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1。基材的准备<ol><li>涂层层状硅SiO 2的基板与自我吸附的共聚物（DMA的NAS地图） : <ol><li>共聚物的合成，化学结构和镀膜工艺，发表在:G. Pirri E. Bontempi，楼大明，布加，乐Depero。吸附DNA微阵列玻片聚合物涂料的表征。肛门。 CHEM。 2004年，76，1352年至1358年。 </li><li>简单地说，准备加入去离子（DI）水5毫升100毫克聚合物聚合物溶液，并添加5毫升40％饱和硫酸铵（（NH 4）2SO 4）解决方案的终浓度达到0.92米</li><li>淹没在摇床30分钟的解决方案，然后芯片离子水彻底冲洗。氩/ N 2气体彻底干燥。 </li><li>烘烤芯片在80 ° C为15分钟。 </li></ol></li><li>商店聚合物涂层基板/芯片在干燥的环境（真空干燥器）中长达3个月，直到探针斑点程序。 </li></ol> 2。制备探针阵列:抗体，抗原，SS /双链DNA和RNA等。 <ol><li>稀释探头（S），以适当的浓度所需的缓冲区。这一步可以有很大的不同，但一个典型的实验中利用在磷酸盐（0.5毫克/毫升的浓度范围为0.1 -1毫克/毫升）的抗原或抗体在pH≈7.4缓冲液（PBS）。 </li><li>将解决方案，在96 - 或384孔板（或标准源板的去污剂的正在使用） </li><li>安装程序发现所需的印刷阵列的参数:表面和正在使用的解决方案，确定相应的斑点参数（停留时间，接近速度等）。确定每人电网，电网布局，复制电网，以及所需的斑点在芯片上的位置的条件复制的数量。将基板和源盘在适当的地点，进行检查，以确保废物和供应瓶准备，并开始印刷运行。 </li><li>完成后斑点在高湿度环境中放置基板一夜之间（4-18小时），允许固定和失活过程进行。 </li><li>洗净基板:三个独立的清洗三分钟以下解决方案:PBS 0.1％吐温（PBST），PBS，终于去离子（DI）水。根据不同类型的实验（湿与干），干燥彻底芯片与氩气，并开始孵化，或放置在流室芯片和组装。 </li><li>停用共聚物表面剩余的NHS的群体，淹没50毫米，30分钟调整至7.4，而在摇床板的pH值在乙醇胺解决方案的芯片。一个主要的含胺化合物，如羟胺或三也可以使用，只要准备的解决方案是安全的使用与蛋白质。用PBS然后去离子水彻底冲洗停用的解决方案。 </li><li>可选步骤（取决于被雇用的表面化学）:块面使用，酪蛋白，牛血清白蛋白的解决方案或水化30分钟牛奶。对于我们使用的聚合物表面化学，我们不执行此步骤，为聚合物骨架行为，以防止非特异性蛋白结合。 </li></ol> 3。数据采集​​和孵化过程:终结点格式<ol><li>做一个解决方案的目标所需的缓冲区的兴趣。在这里，将适当浓度的抗BSA在PBS（例如:1-10毫升10毫微克/毫升抗BSA在PBS溶液）的解决方案。此外，还要确保有一个同等数额的缓冲区（没有目标）为阴性对照孵化。 </li><li>以一个正常化随后收集到的所有数据的硅镜扫描。 </li><li> IRIS系统的准备探针阵列扫描前的孵化步骤，以确定在每一个点（质量）的初始光高度。这将允许在表面上质量的变化进行适当的分析，即具有约束力的水平，孵化后。在这里，将调整为目前的实验，如:曝光时间，视野，繁荣重点等几个参数</li><li>浸没芯片（S），在摇床板1小时准备的解决方案。躁动水平有很大的不同，潜伏期的时间可以有所不同，目标浓度被检测到，流室的传输特性（如果正在使用一个实时检测模式），目标探针对等的亲和力， 。 </li><li>孵化后，重复步骤2.5中所描述的洗涤程序） </li><li>扫描相同的探针阵列，使用"综合注册资讯系统"，并获得预孵化比较孵化后的图像。 </li><li>重复这个过程中，如有必要，不同的孵化步骤，例如在一个正在使用其中一个次要的抗体夹心法格式。 </li></ol> 4。数据分析<ol><li>适合每个虹膜扫描（强度图像序列）收集到的图像，使用该实验室开发的软件亲杜奇探头阵列，包含光学厚度信息的图像。减去（注册）后和预孵化的图像都可以通过一个有约束力的快速定性分析。绑定将出现在那些质量变化观察点的强度变化。进行定量分析，确定每一个点的质量密度相比，平均为每个像素的光学厚度信息，并当场在现货和环外一个直接比较，这两个领域的背景。 </li></ol> 5。代表性的成果: 图1显示了层状硅SiO 2的虹膜具有代表性的抗体阵列发现基板的例子原理。每个抗体合奏发现的不同，针对不同的蛋白质抗原表位的特异性复制。代表了两种不同的全病毒和病毒蛋白为例目标。阴性对照抗体依赖于检测，并可以非特异性和/或病毒特异性。 图2显示了人血清白蛋白（HSA）的结合特定抗体的发现HSA和兔IgG（对照组）点的数组。在这里看到，经过装修和光学测定厚度为前和孵化后的图像，图像可以创建一个绑定数组的区别，定性评估约束力。定量分析表明，一个2.05和0.13纳米意味着光高度变化的HSA和兔IgG点观察，分别为500毫微克/毫升抗血清白蛋白孵育浓度，。 图3显示了IRIS毛皮蛋白约束力的依赖，对蛋白质的浓度和dsDNA低聚物长度测量。顶部的图形显示绝对光高度测量，初步固定的低聚物探头高度和孵化后的毛皮约束力。毛皮浓度的增加（50，100，200纳米）增加DNA探针的结合。低聚物的长度也影响毛皮较长，更有约束力的结果序列的结合。在这里，误差棒代表平均值的标准偏差。下图显示计算毛皮每双链DNA链结合蛋白二聚体。二聚体约束力显着增加，表明非特异性结合的高层次的200纳米的毛皮蛋白质浓度。 <img src="/files/ftp_upload/2694/2694fig1.jpg" alt="图1"/> 图1:通常在实验中检测特定的病毒和病毒成分与"综合注册资讯系统"的复时尚使用的一个例子探头阵列的原理图。 <img src="/files/ftp_upload/2694/2694fig2.jpg" alt="图2"/> 图2。孵化后发现人血清白蛋白人血清白蛋白特异性抗体结合的差图像与此标签系统收集了500毫微克/毫升的浓度。生物材料的质量密度为每一个点是由平均光学厚度在现场信息，然后比较平均的环围绕当场背景。 <img src="/files/ftp_upload/2694/2694fig3.jpg" alt="图3"/> 图3。剧情发现双链与共识的基础上的低聚物长度，低聚物的共识序列位置内，和毛皮蛋白浓度已知序列的低聚物的毛皮蛋白质相互作用的数据绑定。绑定到每个发现序列（顶部）的毛皮蛋白绝对量的信息可以被用来估计绑定到每种类型的低聚物（底部）的毛皮二聚体。

<ol>
	<li>&#214;zkumur, E., Needham, J. W., Bergstein, D. A., Gonzalez, R., Cabodi, M., Gershoni, J. M., Goldberg, B. B., &#220;nl&#246;, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+and+dynamic+detection+of+biomolecular+interactions+for+high-throughput+microarray+applications.">Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications.</a> Proceedings of the National Academy of Sciences. 105, 7988-7992 (2008).</li><li>&#214;zkumur, E., Ahn, S., Yalcin, A., Lopez, C. A., Cevik, E., Irani, R. J., DeLisi, C., Chiari, M., &#220;nl&#246;, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+microarray+imaging+for+direct+detection+of+DNA+hybridization+and+single-nucleotide+mismatches.">Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches.</a> Biosensors and Bioelectronics. 25, 1789-1795 (2010).</li><li>&#214;zkumur, E., Lopez, C. A., Yalcin, A., Bergstein, D. A., Connor, J. H., Chiari, M., &#220;nl&#246;, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+reflectance+imaging+for+a+multiplexed,+high-throughput,+label-free,+and+dynamic+biosensing+platform.">Spectral reflectance imaging for a multiplexed, high-throughput, label-free, and dynamic biosensing platform.</a> IEEE Journal of Selected Topics in Quantum Electronics. 16, 635-646 (2010).</li><li>Lopez, C. A., Daaboul, G. G., Vedula, R. S., &#214;zkumur, E., Bergstein, D. A., Geisbert, T. W., Fawcett, H. E., Goldberg, B. B., Connor, J. H., &#220;nl&#246;, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+multiplexed+virus+detection+using+spectral+reflectance+imaging.">Label-free multiplexed virus detection using spectral reflectance imaging.</a> Biosensors and Bioelectronics. , (2011).</li></ol>

IRIS平台是一个简单而快速的系统，用于收集在一个芯片格式的高通量数据绑定。通过不同功能的蛋白质或DNA探针共价固定在表面上，靶抗原/生物标志物的/ etc。可以捕获从溶液中，在免疫分析。测量这些相互作​​用的跨端点或实时格式的IRIS系统的探头条件允许为高度敏感和定量的信息收集每个交互。这里详细的代表实验只是一个例子，可以使用这种技术研究相互作用的类型。转录因子，病毒抗原，和整个病毒的检测已经证明，以前只是一个代表的类型分析，光圈可以轻松地处理的几个例子。

<ol><li>与500纳米热生长的二氧化硅，硅谷的微电子，客户订单的硅晶片<li>硫酸铵粉末，Sigma - Aldrich公司的猫。 ＃A5132 <li>磷酸盐缓冲液（PBS）10X准备集中，Fisher Scientific这猫。 ＃BP665 - 1 <li>抗体人血清白蛋白（抗- HAS），Sigma - Aldrich公司的猫。 ＃A0433 <li>人血清白蛋白（HSA），Sigma - Aldrich公司的猫。 ＃A9511 <li>氩气或氮气（超高纯度），公司Airgas猫。 ＃AR UHP300或＃倪UHP300 <li>培养皿中，60毫米× 15毫米，Fisher Scientific这猫。 ＃0875713A <li> Scienceware ®真空干燥器，Sigma - Aldrich公司的猫。 ＃Z119016 <li>离心管中，Fisher Scientific这猫。 ＃05-408-120 <li> 96孔板中，低细胞结合，NALGENE NUNC国际猫。 ＃145399 <li> BioOdyssey书法家MiniArrayer，Bio - Rad公司<li>吐温20，Sigma - Aldrich公司＃P1379 <li>乙醇胺，Sigma - Aldrich公司＃398136 <li>羟胺，Thermo Scientific的猫。 ＃26103 <li>多用途的肩，赛默飞世尔科技（Barnstead国际），型号为＃2314，猫。 ＃2314Q <li> Retiga 2000R相机，QImaging猫。 ＃01 - RET - 2000R - FM - 12 <li> AcuLED照明光源，珀金埃尔默，客户订单<li>光学元件（镜片，目标，光圈，导轨，职位等），Thorlabs </ol>

biomolecular detection employing the interferometric reflectance imaging sensor (iris)

敏感的生物分子相互作用的测量已在许多领域和行业，如基础生物学和微生物学，环境/农业/生物防御的监测，纳米生物技术，以及更多的使用。对于诊断的应用，监测（检测）存在的情况下，或有针对性的蛋白质组或基因组的生物标志物在患者的样本中发现的异常表达，可用于确定治疗方法或治疗的疗效。在研究领域，分子的亲和力和特异性的信息是有用的，为充分刻画调查系统。 许多人依靠，以确定分子的浓度或亲疏当前系统使用的标签。这些系统的例子包括，如酶联免疫吸附试验（ELISA），聚合酶链反应（PCR）技术，凝胶电泳分析，质谱（MS）的免疫。一般来说，这些标签的荧光，放射性，或在性质比色法，都直接或间接利益的分子靶点。虽然使用的标签已被广泛接受，并有一定的好处，也有缺点是刺激开发新的无标签的方法测量这些相互作​​用。这些弊端包括实用方面，如增加检测成本，试剂的寿命和可用性，存储和安全问题，浪费时间和努力，在标签，由于标签的过程或标签本身，不同的试剂之间的变异。在科学调研的基础上，使用这些标签也可以引进，如与蛋白质的功能/结构由于存在所附的标签和无法直接测量实时交互影响的担忧的困难。 这里介绍的是使用一个新的无标签的光学传感器，是适合芯片研究，被称为干涉反射成像传感器"（IRIS），用于检测蛋白质，DNA的抗原物质，整个病原体（病毒体）和其他生物材料。 "综合注册资讯系统"已被证明有不同的生物分子相互作用[1-3]高灵敏度，精密度和重现性。其优点包括多元化的成像能力，实时性和端点测量能力，和其他高通量的属性，如减少试剂的消耗和减少检测时间。此外，"综合注册资讯系统"平台使用简单，需要廉价的设备，并利用基于硅的固相检测元件，使得它与许多现代表面化学方法兼容。 在这里，我们目前的"综合注册资讯系统"的使用从探针阵列的制备孵化和测量目标端点格式的结果分析结合。该模型系统将被捕获抗体人血清白蛋白（HSA）的HSA斑基板上的具体目标。

Watch this Scientific Journal Video about Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS at JoVE.com

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

免费的生物分子检测定量，高通量，实时，标签（DNA，蛋白质等）上的二氧化硅<sub&gt; 2</sub&gt;表面可以实现用一个简单的干涉技术，依靠LED照明，最小的光学元件，和一个摄像头。干涉反射成像传感器（IRIS）是价格低廉，使用简单，并适合芯片格式。

生物分子检测的干涉反射成像传感器（IRIS）

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

biomolecular-detection-employing-interferometric-reflectance-imaging

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

14.6K 次观看.  Boston University. 免费的生物分子检测定量，高通量，实时，标签（DNA，蛋白质等）上的二氧化硅 2表面可以实现用一个简单的干涉技术，依靠LED照明，最小的光学元件，和一个摄像头。干涉反射成像传感器（IRIS）是价格低廉，使用简单，并适合芯片格式。

视频: Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

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

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

设计和弹性股软模块化机器人微创手术制造

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

基于琼脂糖组织模拟光学幽灵的漫反射光谱研究

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

多夏巴氏杆菌自主表达采用除抑制技术

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level. The tension threshold for ITS activation is tunable in the range of 10&#8211;60 pN that well covers the dynamic range of integrin tension in cells. On a substrate grafted with an ITS, the integrin tension of adherent cells is visualized by fluorescence and imaged at submicron resolution. The ITS is also compatible with cell structural imaging in both live cells and fixed cells. The ITS has been successfully applied to the study of platelet contraction and cell migration. This paper details the procedure for the synthesis and application of the ITS in the study of integrin-transmitted cellular force.

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

集成张力传感器在亚微米分辨率下的成像整合和细胞力

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...