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>

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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><tbody><tr><td>SPRi apparatus&amp;nbsp;</td><td>Horiba Scientific-GenOptics</td><td></td><td>SPRi apparatus is placed in a temperature regulated incubator at 25 &amp;deg;C.</td></tr><tr><td>Incubator</td><td>Memmert</td><td></td><td></td></tr><tr><td>Syringe pump&amp;nbsp;</td><td>Cavro scientific instruments</td><td>Cavro XLP 6000</td><td></td></tr><tr><td>Micro Elite Degasser&amp;nbsp;</td><td>Alltech</td><td>AT590507</td><td></td></tr><tr><td>6-port medium pressure injection valve</td><td>Upchurch Scientific</td><td></td><td>The volume of injection loop used is 500 &amp;micro;l.</td></tr><tr><td>Femto plasma cleaner (version 7)</td><td>Diener Electronic</td><td></td><td>On-line degassing system with 2 channel.</td></tr><tr><td>Spotter</td><td>Siliflow</td><td></td><td>It is a non-contact piezoelectric spotter.</td></tr><tr><td>SPRi-Biochip</td><td>Horiba Scientific-GenOptics</td><td>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><td>Erythrina cristagalli lectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L5390</td><td></td></tr><tr><td>Arachis hypogaea lectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L0881</td><td></td></tr><tr><td>Myoglobin</td><td>Sigma-Aldrich</td><td>M1882</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>CXCL12&amp;alpha;</td><td></td><td></td><td>Provided by Dr. Hugues Lortat-Jacob; for preparation details, see supporting information in reference 9</td></tr><tr><td>CXCL12&amp;gamma;&amp;nbsp;</td><td></td><td></td><td>Provided by Dr. Hugues Lortat-Jacob; for preparation details, see supporting information in reference 9</td></tr><tr><td>Lactose</td><td></td><td></td><td>Provided by Prof. David Bonnaff&amp;eacute;; for preparation details, see supporting information in reference 9</td></tr><tr><td>Sulfated lactose</td><td></td><td></td><td>Provided by Prof. David Bonnaff&amp;eacute;; for preparation details, see supporting information in reference 9</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5150</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4509</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>274348</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>S0751</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>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

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

生物化学

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

蛋白质的MS检测快速酶法处理与流通型微反应器

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

生物学

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

<meta charset="utf8"></head><body>使用单吸头中的微珠阵列 （BIST） 进行基于凝集素的聚糖自动化分析

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of &lt;10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

<meta charset="utf8"></head><body>作者聚焦:使用全自动系统推进蛋白质糖基化研究

A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

电子舌生成连续的识别模式的蛋白质分析

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此视频中的章节

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

使用电化学传感器的细菌检测和鉴定

蛋白质的MS检测快速酶法处理与流通型微反应器

数字微流体进行自动蛋白质组学处理

全自动离心微流体装置从全血超灵敏蛋白检测

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

使用全自动系统对重组蛋白进行快速糖质定性评估

使用全自动系统对重组蛋白进行快速糖质定性评估

使用全自动系统对重组蛋白进行快速糖质定性评估