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1。海藻酸钠电<ol><li>自定义制作通过补丁使用鳄鱼夹线电极连接电源。铟锡氧化物（ITO）覆盖玻片将作为阳极（工作电极）和铂片作为阴极（反电极）。所以要功能的ITO表面反对反电极和垂直定位的解决方案或水平等，包含表面上沉积的解决方案是将蘸放置电极。 </li><li>蒸馏水1％海藻酸钠和0.5％，碳酸钙3（重量比）混合，然后高压灭菌解决方案，准备海藻酸钠沉积解决方案。建议的解决方案在不使用时要不断搅拌。 </li><li>沉积溶液中浸泡两个电极。可使用海藻酸钠与FluoroSpheres（Invitrogen）的荧光标记的，每程等 14，允许荧光参数CE成像所产生的薄膜。 </li><li>适用于一个恒定的电流密度（A / M 2）2分钟;电压范围内的转移2-3五</li><li>断开电极和删除非交存的解决方案。轻轻冲洗，以去除多余的海藻酸钠薄膜与NaCl（0.145男）。 </li><li>孵育氯化钙 （0.1米）的电影简短（约1分钟），以加强凝胶。冲洗与NaCl（0.145男）和孵化辅以氯化钙2（1毫米）的理想解决方案。 </li><li>使用荧光显微镜（ 图1B）的图像。 </li></ol> 2。共沉积的海藻酸钠沟通的细胞群<ol><li>一个信号发送细胞培养（W3110 WT 大肠杆菌 ），在LB培养基中生长，细胞培养和信号接收器“（MDAI2 + pCT6-LSRR - 放大器 ŕ+ PET-DsRed的 - 根 R），生长在LB培养基+ 50 μg/ mL的卡那霉素和氨苄青霉素，必须一夜之间长大了1ND再增长1光密度（600纳米）的接种。调整接收机的细胞培养的光密度0.4-0.6使用前的LB。 </li><li>准备以1:1的比例到终浓度为1％海藻酸钠，0.5％，CaCO 3的沉积2％的海藻酸钠溶液和1％，碳酸钙3，每个细胞培养的混合，稀释至密度大约一半的细胞养殖密度。 </li><li>使用聚二甲基硅氧烷（PDMS）中（按到Sylgard说明，切成所需大小）和铂电极包含两层ITO电极图案的玻璃幻灯片。一个ITO电极连接到步骤1（海藻酸钠电）与铂的电源。 </li><li>沉浸在电极沉积含有接收机细胞的解决方案。设定恒定电流的电源供应密度3 A / M 2，其中由单电极表面积维度定义，其中，沉积物n将发生。 </li><li>套用目前为2分钟，让海藻酸钠基质细胞共沉积。 </li><li>冲洗步骤1.5中所述的电影。 </li><li>切换到第二，相邻，ITO电极的阳极连接。 </li><li>重复的沉积过程（步骤2.4 - 2.7），但这次推出的解决方案，包含发件人细胞。 </li><li>孵育2电极芯片，包含共沉积的细胞和钙海藻酸钠一夜之间在37°C间在磷酸盐缓冲液（PBS），辅以10％的LB培养基和1毫米氯化钙2。 </li><li>经过孵化，图像使用荧光显微镜（ 图2B）。 </li></ol> 3。壳聚糖电<ol><li>通过弹簧夹电极连接电源。金涂硅芯片将作为阴极（工作电极）和铂金箔作为阳极（反电极）。金电极表面的位置，以便日在反对反电极定位垂直蘸到一个解决方案或水平等，包含表面上沉积的解决方案。 </li><li>准备入水混合壳聚糖片壳聚糖溶液慢慢加入2 M盐酸溶解多糖（最终pH值5.6），一定要遵循由Meyer 等15所概述的程序。 </li><li>放置电极到壳聚糖溶液（0.8％），完全淹没沉积所需的面积。可以使用壳聚糖荧光标记5和6 - （）carboxyrhodamine 6G琥珀酰亚胺酯（Invitrogen公司），为吴等 8，荧光显微镜图像电薄膜。 </li><li>申请一个恒定的电流密度（A / M 2）2分钟。 2-3范围内的电压将转向五，作为工作电极金表面面积接触到的沉积物等功能，计算电流密度n解决方案。 </li><li> DI水冲洗电极以去除多余的壳聚糖。该芯片可以存储在水或PBS（10毫米，pH值7.0）。 </li><li>使用荧光显微镜（ 图3C）的图像。 </li></ol> 4。与功能化的壳聚糖膜的电化学传导<ol><li>共沉积的壳聚糖和葡萄糖氧化酶（GOx）从电流密度在4到图案的电极A / M 2，根据步骤3（壳聚糖电镀）解决方案（1％壳聚糖，680 U / ml的葡萄糖氧化酶，pH值5.6）。 GOX包埋壳聚糖膜将产生。 </li><li>将处理后的电极作为工作电极，铂丝作为反电极，Ag / AgCl电极为参比电极，如在图4A中所述的三电极系统。 </li><li>电极浸入磷酸盐缓冲溶液中含氯化钠（0.1米）（0.1米，pH值7.0）。 </li><li>电化学共轭蛋白质的壳聚糖影片通过一个恒定的电压（0.9伏）使用计时10 60。 </li><li>将在磷酸盐缓冲芯片（0.1M，pH值7.0）和轨道器上的10分钟，以消除任何未反应的NaCl和游离的葡萄糖氧化酶洗。 </li><li>重新连接到步骤4.2到5 mM葡萄糖溶液浸泡所述的三电极系统。采用循环伏安，潜力朝着积极的方向扫至0.7 V使用控制电影，不含葡萄糖氧化酶氧化（ 图4B）扫描见过的金额比较。 </li><li>从电极的葡萄糖溶液和磷酸盐缓冲液（0.1M，pH值7.0）冲洗，然后放入10毫升烧杯中含有8磷酸缓冲液（0.1M，pH值7.0）电极。葡萄糖氧化酶的功能化芯片0.6 V的偏置作为工作电极（ 图4C）。 </li><li>葡萄糖等份添加到缓冲区（每个等份增加血糖浓度4毫米）。 </li><li>稳态电流和血糖浓度的葡萄糖氧化酶的功能化壳聚糖膜之间产生的标准曲线。 </li></ol> 5。蛋白质功能化，利用酶大会<ol><li>与相邻的金载和在PDMS以及ITO电极图案，用玻片。偏见与金电极阴极电沉积壳聚糖如先前所示的潜力。 DI水，然后PBS冲洗电影简要吸管。 </li><li>加入荧光标记的“AI-2合酶”16（用DyLight标签套件）+ 100 U / ml的PBS中酪氨酸酶3μM的蓝色解决方案。 </li><li>在室温下孵育1小时，然后用PBS冲洗胶片。 </li><li>应用ITO电极的阳极共沉积海藻酸钠沉积的解决方案，包含接收细胞（如准备步骤2.1-2.2）的潜力。按照步骤2步骤2.3-2.6（共沉积在海藻酸钠的细胞群）。 </li><li>要生成传输信号（AI-2）酶，冲洗后的影片添加500μM的S-腺苷同型半胱氨酸（SAH）的PBS中的解决方案，辅以10％LB培养基和1mM氯化钙2。覆盖的电极，以防止溶液蒸发和孵育过夜在37°C。这将允许一个接收器细胞的反应，产生红色荧光蛋白（DsRed的）。 </li><li>相邻的电极，可通过调整过滤器捕捉到的AI-2的共沉积的接收细胞（ 图5B）表示合酶和红色荧光的蓝色荧光，荧光显微镜成像。 </li></ol> 6。代表结果施加的电信号可以创建一个电极表面附近，这些刺激可以引发的多糖，如海藻酸钠和壳聚糖凝胶薄膜作为电极表面上的沉积自组装的局部微环境（例如，域和梯度）。因为T他的溶胶-凝胶转变发生在电极表面，由此产生的电影electroaddressed与它相匹配的电极图案（ 图1B，3C）的几何。海藻酸钠和壳聚糖，如的生物相容性电影提供的表面，可与生物元件的功能。使用海藻酸钠，独特的细胞群已共沉积在不同的地址。他们electroaddressment证据后，发送者和接收者的细胞群之间的互动观察。从发件人的细胞和分子autoinducer-2（AI-2）扩散采取了由接收细胞，导致表达DsRed的红色荧光蛋白（ 图2A）。在图2b中，红色荧光观察仅在接收机解决的电极。 壳聚糖胺基提供电以及表面官能适合所需的pH值响应。我们利用这些独特的性质，电化学共轭的生物传感葡萄糖氧化酶（GOX）以电壳聚糖膜。这种酶，然后提供生产过氧化氢 ​​酶反应（ 图4A），然后可以电化学氧化，产生的输出电流通过检测葡萄糖的能力。这种方式，一种化学信号可以被转电。 图4B显示电影GOX电化学共轭在葡萄糖存在的阳极产生强烈的信号，反对那些不含有葡萄糖氧化酶的电影。这些结果表明，葡萄糖氧化酶可以组装到沉积的壳聚糖膜，并保留催化活性。此外， 图4C中产生的阳极电流响应增加血糖浓度的逐步增加。 图4C标准曲线表明，逐步增加在不久的线性FAS进行北恩上依赖的葡萄糖量增加。这些结果表明，这种酶还保留其敏感性，增加血糖浓度的壳聚糖膜后，共轭。检测下限是不是在这里学习，因为它已经本系统的特点在Meyer 等工作。等。 我们也已经证明，设计包含自定义的标签，在五酪氨酸酶控制方式，壳聚糖共价固定的一种酶的利益。具体来说，这一过程的酶酪氨酸酶介导的。所示，由图5A计划（上），这种酶的AI-2合酶包括五酪氨酸的标签。酪氨酸酶作用于酪氨酸标记，O型醌，然后共价结合到壳聚糖的胺氧化残留的酚组。 AI-2的酪氨酸酶的组装合成酶的壳聚糖膜功能化的证据是观察图5B </st荣&gt;，其中AI-2合酶已被荧光标记的蓝色。因为AI-2合成酶底物S-腺苷同型半胱氨酸（SAH）的作为发件人细胞的相同的方式在产生AI-2，其接近接收细胞中存在的SAH共沉积也会使接收机细胞荧光表达DsRed的回应（ 图5A（下））。接收细胞的红色荧光（ 图5B），再次证明了由于AI-2的扩散从一个到另一个地址之间的互动，并进一步表明，酶固定化壳聚糖保留一次活动共价结合。 <img alt="图1" src="/files/ftp_upload/4231/4231fig1.jpg" /> 图1海藻酸钠电。 （一）机制海藻酸钠电：作为一个电极阳极偏颇，电解水在其表面发生，产生局部的低pH值。碳酸钙粒子反应与盈余质子，释放钙离子的颗粒溶解。离子在海藻酸钠高分子链的存在，成为螯合在的“eggbox”网络，形成了电极的交联水凝胶。海藻酸钠为增加电极的距离，有一个更大的趋势继续在溶液中，由于减少钙离子存在。 （二）使用一个L形图案的ITO电极电沉积海藻酸钠。一个硅橡胶固定在电极包含1荧光标记的绿色海藻酸（1％）和3（0.5％）碳酸钙沉积的解决方案。经过2分钟的电。在3A /米2的电流密度，的electroaddressed海藻酸钠水凝胶荧光显微镜成像。 <img alt="图2" src="/files/ftp_upload/4231/4231fig2.jpg" /> 图2。共沉积的细胞群。 （a）计划显示之间的相互作用2 大肠杆菌大肠杆菌菌株：一个人口产生autoinducer-2（AI-2），一种信号分子，被称为“AI-2发件人。”其他人口，被称为“AI-2接收机，是一个AI-2的记者收到来自该发件人的扩散AI-2后，它表达红色荧光蛋白DsRed的。 （二）左边的电极和电极与发件人人口的AI-2对红色荧光图像共沉积与海藻酸钠海藻酸钠共沉积在右边电极AI-2接收机的人口。放大视图显示只有AI-2接收机DsRed的表达。 <img alt="图3" src="/files/ftp_upload/4231/4231fig3.jpg" /> 图3壳聚糖电。 （a）计划显示壳聚糖的pH依赖型电。在阴极偏置电极的电解水导致局部的高pH值（本地化的显微阴极附近pH值指示剂的颜色变化），刺激本地区的溶胶 - 凝胶壳聚糖过渡。 （乙）胺壳聚糖提出的给我牛逼的pH响应性能。胺，pH值高于6.3（PKA）的壳聚糖去质子化，便利从质子的可溶性形式，其不溶性的凝胶形式的过渡。 （三）有图案的金电极用于电沉积壳聚糖。电极，阴极连接电源，浸入到荧光标记壳聚糖（0.8％）沉积解决方案绿色。经过2分钟的电。在电流密度为4 A / M 2，的electroaddressed壳聚糖膜荧光显微镜成像。 <img alt="图4" src="/files/ftp_upload/4231/4231fig4.jpg" /> 图4。与官能的壳聚糖膜的电化学传导。 （一）示意图显示的三电极系统设置。官能壳聚糖膜作为工作电极，铂丝为对电极和Ag / AgCl电极为参比电极。电化学传导葡萄糖收益通过日Ë酶和电化学反应产生过氧化氢可以被氧化和在工作电极检测显示。 （二）（CV）与壳聚糖含有电化学结合葡萄糖氧化酶（GOx）膜电极循环voltammagram一个5毫米的葡萄糖溶液中显示了强大的阳极信号。不含GOX A片作为对照，并没有显示任何信号在同一个解决方案。 （C）之间的阳极电流和血糖浓度的标准曲线显示附近的线性关系（每个等份增加血糖浓度由4毫米，也增加了电流幅度在分步进行的方式嵌入图）。 <img alt="图5" src="/files/ftp_upload/4231/4231fig5.jpg" /> 图5。蛋白质官能使用酶的组装。 （一，上）计划酪氨酸标签“的AI-2合酶”共价拴到壳聚糖膜酪氨酸酶的组装。酪氨酸残基成为OXIDized 为 O-醌酪氨酸酶的行动和反应可能与胺壳聚糖膜组，形成了共价键。 （，低），AI-2合酶产生AI-2从基板（SAH）;接收细胞的报告生成的AI-2 DsRed的荧光表达。 （二）黄金壳聚糖膜的荧光图像显示，用蓝色标记的AI-2合成酶功能。 AI-2接收机细胞相邻，共沉积在ITO海藻酸钠。除了酶解底物以及孵化后，AI-2接收细胞表达DsRed的。

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生产和自由地进入这篇文章是由美国国防威胁降低局主办。

我们的程序，证明了生物高分子薄膜电和功能化，一个过程，我们长期biofabrication。通过与细胞和生物分子的功能化，我们创造能力的生物表面相互作用彼此和他们正在组装后，电极地址​​。第一步，电，通过生物聚合物，海藻酸钠和壳聚糖在我们的研究引发了自组装的地方，在响应的电信号。如前所述产生pH梯度可以通过控制电流密度和沉积时间，在电影的尺寸和性能6,17提供额外的控制。我们发现，各种电流密度和沉积时间的组合可以用于表1所示的电极。而其他电极的使用是可行的，调整的过程是必要的。与其他成膜技术的electrodeposi过程TION是简单，快速和无试剂。有没有需要昂贵的设备和艰苦的准备工作，广泛剧目。重要的是，这个过程可以承受轻微的实验偏差，并可以很容易地开始，如果发生问题。 壳聚糖是能够应对高阴极pH梯度，由于它赋予了伯胺含量高的重要的功能特性。在高pH值（大于它的pKa〜6.3）胺是去质子化和壳聚糖变成不溶性，使膜的形成。薄膜沉积之后，将继续附着在电极。然而，能力存在分层他们，如果需要的话。只要溶液的pH值不低于的pKa的电影将保持稳定。酸性溶液中质子化胺和随后的静电排斥力膨胀的凝胶，直到它溶解18。也就是说，装配/拆卸过程是可逆的需求和异体WS去除薄膜和电极的重用。方便，发生在溶胶 - 凝胶转变的pH值范围是接近，在大多数生物元件的功能优化。这使得6装配过程中保留功能的过程中理想。 海藻酸钠膜的形成，促进了阳极电解水，以及存在的碳酸钙7。本地化的低pH值在阳极溶解导致释放钙离子的碳酸钙。这些离子螯合海藻酸钠，在电极表面形成交联网络。海藻酸钠薄膜是由其他化合物，如柠檬酸或EDTA螯合，它可以用来解散电影让底层电极的再利用，钙离子的竞争尤其是可逆的。因此，海藻酸钠薄膜时，受到生理条件相对脆弱，因为钙离子容易小号cavenged从凝胶基质，削弱了其结构和促进电影分层或再溶解。为了克服这种局限性，我们已在1 M 2氯化钙电影的孵化步骤，以加强凝胶。此外，我们建议，在500微米3毫米的浓度氯化钙补充电影的孵化液（手机媒体等）。 第二个主要过程是有关的生物元件的沉积薄膜的功能化。这样就可以实现在两个方面，首先是电化学共轭，策略，可以快速，无试剂的蛋白质组装与特殊空间控制10。然而，这种方式的官能是有限的氯离子扩散-离子通过膜电极以及次氯酸扩散，所产生的活性中间体，背部成溶液。通过电化学活性分子的能力通过电影，让化学和生物信号转成容易阅读的电气信号15。我们已经表明作为第二个战略壳聚糖酶官能酪氨酸酶介导的耦合，通过共价连接AI-2合酶证明。这一策略使功能化进程的控制和选择性的依赖-在一个特定的试剂，酪氨酸酶，它的行为红皂白地对蛋白质含有酪氨酸标签9。 我们展示的多址系统的实用性和生物相容性，在一个芯片上复制自然途径。首先，我们举办了两个细胞群（即“发件人”和“接收”）在不同的地址，并显示他们在相邻电极相互作用提供AI-2和产生荧光反应。这个概念也已经证明，郑等人 。在微芯片14。我们也模仿的互动，而是使用一种酶的合成AI-2交付。以这种方式，一种人工合成的细胞内途径，AI-2的合成，复制通过biofabrication和运作，因为它会在溶液中。 在这两种情况下，多个地址大会提出了挑战，因为每个沉积解决方案必须引入到整个电极​​阵列，电即使只打算在一个地址，避免地址之间的非特异性结合。温和而彻底清洗，可去除大部分残余的解决方案从非偏电极;使用微通道的流动可能进一步减少非特异性。特别是对相邻biofabrication壳聚糖和海藻酸钠地址，我们建议先沉积的壳聚糖膜，随着biofunctionalization步骤，并在此之后，电海藻酸钠。虽然我们这里没有这样做，我们发现，阻断与惰性蛋白（如MIL壳聚糖膜K，牛血清白蛋白等），大大减少不必要的分子壳聚糖的胺基表面的非特异性结合。 我们已建立的细胞和生物分子的复杂安排的“蓝图”为图案的电极，在微机电设备往往发现，实用。用途电微机电设备中的壳聚糖可以远远超出的例子，这里提到的19。壳聚糖可存放各种微观几何形状-如在微和非平面表面20,15。薄膜也可与其他聚合物和各种蛋白质的DNA，纳米粒子，氧化还原活性分子和新的性能21,22,23修改。在微机电设备，壳聚糖膜已用于药物传递，氧化还原和小分子检测，生物催化，及细胞的研究20,23,24,25。同样，海藻酸钠被广泛用来作为细胞包埋基质，并已探索可逆流体遏制细胞群，并在电影免疫分析26,27,28。组织工程的应用复合薄膜制备使用海藻酸钠电，如羟基磷灰石骨科植入物29组件。 在示威我们biofabrication的，我们已经证明生物成分之间的相互作用和整个生物电子界面同样适用，这带来达到整合所有品种的相互作用，在复杂的片上信号传输性能的前景。因此，biofabrication的，可能有助于减少“最小特征尺寸”作为设备制造直接后续微细加工的迅速发展，往往由消费类电子产品的动机。也就是说，未来的新一代设备，实际上可能包括不稳定的生物提供自然的精湛的组装和识别能力，甚至更小的长度SCA组件莱比人造系统。我们设想在短期分析仪器，环境传感器，甚至生物相容性植入设备的应用。

<table border="1"><tbody><tr><td> 组件的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>电源供应器</td><td>吉时利</td><td>源2400 </td></tr><tr><td>三电极电位</td><td> CH仪器</td><td>电位/恒电流600D </td></tr><tr><td> RE-5B银/氯化银参考电极与柔性接头</td><td>巴斯</td><td> MF-2052 </td></tr><tr><td>镀金硅片，硅500um，12NM铬，区的120nm，二氧化硅绝缘</td><td>自定义制作</td><td></td></tr><tr><td>铟锡氧化物镀膜玻璃幻灯片，长方形，8-12欧姆抗性</td><td> Sigma-Aldrich公司</td><td> 578274 </td></tr><tr><td>白金片/铝箔（0.002） </td><td> surepure Chemetals </td><td> 1897年</td></tr><tr><td>苗条的2号线“鳄鱼夹</td><td> RadioShac​​k公司</td><td> 270-346 </td></tr> &lt;TR&gt; <td>多堆叠香蕉插头跳线</td><td> TSElectronic </td><td>的B-36-02 的B-24-02 </td></tr><tr><td> SYLGARD 184硅酮弹性体套件</td><td>道康宁</td><td> NC9020938 从菲舍尔</td></tr><tr><td> fluorescecence体视显微镜</td><td>奥林巴斯</td><td> MVX10高威</td></tr><tr><td> cellSens标准</td><td>奥林巴斯</td><td>版本1.3 </td></tr></tbody></table> 表1。电泳和荧光可视化设备。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>中等分子量壳聚糖， </td><td> Sigma-Aldrich公司</td><td> 448877 </td></tr><tr><td>盐酸，ARISTAR。 ACS，NF催化裂化级</td><td>厂商VWR </td><td> BDH3030 </td></tr><tr><td>氢氧化钠溶液。 10.00N </td><td>厂商VWR </td><td> VW3247 </td></tr><tr><td>褐藻酸，钠盐</td><td> Sigma-Aldrich公司</td><td> 180947 </td></tr><tr><td> multifex-MM的沉淀 碳酸钙，70nm的颗粒</td><td>专业 矿业公司</td><td> 100-3630-3 </td></tr></tbody></table> 表2。壳聚糖和海藻酸钠溶液试剂。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>氯化钙，二水</td><td>托德·贝克</td><td> 0504 </td></tr><tr><td>氯化钠，认证 ACS的结晶</td><td>菲舍尔 科学的</td><td> S271 </td></tr><tr><td>磷酸二氢钾，无水</td><td> Sigma-Aldrich公司</td><td> P9791 </td></tr><tr><td>磷酸氢二钾，无水</td><td> Sigma-Aldrich公司</td><td> P3786 </td></tr><tr><td>磷酸盐缓冲液</td><td> Σ- 奥尔德里奇</td><td> P4417 </td></tr></tbody></table> 表3。其他解决方案组件和缓冲试剂。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司/源 </td><td> 目录编号 </td></tr><tr><td> 黑曲霉葡萄糖氧化酶</td><td> Sigma-Aldrich公司</td><td> G2133 </td></tr><tr><td>酪氨酸酶蘑菇</td><td> Sigma-Aldrich公司</td><td> T3824 </td></tr><tr><td> LB肉汤，米勒（颗粒状） </td><td>菲舍尔科学</td><td> BP9723-2 </td></tr><tr><td> “AI2的合酶”（HGLPT） </td><td>实验室股票16 </td><td></td></tr><tr><td> W3110野生型细胞</td><td>实验室的股票30日 </td><td></td></tr><tr><td> MDAI2 + pCT6-LSRR - 放大器 ŕ+ PET <eM&gt; DsRed的- 根 细胞 </td><td>实验室的股票30日 </td><td></td></tr><tr><td> FluoroSpheres：直径1微米，EX / EM：505/515 </td><td> Invitrogen公司</td><td> F8765 </td></tr><tr><td> （5 - 和6）carboxyrhodamine 6G琥珀酰亚胺酯，前/ EM：525/560 </td><td> Invitrogen公司</td><td>的C-6157 </td></tr><tr><td> DyLight抗体标记试剂盒，405 </td><td> Thermo Scientific的</td><td> PI-53020 </td></tr></tbody></table> 表4。酶，细胞和其他官能试剂。

bridging the bio-electronic interface with biofabrication

在实验室上的一个芯片技术的承诺，通过降低成本，更好的灵敏度，可携性，和更高的吞吐量革命性的研究和医药的进步。生物组成部分纳入到生物微机电系统（微机电），表现出很大的潜力，为实现这些目标。微型电子芯片允许微米尺度的功能，以及传感和驱动的电气连接。功能性生物成分，使该系统的分析物，酶的功能，全细胞功能的具体检测能力。标准的微细加工工艺和生物分析技术已成功地利用几十年来在计算机和生物产业，分别为。然而，在一个芯片上的实验室环境的组合和接口，带来了新的挑战。有一个技术，可以建立一个电极之间的界面和生物COMPON的呼叫耳鼻喉科，是温和，很容易制作和图案。 biofabrication，这里描述的是这样一个方法，已显示其易于组装在芯片上的功能已启用的多功能生物成分纳入大有希望。 biofabrication采用自下而上分层组装生物材料和生物机制（自组装，组装酶）。虽然我们的实验室已经证明，在许多格式1,2,3这些概念，我们在这里展示装配工艺的基础上由多个应用程序的基于信号的相互作用的电。装配过程中，由生物相容性如DNA，酶，或4,5活细胞的生物元件的电极和随后官能刺激响应聚合物薄膜的电。电需要在有偏见的电极表面电解pH梯度优势水6,7，。壳聚糖和海藻酸钠是刺激敏感触发响应施加的电信号，可以自组装成凝胶薄膜的生物聚合物。这些水凝胶的厚度是决定在何种程度上的pH梯度电极延伸。这可以使用不同的电流密度和沉积时间6,7修改。该协议将描述如何壳聚糖膜沉积和生物成分丰富的伯胺组目前在胶片上，或者通过酶或电化学方法9,10共价连接功能。海藻酸钠薄膜和活细胞中的包封也将讨论11。最后，实用biofabrication，表明通过基于信号的交互的例子，包括化学，电气，细胞到细胞，酶对细胞信号的传输。 既电镀与功能可以接近生理条件下进行，没有需要的试剂，从而免受恶劣条件下的活性生物成分。此外，壳聚糖和海藻酸钠长期被用于生物相关的用途12,13。总的来说，biofabrication，一种快速检测技术，可以简单地在工作台上进行，可用于创建功能的生物元件的电极微米尺度模式，可用于各种实验室在单芯片应用。

Watch this Scientific Journal Video about Bridging the Bio-Electronic Interface with Biofabrication at JoVE.com

Bridging the Bio-Electronic Interface with Biofabrication

本文介绍了biofabrication方法：在存在偏颇创建可与细胞或蛋白质功能的生物相容性薄膜电极刺激响应多糖沉积。我们展示了一代电影以及他们的基本用途，为创建互动实验室在一个芯片应用biofunctionalized表面台式战略。

生物电子界面衔接与Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

bridging-the-bio-electronic-interface-with-biofabrication

Research

JoVE Journal

Bioengineering

16.7K 次观看.  University of Maryland. 本文介绍了biofabrication方法：在存在偏颇创建可与细胞或蛋白质功能的生物相容性薄膜电极刺激响应多糖沉积。我们展示了一代电影以及他们的基本用途，为创建互动实验室在一个芯片应用biofunctionalized表面台式战略。

视频: Bridging the Bio-Electronic Interface with Biofabrication

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

粘附频率含量为原位跨交界的分子相互作用，在细胞与细胞界面的动力学分析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

科研

生物工程

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

对干燥空气-LC接口Megamolecular生物大分子的自我整合方法

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

利用合成生物学工程师活细胞接口，具有可编程材料

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

成年大鼠复合再生外周神经接口（C-RPNI）的制造

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

温度控制组件和液滴接口比莱耶的特征

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

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 ...