这项工作是由联盟的NanoHealth预中心奖（W81XWH-11-2-0168）和得克萨斯州癌症奈米中心（1U54CA151668-01）。

1。芯片制造<ol><li>水解硅酸盐前驱体溶液开始创建芯片的涂层解决方案。混合17毫升乙醇酸乙酯（TEOS）14毫升，6.5毫升去离子水和0.5毫升6M盐酸强烈搅拌下（1200转）使用搅拌热板。这一解决方案，在80°C加热2小时，保持搅拌不变。 </li><li>准备所需的三块coploymer的（聚醚F127，L121和P123）在室温下具有较强的搅拌加入10毫升乙醇的聚合物溶液。完成三嵌段共聚物溶液在室温下强烈搅拌2小时，加入7.5毫升硅酸盐溶液（1.1步）的混合物。这代表了最终的涂层解决方案。 </li><li>适用于1毫升的涂层解决方案，通过旋涂4英寸硅晶片在1500转的速度为20秒。然后加热在80℃12小时。 </li><li>加热膜去除有机苏通过rfactant温度1°C间每分钟提高到425°C，然后烤了5小时。 </li><li>预处理与氧电浆灰化（等离子体舍 - 三月等离子体系统）的介孔二氧化硅（MPS）的芯片表面。 （O 2的流量：80 SCCM，功率：300 W，时间：10分钟）。 </li><li>可选的表面化学改性：在3％甲醇的有机硅烷silanate芯片：DI水（19:1）为72小时，在常温中的N 2手套箱解决方案。甲醇和去离子水冲洗顺序。在110°C风扇操作烤箱15分钟治愈的筹码。 </li></ol> 2。样品前处理<ol><li> TFA和乙腈等，最终浓度是0.01％和5％三氟乙腈每个血清样品。涡旋混合。 </li><li>在室温下表涡摇床摇30分钟，这些样本。 </li></ol> 3。血清分离<ol><li>预焙芯片超过晚上在烤箱在160°C。另外，STORË在干燥器的芯片，直到准备使用由环境空气中的水，以防止表面的水化。 </li><li>使用压缩空气除尘任何可能对芯片表面的颗粒。 </li><li>削减预制CultureWell腔玻璃罩，购买包括所需的油井数量。清洁玻璃罩100％的乙醇，然后MPS芯片表面的地方。按钳玻璃罩带芯片，以确保完全密封。 </li><li>吸取10μL血清样品以及每3毫米。在室温的湿盒孵育30分钟。 </li><li>从井中吸取了血清和丢弃。吸取10μL去离子水，每孔洗去较大的蛋白质。重复4次。 </li><li>吸取5μL洗脱缓冲液（0.1％TFA的50％乙腈），每个样品好。吸取向上和向下的30倍，而在井周围枪头混合洗脱缓冲。 （仅适用于1-2个样品的洗脱缓冲液洗脱缓冲时间，以防止从如此迅速蒸发）。 </li><li>混合后，在离心管中吸取所有的洗脱缓冲液和地点，直到准备执行的MALDI-TOF分析。 </li><li>为了模拟生物样品的复杂性和评估，在收获与我们的芯片分馏系统的低分子量物种的富集效应，我们选择，并装配有一个广阔的计数26个不同种类的蛋白质和多肽的标准混合物分子量（900-66 500大），PIS（4.0-10.2）和浓度（0.5-8 pmol /μL的）范围（见表1中的蛋白质列表）。 </li></ol> 4。肽的MALDI-TOF分析<ol><li>现货0.5μL的样品MALDI靶盘，并晾干。 </li><li>现货0.5μL的矩阵（α-氰基-4 - 羟基肉桂酸（CHCA），5克/升）或饱和溶液中50％的乙腈含0.1％TFA和反式-3,5 - 二甲氧基-4-羟基酸（SA）允许合作的结晶。&lt;/ LI&gt; <li>现货0.5μL，每个校准点的校准解决方案，并晾干。 </li><li> MALDI-TOF质谱仪插入到目标板。本机应设置正反射模式，每个样品拍摄了4200和3000的激光强度。选定的质量范围应该是800到5000与2000年大目标的大规模的大。 </li><li>执行相同的线性模式的MALDI-TOF分析，但改变质量范围达900至10000或3000至70000大和5000大目标的质量。 </li></ol> 5。数据分析<ol><li>原始光谱进行了处理与ConvertPeakList软件出口预处理SpecAlign软件和数据。所有光谱对齐使用的PAFFT相关方法，并在每个相应的频谱正常化的总离子流图（TIC）的强度。所有光谱进行平滑和去4和0.5倍，分别为消噪。 0.5，21大窗口和高度的基线检测峰比1.5，负值分析前被拆除。 </li><li>层次聚类Cluster 3.0和化秉持软件。 MALDI MS数据（M / Z峰强度）数转换，规范化，中位数为中心。 Pearson相关系数是用来计算样本之间的距离，并进行完整的连锁聚类。 </li><li>一个独立的学生t检验用于组（N = 2组）之间的比较，为每个检测到的质谱峰前无监督聚类分析。被认为是显着的差异收获的多肽和蛋白质之间选择不同的介孔蛋白质芯片（大毛孔与毛孔小）一个P值0.02或更低。 </li></ol> 6。代表结果 如图1所示，在这项研究中，我们制作的各种nanotextures一系列介孔二氧化硅薄膜，并全面探讨IR使用选择性的捕获和丰富从人体血清低分子量多肽和蛋白质。 图2a和 b显示未处理的血 ​​清样品的质谱范围在900至10,000道尔顿的肽和蛋白质范围在3000〜7大分别。这些谱说明在低分子量区域的信号抑制由于存在以及电离，非常丰富，超高分子量（高分子量）的蛋白质，如白蛋白。 图2c和 d描绘的血清样品的质谱后，由公安部L121分馏（孔径为6 nm）。大分子大部分已经耗尽，导致中的低分子量成分显着富集。作为对照，同一血清样品被应用到无孔纯硅表面，LMWP回收评估公安部薄膜的特异性。可以看出， 图2e和 f没有PEP的显着收获潮汐或蛋白质从毛细孔二氧化硅。因此可以断定，这是介孔结构，不构成的LMWP富集的占主导地位的因素在硅表面的亲和力。 通过使用共聚物疏水块长度不同，可以实现精确控制孔径的变化。通过设计的蛋白质和多肽的混合物，孔径对低分子量多肽和蛋白质恢复疗效的影响进行了研究，利用MPS准备从四个聚醚表面活性剂（F127，P123，L121，L121加溶胀剂）薄膜具有不同的体积比的亲水性和疏水性成分，形成孔径为3.7纳米，5.2纳米，7.4纳米，9.0纳米。此孔径的范围，导致复苏的肽和蛋白质来自同一血清样本的大小和形状排斥（ 图3）通过不同的剧目。在高的分子重量d蛋白质谱emonstrate每种类型芯片的分子切断。此外高分子量蛋白的大小依赖耗竭，芯片上的标准解决方案的分馏显示一个差与孔径相关的低分子量物种的选择性富集。双向的层次聚类图3b中显示的低分子量标准富集规律与不同的MSC获得。即使所有的肽以下芯片的分子切断，有孔径和被困物种的分子量之间的正相关。毛孔粗大，多达9个纳米，硕士优先收获更大的多肽，而较小的肽毛孔变小的芯片，更有效地恢复。通过调整模板聚合物的浓度进行了结构转型的孔安排。模板聚合物浓度的增加导致减少各相之间的界面曲率水，共聚物，硅酸盐，从而启动相互关联的进展，从一个球形，圆柱形结构。聚醚F127的，其分子量高，具有这种结构的周期性的高度。 F127的浓度增加，在开始的解决方案，不同的MPS薄膜周期性纳米结构可以得到3D纳米二维的纳米结构。三维立体蜂窝状六角形纳米结构，具有较理想的纳米孔的互联互通和更方便的纳米孔形态，表现出优越的性能，选择性比二维六角结构丰富的低分子肽，即使他们有着类似的孔径分布和血清相同的分子切断分馏（ 图4）。我们还精简了公安部芯片的共轭有机硅烷通过引入氧电浆灰化预处理芯片表面。以定性研究上选择的静电作用IVE芯片上富集，我们使用的蛋白质和多肽的混合物。在图5的质谱分析蛋白质的标准解决方案L121准备与总薪级芯片和分馏，化学官能团的共轭。带正电和带负电荷的肽和低分子量蛋白质阴离子和阳离子芯片上分别抓获。 图5a显示多个公安部芯片的恢复具有积极的净电荷的肽的定量比较。没有任何修改（最初有轻微的负电荷）的带负电荷的芯片和芯片表现出显着较高的富集比，APTES（-NH 2）修改了芯片的肽。相反，带正电的公安部芯片拥有特殊的恢复能力与负净电荷的肽，在图5b所示。 α-内啡肽，而并没有表现出显着的变化ðUE到其周围6有价证券。 <img alt="图1" src="/files/ftp_upload/3876/3876fig1.jpg" /> 图1。公安部芯片分馏和LMW富集的原则。后样品表面上发现，低分子量蛋白质和多肽被困进入毛孔较大的品种，而保持毛孔外，并在洗涤步骤中删除。丰富的分数，然后洗脱，并用MALDI分析。 <img alt="图2" src="/files/ftp_upload/3876/3876fig2.jpg" /> 图2。肽富集使用的介孔二氧化硅薄膜芯片。在低质量范围（900至10 000大）和高的质量范围（3000至70 000大）前（A，B）（C，D）的介孔二氧化硅薄膜的血清处理（后电离质谱型材L121，为6 nm）。使用空白无孔硅表面（E，F）时，分子复苏显着降低。 <img alt="图3" src=“/ files/ftp_upload/3876/3876fig3.jpg”/&gt; 图3。分子切断和大小依赖的MPS芯片的富集。 （一）放大视图展示的特征分子切断关联孔径每个公安部芯片电离光谱。 （二）的肽组合功能不同的芯片之间的双向分层聚类。红色或黄色的强度表示相对肽浓度。毛孔变大提高了收获更大的多肽（从3600到8500大），而小肽（从900到3500大）优先恢复从毛孔变小的芯片。 <img alt="图4" src="/files/ftp_upload/3876/3876fig4.jpg" /> 图4。公安部薄膜和不同的纳米结构选择性恢复的物理表征。 XRD图（A，B，C），透射电镜（插图A，B，C），聚醚F127，在不同的前驱体溶液浓度：4.0×10 -3 M（A），6.0×10 -3米（B），8.0×10 -2 M（C）。 （四）说明检测三维立体三维六角形F127的蛋白质芯片（幼童和六角，分别）的选择性肽恢复强度的条形图。不同结构的修改提出了选择性富集。 <a href="http://www.jove.com/files/ftp_upload/3876/3876fig5_lg.jpg"><img alt="图5" src="/files/ftp_upload/3876/3876fig5.jpg" /></a> 图5。负责具体的芯片具有不同的表面功能的恢复。选择性地捕获的官能芯片上的肽检测的MS强度的条形图。根据他们的ISO-电点，肽是积极或消极的被控在pH值7.0。 （一）（正肽（1）DES-ARG1缓激肽，（2）缓激肽，（3）P物质酰胺，（4）（5）神经降压素，促肾上腺皮质激素（1-17），（6）促肾上腺皮质激素（7 - 38））上的负电荷的表面特别丰富面孔。 （二）（7）负肽（Glu1-乙纤维蛋白，α-内啡肽，（9）（8）促肾上腺皮质激素（18-39），（10）胰岛素，（11）（12）表皮生长因子，GFII胰岛素样）带正电的表面上特别丰富。 <a href="http://www.jove.com/files/ftp_upload/3876/3876fig6_lg.jpg"><img alt="图6" src="/files/ftp_upload/3876/3876fig6.jpg" /></a> 图6。芯片分馏血清稳定。 （一）代表的MALDI型材低分子量多肽和蛋白质的洗脱后立即血清分馏（顶部），或在室温下（底）片上存储的后3周。 （二）从上到下，每个重复检测的质谱峰平均强度的线性回归分析相比，复制新鲜分馏的血清和分馏血清后3wk公安部芯片储存在室温下的1。方程，CV和决定系数（R）表示。

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我们什么都没有透露。

越来越多的证据，低分子量的循环系统蛋白质的地区是为疾病的早期检测，诊断标志物的丰富来源。在这种技术中，我们提出了一系列介孔硅芯片与不同孔径，孔选择性丰富的多肽和低分子量蛋白的结构和修改。来评价蛋白的稳定性，MPS芯片孵育，洗涤后干燥，人血清，储存在室温下3wk。获得的蛋白/多肽模式与那些新鲜分割血清（ 图6a），由图6b显示的统计分析的结果证实，可比。估计在原油血清的12.7％和14.2％为分割样本的平均CV测得的峰值信号的变化。边际变化可能是由于电离仪器的内部变异和建议的ON-芯片预处理和存储，并没有引起任何重大改变的MS蛋白质图谱。非多孔硅上已进行相同的实验。干血清储存后恢复硅表面上是分割前的MALDI分析的MPS芯片。穷人的质谱分析得到证实孔表面的稳定优势。在类比与先前推测的机制，我们推测，被困的孔洞内的低分子量物种退化通过蛋白酶的大小排斥保留，或在密闭空间内的孔洞，其蛋白活性的立体抑制。 MPS的芯片为基础的方法可能是一个复杂生物流体研究低分子量的多肽和蛋白质分析的强大工具。 MPS的芯片制造成本低廉，并允许扩大生产，以达到同时处理大量的样品，提供有利的特点，探索筛选和生物标记发现。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>酸乙酯</td><td> Sigma-Aldrich公司</td><td> 131903 </td><td> 98％ </td></tr><tr><td>旋转涂布机</td><td>布鲁尔科学</td><td>中欧和东欧200X </td><td></td></tr><tr><td>等离子体灰粉</td><td>诺信三月</td><td> AP-600 </td><td></td></tr><tr><td>光谱椭偏仪</td><td> JA Woollam有限公司</td><td>的M-2000DI </td><td></td></tr><tr><td>的MALDI-TOF </td><td>应用生物系统公司</td><td>航海家-DE-STR基因座</td><td></td></tr><tr><td> α-氰基-4 - 羟基肉桂酸</td><td> Aldrich公司</td><td> C8982 </td><td>矩阵的MALDI-TOF </td></tr><tr><td>反式-3,5 - 二甲氧基-4 - 羟基肉桂酸</td><td> Aldrich公司</td><td> 85429 </td><td>矩阵的MALDI-TOF </td></tr><tr><td>搅拌热板</td><td> Thermo Scientific的</td><td> 11-475-30Q </td><td></td></tr><tr><td> CultureWell腔玻璃罩</td><td> Sigma-Aldrich公司</td><td> GBL103350 </td><td> 3毫米直径。 ×1 mm深度3-10μL，无菌</td></tr><tr><td>聚醚F 127 </td><td>巴斯夫</td><td></td><td> PEO的106-70的PPO-PEO的106 </td></tr><tr><td>聚醚L121 </td><td>巴斯夫</td><td></td><td> PEO的5-70的PPO-PEO的5 </td></tr><tr><td>聚醚，P123 </td><td>巴斯夫</td><td></td><td> PEO的20 - 70的PPO-PEO的20 </td></tr></tbody></table> 表1。物理化学性质和设计浓度（分子量和Iso电点）选定的多肽和蛋白质的标准。

low molecular weight protein enrichment on mesoporous silica thin films for biomarker discovery

循环标志物的识别持有的非侵入性方法，早期诊断和预后，以及监测治疗效率的潜力巨大。1-3循环的低分子量蛋白质（LMWP）从组织和细胞中棚或小的蛋白质组成从较大的蛋白质水解降解的多肽片段，一直伴随着患者的病理状态，并可能反映了疾病的状态。尽管存在这些潜在的临床应用4,5，使用质谱（MS）分析从LMWP生物体液中已被证明是非常具有挑战性，由于血清中的蛋白质和肽浓度大的动态范围。没有样品前处理6，一些不起眼的高度较丰富的蛋白质检测血清/血浆中低丰度物种。目前蛋白质组学为基础的方法，如二维聚丙烯酰胺凝胶-ELectrophoresis（2D-PAGE）和鸟枪蛋白质组学的方法是劳动密集型，低吞吐量和临床应用提供有限的适用性。7-9因此，需要一个更有效的策略LMWP从血液中分离，并允许临床样品的高通量筛选。 在这里，我们提出一个快速，高效，可靠的介孔氧化硅芯片为基础的多分馏系统专门针对和丰富LMWP。10,11孔二氧化硅（MPS）与可调功能在纳米薄膜制备嵌段共聚物模板途径。使用不同的聚合物模板和聚合物浓度的前驱体溶液中，各种孔径分布，孔隙结构，连通性和表面特性的测定和应用低质量的蛋白质的选择性恢复。丰富肽选择性解析成不同的子类根据其理化性质将昂斯恢复和检测低丰度物种的效率。在质谱和统计分析相结合，我们表现出的介孔二氧化硅薄膜的纳米特性和低质量的蛋白质收获的特异性和疗效之间的相关性。此处给出的结果揭示潜在的纳米技术为基础的技术，提供一个强大的替代LMWP收获的传统方法，从复杂生物流体。因为能力来调整材料的性能，低成本生产的能力，样品收集，并进行分析，大大降低了样品的要求简单和迅速，这种新型的纳米技术将大大影响蛋白质生物标志物研究和临床蛋白质组学领域评估。

Watch this Scientific Journal Video about Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery at JoVE.com

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

我们从人体血清低分子量蛋白质和多肽的选择性恢复对介孔二氧化硅薄膜为基础的技术开发。我们的孔芯片的物理化学性能进行了微调，在肽富集提供实质性的控制，因此分析用于诊断目的的血清蛋白质。

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

low-molecular-weight-protein-enrichment-on-mesoporous-silica-thin

Research

JoVE Journal

Bioengineering

13.3K 次观看.  The Methodist Hospital Research Institute. 我们从人体血清低分子量蛋白质和多肽的选择性恢复对介孔二氧化硅薄膜为基础的技术开发。我们的孔芯片的物理化学性能进行了微调，在肽富集提供实质性的控制，因此分析用于诊断目的的血清蛋白质。

视频: Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

开发自定义的中国仓鼠卵巢宿主细胞蛋白检测使用声膜微粒子技术

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

科研

生物工程

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

娇嫩的组织操纵定制的软机器人气动设备手爪基于杆制造

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...