所有样品都通过延时图像进行监测, 以审查生长时间。在这里, 5 插座样品在接种的 10 h 之内连接了二个电极。其中最快的生长在 2 h 以下, 最长为 10 h, 平均生长时间在所有5样本 7 h 24 min。四的控制样品产生了一个连接原生质管和一个传播的接种电极, 但干燥之前, 它作出了必要的连接。最快的控制样本, 使其连接在19小时内, 而最慢的采取了36小时, 平均增长时间的控制样本26小时15分钟。这些数据表明, 使用所提出的方法器生长时间明显减少。
阻的 i-v 轮廓是其最具定义的特征。因此, 我们对样品进行了 i-v 测量, 以产生具有代表性的结果。在这里, 在160步电压正弦波的每个点上进行瞬时电流测量。每个电压步有一个静态驻留时间2秒. 使用230可编程电压源和617可编程静电计进行电气测量。这些设备被选中, 因为它们有能力在高分辨率下进行电压测量和计量。实验是在室温下在一个未点燃的房间进行的。
图 6显示了从P. 多头器上的测试产生的典型的 i-v 曲线。图 6c和6d显示了来自在 Petri 盘中实现的组件的代表度量的图形。结果表明, 虽然在同一个样本上测量的曲线是相似的, 但迟滞变化与 sample-to-sample。这种变化包括夹点的位置, 正负叶的大小, 以及在正负电压域中测量的对称性。因此, 用培养皿法测量器的 i-v 曲线不是 "理想" 阻的足迹, 因为夹点不在零电压和电流。图 6a和6b显示了来自容器中的器的具有代表性的度量的图形。这些滞后环的夹点位置和叶大小在不同电压范围和 time-steps 的离散样本曲线和 sample-to-sample 曲线上都是相对一致的。因此, 插座 i-v 曲线更让人联想到一个 "理想" 的阻的足迹, 其中捏点总是奇异的, 几乎始终如一地在零电压和电流。然而, 虽然迟滞形态与 sample-to-sample 相似, 但在样品的整体抗性上存在差异。.
在最初的 I-V 测量完成之后, 每个样品每天进行一次测试, 直到它们没有记忆曲线。在4控制样品中, 2 在最初测试的2天内干涸, 其余2则继续记录被挤压的曲线, 再持续2天。容器样品保持了他们的 memristance 至少7天, 与3样品超过那。随着时间的推移, 每个贮器样品的原生质管变得更厚, 整体电阻下降, 一些样品在 x 10-04范围内测量, 10 V 在其早期测试中对 x 10-05运行。
读者通过 Braund14引用该文章, 以得到对所提供的容器进行广泛测试的结果。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56076/56076fig1.jpg"> 
图 1:多头的疟原虫的2天文化的照片.<a href="//ecsource.jove.com/files/ftp_upload/56076/56076fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56076/56076fig2.jpg"> 
图 2:在将STL 文件加载到切片软件后的一个截图.<a href="//ecsource.jove.com/files/ftp_upload/56076/56076fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56076/56076fig3.jpg"> 
图 3: 将电极 STL 文件加载到切片软件后的屏幕截图.<a href="//ecsource.jove.com/files/ftp_upload/56076/56076fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56076/56076fig4.jpg"> 
图 4: 用于打印电极 STL 模型的设置配置的截图。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56076/56076fig5.jpg"> 
图 5:两张照片描述P. 多头器在一个 Petri 盘中实现 (左), 并使用本文中介绍的方法 (右).<a href="//ecsource.jove.com/files/ftp_upload/56076/56076fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56076/56076fig6.jpg"> 
图 6:四 i-v 图是由两个在容器 (a, b) 中生长的器产生的, 两个在培养皿中实现 (c, d)。<a href="//ecsource.jove.com/files/ftp_upload/56076/56076fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56076/56076fig7.jpg"> 
图 7:显示容器被用来长出不同长度的管子的照片
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56076/56076fig8.jpg"> />>图 8: 显示已与腔室断开连接的原生质管的照片.

Watch this Scientific Journal Video about A Method for Growing Bio-memristors from Slime Mold at JoVE.com

A Method for Growing Bio-memristors from Slime Mold

一种从煤泥霉菌中生长生物器的方法

本文介绍了一种改进的生物器的方法, 从疟原虫的多头多头。这种方法已被证明可以减少生长时间, 增加元件寿命, 标准化电观测, 并创造一个可集成到传统电路中的保护环境。

Our research is aimed at gaining a better understanding of the electronic properties of organisms in order to engineer novel bioelectronic systems and ...

a-method-for-growing-bio-memristors-from-slime-mold

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.9K 次观看.  Plymouth University. 本文介绍了一种改进的生物器的方法, 从疟原虫的多头多头。这种方法已被证明可以减少生长时间, 增加元件寿命, 标准化电观测, 并创造一个可集成到传统电路中的保护环境。

视频: A Method for Growing Bio-memristors from Slime Mold

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

科研

遗传学

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

海洋微生物的趋化反应的微观尺度的营养层

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

生物学

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

生物工程

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

神经科学

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

生成受控动态化学景观，研究微生物行为

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

数百个 C.白化病 细胞生物原子力显微镜测量自动化

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

利用软光刻改变根面微结构的仿生复制

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

3D 打印在微型神经元细胞培养设备中的应用

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

作者聚焦:用于分析大脑功能的模块化神经元网络

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

微尺度神经元细胞培养装置的双光子聚合3D打印

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

一种从煤泥霉菌中生长生物器的方法

摘要

探索更多视频

此视频中的章节

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

海洋微生物的趋化反应的微观尺度的营养层

协议生物膜形成的流光在微流体器件与微柱

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

生成受控动态化学景观，研究微生物行为

数百个 C.白化病细胞生物原子力显微镜测量自动化

利用软光刻改变根面微结构的仿生复制

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

微尺度神经元细胞培养装置的双光子聚合3D打印

一种从煤泥霉菌中生长生物器的方法

摘要

探索更多视频

此视频中的章节

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

海洋微生物的趋化反应的微观尺度的营养层

协议生物膜形成的流光在微流体器件与微柱

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

生成受控动态化学景观，研究微生物行为

数百个 C.白化病 细胞生物原子力显微镜测量自动化

利用软光刻改变根面微结构的仿生复制

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

微尺度神经元细胞培养装置的双光子聚合3D打印

数百个 C.白化病细胞生物原子力显微镜测量自动化