桑迪亚是multiprogram的实验室桑迪亚公司是洛克希德 - 马丁公司，美国能源部下属的国家核安全管理局根据合同DE-AC04-94AL85000经营。作者们承认，资金由美国桑迪亚国家实验室的实验室指导研究和开发（LDRD）计划。

1。微针的制备<ol><li>使用三维造型软件SolidWorks（Dassault Systemes的SA，韦利济，法国），设计一个金字塔形的空心微针阵列（ 图1）。3-5 </li><li>为支持使用魔法的RP 13软件（Materialise的内华达州，比利时鲁汶）微针阵列结构设计。支撑结构，使树脂从设备在制造过程中流失，并提供了一​​个建立基地的微针。一个例子支持结构如图1所示。 </li><li>链接的支持和微针阵列的文件上传到的Perfactory RP软件，格拉德贝克（EnvisionTEC有限责任公司，德国），它控制的制造工艺。在这个软件包中，选择要制作的微针阵列的数量，并确定设备制造板块的位置。 </li><li>校准在紫外线模式运行180兆瓦的Perfactory迅速公关ototyping制造系统，并确认在能源偏差在±2兆瓦。 </li><li>一旦制作完成，从底板的微针阵列，并在异丙醇发展为15分钟。干燥压缩空气阵列，并在室温下固化在Otoflash Postcuring系统，格拉德贝克（EnvisionTEC有限责任公司，德国）50秒的微针，以确保完全聚合。 </li><li>验证通过显微镜微针的制作和验证，每个微针孔是空心的和通畅。完全捏造的微针是在图2所示。 </li></ol> 2。碳糊电极阵列的制备<ol><li>使用60 W型6.75 CO 2的光栅/矢量激光系统（通用激光系统公司，斯科茨代尔，亚利桑那州），以减少漏洞暴露在平坦的软电缆（21039-0249），这是底层单独寻址连接铜线从商业来源（MO法连接器公司，莱尔，白细胞介素）（ 图3（A和B））。放置在正确对齐它们对激光烧蚀板夹具的柔性扁平电缆。使用光栅方法，创建500微米直径腔软电缆的绝缘部分。在CorelDraw中创建消融模式（Corel公司，渥太华，安大略省）和发送的激光系统。 </li><li>在40 psi清洁喷枪，喷雾丙酮改性的柔性扁平电缆。完成清洁异丙醇和去离子水冲洗。在显微镜下观察，确认无绝缘膜保持在暴露的铜带。 </li><li>下一步是创建一个碳糊包装控股腔。聚酯薄膜胶带（0.002“厚度对压力敏感的丙烯酸胶粘剂的单面涂层）消融电极条，在消融电极条为导向，并在3000磅2分钟，以确保正确的连接压缩相同的模式。此CA本身，腔直径为750微米。 </li><li>一个额外的一层聚酯薄膜胶带（0.004“对压力敏感的丙烯酸胶粘剂的两面涂层厚度），随后消融在相同的模式为单面胶带和债券的微针阵列的碳糊电极阵列对齐后用。 </li></ol> 3。合成功能的碳浆料和电极腔包装<ol><li>葡萄糖敏感的碳糊是基于以前的配方，并获得10毫克的葡萄糖氧化酶，聚（亚胺）和2.2毫克，直至获得均匀的混合物混合16。这种混合物，碳粉60毫克铑（添加5％负载）。添加40毫克矿物油，随后混合。膏是存放在4°C，直到使用;膏用于配制后一周。 </li><li> pH值敏感的碳糊混合30％（W / W）的矿物油和70％（W / W）石墨带够得到owder。包粘贴到电极腔，如3.4节所述。使10毫米快速蓝RR重氮盐在0.5 M磷酸（4 benzoylamino-2 ,5-dimethoxybenzenediazonium氯半氯化锌盐）17。解决方案，包装糊电极置于这个解决方案的20μL下降30分钟，以自发的化学吸附快速蓝公关重氮盐。用去离子水和存储在缓冲区或去离子水在不使用时冲洗。 </li><li>乳酸敏感碳糊是基于以前的配方，是由碳粉和2.5毫克的乳酸氧化酶的混合2.5毫克铑，超声5分钟和5分钟的涡旋之间交替五旋转18。 </li><li>修改糊包装成准备扁平电缆是通过运用各自在电极腔膏。作为一个镘包ţ使用薄薄的塑料片（如塑料权衡船的边缘）他粘贴表面光滑，直到实现。第二个清洁称重船重复直到过剩粘贴删除。用去离子水冲洗。示意图显示激光消融创造腔，碳糊包装， 如图3和微针集成（第2和第3条所述）。 </li></ol> 4。检测和传感器校准<ol><li>乳酸检测完成在-0.15至五测量传感器的计时响应和记录目前在15秒后在0.1 M磷酸缓冲液（pH值= 7.5）。 图4（一）含有乳酸检测的电化学反应原理。 </li><li>以类似的方式进行血糖检测，通过测量计时在-0.05至五传感器的响应和录制后15秒，在0.1 M磷酸缓冲液（pH值7.0）的电流。 图4（b）包含的电化学反应原理ðetection葡萄糖。 </li><li> pH值监测运行从-0.7 V至0.8 V，100 mV / s的循环伏安扫描和记录的氧化峰电位的地位。检测pH值，氧化还原反应的原理如图5所示。 </li><li>葡萄糖和乳酸传感器的校准曲线可以由各自的分析物的连续增加;计时测量后每个待测除了执行在5.1和5.2节所述。另外，固定潜在的计时测量，可在搅拌下，同时允许在待测电流稳定除了彼此之间有足够的时间（10-100秒）。 </li><li> pH校准曲线可以创建通过一系列已知的pH值，氧化峰电位测量的位置，从5日至8 1.0 pH单位递增，并记录，如5.3节所述的循环伏安。 </li></ol> 5。代表回复sults 获得计时曲线粘贴填充修饰碳微针的静态解决方案（例如，血糖检测或乳酸检测）时，电流会立即根据各自的检测潜在的应用减少。它最终将衰减到一个稳定的状态值。一个有代表性的结果如图6所示;乳酸微针从2毫米增加乳酸和录音得到这个结果。该解决方案必须简单搅拌每个乳酸此外后。 15秒后电流上升后增加乳酸浓度;电流响应可以被用来确定未知溶液中的乳酸浓度。另外，连续监测可以用来搅拌的解决方案（或在流动性的解决方案）为增加血糖浓度（ 图5）的解决方案。再次，在当前的增长后增加ţ他的血糖浓度，可以使用规范的血糖反应到一个未知的解决方案。每穗后，必须有足够的时间允许，以便稳定的解决方案。在0.1 M磷酸缓冲液的pH值敏感的微针的循环伏安曲线显示了四个不同的pH值的解决方案，从5日至8合1的pH值在图6中的单位增量。氧化峰电位随pH值的变化，这种现象被用来作为pH值的指标。 <img alt="图1" src="/files/ftp_upload/4067/4067fig1.jpg" /> 图1。STL文件在SolidWorks（A）和打印屏幕，它显示了支撑结构（二）创建的微针阵列的图像。 <img alt="图2" src="/files/ftp_upload/4067/4067fig2.jpg" /> 图2。扫描电子显微镜的微针阵列（A）和（二）内这个数组的单个微针。 图3。柔性扁平电缆装配示意图。涉及的步骤，包括修改的柔性扁平电缆（A），烧蚀图案界（二），开始消融的聚酯薄膜层，这是充满了碳糊（三），以及添加第二聚酯薄膜烧蚀层和交配微针阵列（D） <a href="http://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/4067/4067fig4.jpg" /> 图4。校准乳酸敏感粘贴在0.1 M磷酸缓冲液（pH = 7.5），15秒的计时，-0.15至五扫描。每个电流对应的乳酸2毫米除了增加。 <img alt="图5" src="/files/ftp_upload/4067/4067fig5.jpg" /> 图5。 </s豪华型&gt;葡萄糖敏感的运行计时扫描，在-0.05 V.减少响应在每个对应的葡萄糖2毫米除了粘贴校准。在校准过程中搅拌0.1 M磷酸缓冲溶液（pH值= 7.0）。在这项研究中使用外部Ag / AgCl电极和Pt参考和反电极。 <img alt="图6" src="/files/ftp_upload/4067/4067fig6.jpg" /> 图6 pH值敏感的碳循环伏安（CV）的粘贴在pH值5-8 1个pH单位的增量（水鸭= 8.0 pH值，绿色= pH值7.0，紫色= pH值6.0，红色= pH值5.0），0.1 M磷酸缓冲。第五CV与Ag / AgCl电极的参考和铂丝对电极用于分析。

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没有利益冲突的声明。

基于这种微针传感器的设计的多个方面被认为是前设备制造。为了使用这种传感器，实时检测传感器的响应时间必须是低的，在这个协议中，每个测试传感器具有响应时间低于十五秒。其选择性体内环境，其中包含可以干扰与电极反应的电化学生物分子内也选择在本协议中使用的浆料。除了粘贴组成，经营潜力的选择，以尽量减少干扰电活性的影响。微针阵列的制造成功，需要选择一个合?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>扁平电缆</td><td>莫仕</td><td> 3302/10SF </td></tr><tr><td> 0.003“侧双面胶带</td><td>聚酯薄膜</td><td></td></tr><tr><td> 0.004“双面胶带</td><td>聚酯薄膜</td><td></td></tr><tr><td>乳酸氧化酶</td><td>西格玛</td><td> L0638 </td></tr><tr><td>葡萄糖氧化酶</td><td>西格玛</td><td> G7141 </td></tr><tr><td>铑碳</td><td>西格玛</td><td> 206164 </td></tr><tr><td>石墨粉末</td><td>西格玛</td><td> 385031000 </td></tr><tr><td>聚（亚胺） </td><td>阿库罗斯福冈</td><td> 178570010 </td></tr><tr><td>矿物油</td><td>西格玛</td><td> M5904 </td></tr><tr><td>葡萄糖</td><td>西格玛&lt;/ TD&gt; <td> G8270 </td></tr><tr><td>乳酸</td><td>西格玛</td><td> L1750 </td></tr><tr><td>快速蓝RR盐</td><td>西格玛</td><td> F0500 </td></tr><tr><td> E-壳牌300 </td><td> EnvisionTEC </td><td></td></tr><tr><td> E-壳牌200 </td><td> EnvisionTEC </td><td></td></tr><tr><td>银/氯化银参比电极</td><td>巴斯</td><td> MF-2052 </td></tr><tr><td>铂丝</td><td>巴斯</td><td></td></tr><tr><td> PGSTAT12 AutolabPotentiostat </td><td> EcoChemie </td><td></td></tr><tr><td> perfactory反相</td><td> EnvisionTEC </td><td></td></tr><tr><td> ottoflash Postcuring系统</td><td> EnvisionTEC </td><td></td></tr><tr><td>磷酸</td><td>费舍尔</td><td> A366-4 </td></tr><tr><td> 60W型号6.75 CO 2的光栅/矢量激光系统</td><td> UNIVERSAL激光系统</td><td> PLS6.75 </td></tr><tr><td> CorelDraw中</td><td> Corel公司</td><td></td></tr><tr><td> Solidworks的</td><td> Dassault Systemes的</td><td> 2009 </td></tr><tr><td>魔法RP13 </td><td> Materialise的</td><td></td></tr></tbody></table>

hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

一种微创复为有关生物分子进行快速分析监测系统的发展，可以提供个人患有慢性疾病的简便评估其直接的生理状态。此外，它可以作为一个复杂的，多因素的医疗条件的分析研究工具。为了实现这样一个多分析传感器，它必须是微创，组织间液的采样必须出现无疼痛或伤害用户，并分析必须迅速以及选择性。 最初发达，无痛苦的给药微针已被用于提供疫苗和药物制剂（如胰岛素）通过皮肤1-2由于这些设备访问的间隙，用微电极集成的微针可以作为透皮电化学传感器。选择性检测葡萄糖，谷氨酸，乳酸，Hydrogen过氧化氢 ​​和抗坏血酸已被证明使用碳纤维，修饰碳糊，铂金涂层的聚合物微针作为换能元件的集成微针电极的装置。3-7,8 这个微针的传感器技术，使一个新的和先进的分析方法，在原位，同时检测多种分析物。复用提供了复杂的微环境监测，否则很难在一个快速，微创的方式来描述的可能性。例如，这项技术可以被利用外同步监测，血糖，乳酸和pH值，9是重要的代谢疾病状态7,10-14指标（如癌症扩散）和运动诱发性酸中毒15。

Watch this Scientific Journal Video about Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing at JoVE.com

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

本文详细介绍了建设一个复用微针为基础的传感器。该设备正在研制多种分析快速和选择性地在现场采样和电化学分析。我们设想，临床医学和生物医学研究，这些微针为基础的传感器使用。

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 ...

hollow-microneedle-based-sensor-for-multiplexed-transdermal

Research

JoVE Journal

Bioengineering

14.2K 次观看.  University of North Carolina and North Carolina State University. 本文详细介绍了建设一个复用微针为基础的传感器。该设备正在研制多种分析快速和选择性地在现场采样和电化学分析。我们设想，临床医学和生物医学研究，这些微针为基础的传感器使用。

视频: Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

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, ...

科研

生物工程

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

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.

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 ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

阻隔组织破坏与有机电化学晶体管传感

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

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 ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

中空纤维生物反应器的<em&gt;在体内</em&gt;般的哺乳动物组织培养

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

基于植物的提取胶囊微胶囊化应用

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...