The authors would like to acknowledge Ph.D. grant of LANEF in Grenoble for support of Laurie-Amandine Gar&#231;on. This work was financially supported by the French National Research Agency (ANR-grant 06-NANO-045).

1，准备各种解决方案和蛋白样品<ol><li>制备100毫升含有50mM的NaH 2 PO 4，50 mM氯化钠的磷酸盐缓冲液（PBS-G）和10％甘油在pH为6.8。 </li><li>准备加入250ml含有10mM HEPES，150 mM氯化钠，0.005％吐温20，在pH 7.4的HEPES缓冲液等。 </li><li>制备积木1（BB1），乳糖和积木2（BB2）硫酸化乳糖（ 图1）的储备溶液在0.2 mM的PBS中-G。 </li><li>准备1毫升蛋白质溶液中的HEPES:一个脊柱。花生凝集素（AHL），在500nm时，肌红蛋白在1微米，溶菌酶在500nm左右。 </li><li>在超纯水准备20毫升1％的SDS。 </li></ol> 2，制备CoCRR阵列<ol><li>清洁棱镜48小时在这些条件下用等离子清洗机使用3分钟前的金表面:75％氧，25％的氩气，0.6毫巴，功率40瓦。 </li><li>准备11混交林及纯solutioBB1和BB2与[BB1] /纳秒（[BB1] + [BB2]）由10％的在0.1 mM的PBS中-G共BB浓度增量在0和100％的比率。 </li><li>存款用一式四份的非接触性去污剂对每个比率为44点总数（ 图2）上的棱镜面这些纯与混合溶液8 NL液滴。 注:10％甘油的PBS-G加入是很重要的，以减少溶剂蒸发和BB浓度的沉积后的变化。 </li><li>放置在棱镜含有1ml超纯水中的陪替氏培养皿内，并把它O 2 / N在RT BB1和BB2的金表面的自组装体。这里，假定在该混合自组装膜的平均表面组成反映了沉积的混合溶液的组成（ 图3）11。 </li><li>用超纯水彻底清洗棱镜，然后烘干的氩气流下。 </li></ol> 3，蛋白质感测SPRI <ol><li>设置incubatoR IN其中SPRI装置被放置在25℃，以避免蛋白质感测期间由温度引起的变化的折射率变化。 </li><li>将在10微升聚醚醚酮非官能冲洗棱镜醚酮（PEEK）流动池连接到计算机控制的注射泵，脱气机和6端口中压进样阀（ 图4）。在流动系统中注入新鲜过滤和脱气缓冲液HEPES。 </li><li>除去漂洗棱镜，插入包含CoCRR阵列中的流通池的棱镜。 HEPES运行在100μL/ min的流速。用CCD照相机的帮助下通过使运行缓冲液快速去除存在于棱镜表面的气泡。 </li><li>画一个圆的直径相同的利息基于阵列的良好对比度的图像，并与集成软件在SPRI系统的帮助下，区域定义研究区域的每个点。 </li><li>追踪等离子体曲线，它代表反映ivity曲线中的入射角，对于所有的点的功能。 </li><li>选择的工作角度（位置在该动力学曲线将被记录）的反射率曲线的最大斜率。旋转扫描镜来解决这个选择的工作角度动力学测量。 </li><li>继续通过流动系统运行的HEPES直到反射信号对于所有的点是稳定和恒定。 </li><li>注射1毫升注射器进样环（500微升）与进样阀上的位置"负荷"AHL的解决方案。然后把进样阀定位"注水"。用于所有的蛋白注入流速为100微升/分钟。 </li><li>通过在所有地点同时监测对时间的反射率的变化开始动力学测量。 </li><li>在蛋白注射结束时，冲洗用运行缓冲液为8分钟的阵列。最后，注射1毫升1％的SDS去重新创造它。 </li><li>重复同样的步骤对另一个proteins。 </li></ol> 4，数据处理和分析<ol><li>以下条目的蛋白质溶液进流动池，分子结合时，诱导的等离激元的曲线的移位和反射率的变化。图像采集软件转换所测量的光强度值灰度等级，给SPR图像图5A，并产生反射率与时间的关系的变化，从而传感（ 图5B）。 </li><li>用数学计算软件的每个蛋白质注射兑[BB1]年底绘制的反射率（R％）/（[BB1] + [BB2]）的比值来生成二维不断发展概况（CEP），每个样品（ 图5C）。 </li><li>添加[BB1] /（[BB1] + [BB2]）中的传感比率（ 图5B），以生成三维连续进化景观（CEL）的每个蛋白质（ 图5D）。 </li></ol>

<ol>
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The authors have no conflicts of interest to declare.

<html>专用于结构本等的最重要的步骤，以确保良好的系统的再现性。例如，清洁的棱镜的金表面在使用前用标准程序，在BB1和BB2的11纯与混合溶液中加入甘油10％时在棱镜的金表面黑带的自组装，以消除溶剂蒸发，沉积多次重复为每个[BB1] /（[BB1] + [BB2]）的比例， 等等 。作为用于蛋白检测由SPRI，工作角度的选择是至关重要的。通常，它被固定在反射率曲线的最大斜率。此外，它是使用标准化的实验条件下对每个蛋白检测，如工作温度，流量等非常重要。每种蛋白注射后，CoCRR阵列必须再利用再生用适当的解决方案，允许该系统的完全再生，但无受体的任何损害。 e_content"&gt;为了证明开发等，不仅有效于如先前报道9糖结合蛋白的研究，而且对于普通的蛋白质，1凝集素AHL连同两个非糖结合蛋白肌红蛋白和溶菌酶被使用，它们被选择为具有各种电荷在HEPES（pH 7.4）中:AHL（等电点（pI）6.0），肌红蛋白（等电点7.2）和溶菌酶（pI值11）值得注意的是， 如图6所示，每个蛋白质具有独特的响应模式。AHL稍微负在HEPES充电。及其CEP表明，它具有与带负电荷的BB2富CoCRRs更强的相互作用。最有可能的，这是由于该蛋白的带正电荷的结构域和BB2的富CoCRRs之间的相互作用对于肌红蛋白，它是在HEPES全局中性，不存在与所有CoCRRs获得的信号之间太大的差别。当比较肌红蛋白在CEP到AHL和溶菌酶的CEP证书，甚至在较高的浓度的信号是多路WER。如溶菌酶，这是正在HEPES充电，用含有纯BB2的CoCRR获得的最大信号。 由于SPRI的能够监测实时的吸附和解吸的动力学，对已生成的三维连续的演化风景每种蛋白的优点。这种随时间变化的识别模式的使补充分化参数用于蛋白质分析。特别是，它可以是用于分析两种分析物呈现相似的亲和力为一组CoCRRs的但不同的在它们的相互作用的动力学极其有用的。 如图7所示，的CEL很容易分辨。这些结果表明，三种蛋白质与CoCRRs的相互作用依赖于它们的表面特性，如疏水性，中性和带电的氨基酸残基的分布。既CEP和CEL可以用作"指纹"的蛋白鉴别和prospec略去鉴定。因此，ET是有效的普通蛋白质的分析。 这里所描述的协议使用组合的方式来构造等进行蛋白质分析。不同于使用一些分子，其在结构上是复杂的和费力的，合成的其他方法中，只有两个小分子来制备的交联反应活性的受体阵列能分化蛋白与在本研究中良好的分辨率。另外，根据以前的调查中，ET是稳定的，可重复的和可重复使用的9。此外，SPRI允许监测结合事件的实时和产生新的连续的识别模式具有前所未有的可靠性。在不远的将来，具有互补的物理化学性质的附加黑带将引入以增加CoCRR的多样性中的阵列中的液体或气体的复杂混合物的分析。

精确和快速蛋白检测方法是在医学诊断学和蛋白质组学非常重要。经典的蛋白检测阵列，如生物芯片，是基于"锁和钥匙"的识别原则和需要特定的受体，如适体，抗体，或其模拟物。 近年来，差分检测灵感来自于人类的嗅觉和味觉已经成为一种替代1。这种电子鼻/舌（EN / ET）的方法是基于差分分析物结合到交叉反应的受体（CRR），它们，这并不需要是高度特异性的或选择性的靶标分子的阵列从而允许克服费力开发高选择性受体的过程。这是所有的受体结合反应，创建一个独特的模式为每个样品，就像指纹一样，允许其身份。 在ELEC发展的两个关键挑战tronic的鼻/舌为有效蛋白质检测是生产具有到中间结构上相似的分析物和对于该结合事件的适当的转导系统分辨能力感测元件。到现在为止，研究已经报道的各种方法，以阵列发展2。例如，在一项研究中的蛋白的基于阵列的识别，使用从四carboxyphenylporphyrin衍生物制备CRRs通过偶联的羧基以不同的氨基酸或二肽，以提供差动受体具有疏水核的蛋白质和不同的带电周边的亲和力开发传授鉴别约束力。使用本系统时，不同的蛋白质和蛋白质的混合物进行鉴定相互作用时测量受体的荧光猝灭与待测物3,4。在另一项研究中，29 CRRs含三肽和硼酸部分库的组合合成的方式在hexasubstituted苯支架被用于检测蛋白质与指示剂摄取比色检测5,6研制。通过这样的设计，每个受体显示出与协助从糖蛋白的差异的基础上，在肽武器的变异蛋白质，和硼酸差的结合能力。最近，一种结合有阴离子性荧光聚合物聚（对苯亚乙炔）（PPE）的不同阳离子官能化的金纳米粒子组成的阵列已经创建了用于检测和鉴定蛋白质7。和蛋白质分析物之间的竞争性结合淬火再生荧光PPE /金纳米粒子复合物，从而产生不同的识别模式的蛋白质。在这项研究中，所述官能化的纳米颗粒 - 蛋白相互作用通过改变纳米颗粒的端基的物理化学性质进行调整。此外，研究结果表明，该方法是有效的在复杂和富含蛋白质的培养基，例如蛋白质分析人血清在生理学相关浓度，从而显示出在性能分析实际样品用于诊断疾病状态8等的潜力。 虽然非常有前途的，这些系统有一些固有的局限性。它们需要设计和从5到29 CRRs用相当复杂的结构合成。此外，嗅觉系统，该系统复位下列各测量不同的是，蛋白质检测需要制备每个样品的阵列。最后，监控实时结合事件是极其困难的。 在此背景下，提出了一种组合方法通过使用少量的简单和方便的分子具有不同的物理化学性质的（亲水性，疏水性，带正电，带负电荷，中性， 等等 ）作为构建块（BBS）9。上的金表面通过混合不同黑带和控制的比例，并允许该混合物的自组装棱镜，组合表面为特色的结合蛋白，相应的属性数组创建。值得注意的是，在该系统上的自组装单层允许容易地调谐的范围内的表面性质的在高度发散的方式，可实现多种组合的交叉反应性的受体（CoCRRs）是快速和有效地生产。使用光学检测系统进行蛋白质检测，表面等离子体共振成像（SPRI）。简单地说，从LED的广泛光束单色偏振光照射棱镜表面上，整个CoCRR阵区。高分辨率的CCD摄像机可以穿过CoCRR阵列的所有点的实时差分图像。它捕获所有的局部变化在CoCRR阵列提供有约束力的事件和动力学过程10的详细信息的表面。同时，随着图像处理软件的帮助下，对应点的SPR图像自动转换成反射versu的变化的时候，产生了一系列的运动结合曲线称为传感。因此，SPRI允许无标签，同步，并行和实时观测结合的事件。此外，将获得的CoCRR阵列是可再生的和可重复使用的用于蛋白质分析。 这个协议描述了电子舌的结构通过仅使用两个小分子作为构建块和表示其为基于与SPRI获得连续的识别模式常见的蛋白质分析中的应用。

<table border="0" cellpadding="0" cellspacing="0" width="1511">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:225px;">Catalog Number</td>
			<td style="width:799px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPRi apparatus&#160;</td>
			<td>Horiba Scientific-GenOptics</td>
			<td style="width:225px;"></td>
			<td>SPRi apparatus is placed in a temperature regulated incubator at 25&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Memmert</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump&#160;</td>
			<td>Cavro scientific instruments</td>
			<td style="width:225px;">Cavro XLP 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Micro Elite Degasser&#160;</td>
			<td style="width:203px;">Alltech</td>
			<td style="width:225px;">AT590507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-port medium pressure injection valve</td>
			<td>Upchurch Scientific</td>
			<td style="width:225px;"></td>
			<td>The volume of injection loop used is 500 &#181;l.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Femto plasma cleaner (version 7)</td>
			<td>Diener Electronic</td>
			<td style="width:225px;"></td>
			<td>On-line degassing system with 2 channel.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spotter</td>
			<td style="width:203px;">Siliflow</td>
			<td style="width:225px;"></td>
			<td>&#160;It is a non-contact piezoelectric spotter.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SPRi-Biochip</td>
			<td>Horiba Scientific-GenOptics</td>
			<td align="right" style="width:225px;">36000067</td>
			<td>The prism is made of a high refractive index glass prism coated with a thin gold film (45 nm) and specially developed for imaging purposes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">erythrina cristagalli lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L5390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">arachis hypogaea lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L0881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">myoglobin</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">M1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lysozyme</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#945;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#947;&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sulfated lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">glycerol</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">G5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SDS</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">tween 20</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td align="right" style="width:225px;">274348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">HEPES</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S0751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium chloride</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S3014</td>
			<td></td>
		</tr>
	</tbody>
</table>

electronic tongue generating continuous recognition patterns for protein analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

<html>探测电子舌的常见蛋白质分析的能力，三种蛋白分别采用:AHL，肌红蛋白和溶菌酶。对于每种蛋白，明显的二维连续演化信息，CEP，是由ET产生，如示于图6。 此外，由于SPRI，其能够监测实时的吸附和解吸的动力学，对于每个蛋白质与时间相关的连续的识别模式，称为三维连续进化景观（CEL），被生成的。在图7中，示出了这三种蛋白质的特性的CEL。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig1highres.jpg" width="500" /> 在这个协议中使用的两个积木的图1的化学结构以制备CoCRR阵列。51901 / 51901fig1highres.jpg"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig2highres.jpg" width="500" /> 图2:采用非接触式压电去污剂，在各一式四份比例沉积44滴BB1和BB2的纯洁和混合解决方案，经过光棱镜，棱镜放在旁边的1.5毫升离心管中。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig3highres.jpg" width="500" /> 在金河畔含有制备与BB1和BB2的各种比例的纯与混合的解决方案，11差动受体并使混合物于CoCRR阵列的图3示意图自组装面的棱镜。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig4highres.jpg" width="500" /> 图的SPRI检测系统结合了微流体系统的4示意图。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig5highres.jpg" width="500" /> 2D不断发展概况和3D不断演变的景观:用于生成两种具有发达的电子舌的连续识别模式的数据处理图5示意图。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig6highres.jpg" width="500" /> 图6提供后续三纯的蛋白质进化的配置文件:AHL（500纳米），肌红蛋白（1微米），溶菌酶（500纳米），他们通过在蛋白注射液兑[BB1年底绘制反射率（R％）的变化产生了] /（[BB1] + [BB2]）的比值。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig7highres.jpg" width="500" /> 图7。景观味:由电子舌产生的AHL，肌红蛋白和溶菌酶特点3D的CEL。

Watch this Scientific Journal Video about Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis at JoVE.com

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

电子舌生成连续的识别模式的蛋白质分析

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

electronic-tongue-generating-continuous-recognition-patterns-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.8K 次观看.  Institut Nanosciences et Cryogénie, CEA-Grenoble. A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination. 

视频: Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

不对称受体模式研究细胞滚动轨迹

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

科研

生物工程

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

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 ...

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

在芽殖酵母连续高分辨率显微观察复制性衰老

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

制作定量，皮肤伤口管理一个形皮肤般的电子系统与表征

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

关于纺织品有机电子设备的简单和可扩展的制作方法

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...