制造微流体通道在一个循环的聚烯烃（Zeonex ® 690R，吉恩化工有限公司，路易斯维尔，肯塔基州）。 Zeonex有136℃的玻璃化转变温度（Tg）和紫外线透明的，这是光在巨石形成多孔的用于化学至关重要。微结构微热压印一个尼科电铸主模，它具有负（反）微流体平台的功能。热压印完成如下： <ol><li>模具和聚合物衬底之间放置两个平行的金属压板。 </li><li>该压板加热压花温度（166℃）， ​​这是30 º C以上的Zeonex 的 Tg。 </li><li>的压板，然后同时按下约5分钟，并保持在250磅的压力。 </li><li>模具与基板，然后取出从加热盘，让其冷却1-2分钟，然后手动相互分离。 </li><li> 1.5毫米直径的井钻探样品的引进和收集渠道的两端。 </li><li>压纹塑料基板，然后用另一块的Zeonex热粘合，形成封闭的微流体通道。浮雕基材和覆盖件可紫外臭氧或热粘合前处理，以提高密封效率和确保封闭的渠道结构（巴塔和Klapperich，2007年）的保真度的等离子体。 </li></ol>塑料微流体通道内固相萃取（SPE）柱的制备固相制备是一个两步的过程。起初，编造微表面改性的塑料装置，以提高固相萃取柱的附着力。通过表面聚合接枝是用于功能化的聚合物微通道的墙壁。 嫁接过程如下： <ol><li>微充满了1:1的混合物的环氧丙烯酸酯（EDA）和甲基丙烯酸甲酯（MMA）用3％的二苯甲酮，这是一个抽象的光引发剂的氢。 EDA，甲基丙烯酸甲酯和二苯甲酮均购自Sigma - Aldrich公司，圣路易斯，密苏里州。 </li><li>然后，该芯片是紫外线照射，在254 nm紫外线波长和紫外线照射仪（CL - 1000紫外交联剂，UPV公司，高地，加州）200兆焦耳 /厘米2的能量为10分钟。嫁接发生拴表面的聚合物链，获得的庞然大物在第二步准备使用的聚合混合物中通过的H -抽象的聚合和结果。 </li><li>从通道中删除多余的单体与15μL甲醇使用真空抽吸冲洗。 </li></ol>后接枝过程中，微孔聚合​​物巨石形成的微通道内，坑害二氧化硅粒子对DNA的提取。 多孔的一块准备在原地如下： <ol><li>表面改性的通道都充满了一个庞然大物的易制毒化学布玛（15％WT），EDMA（10％WT），1 - 十二醇（52.5％WT），环己醇（22.5％WT），DMPAP组成的混合物（1％WT与尊重单体）和0.7微米的二氧化硅微粒（尊重的预聚物的混合物总量的15％）。硅胶微球均购自Polysciences，公司（睡眠PA）和Sigma - Aldrich公司，圣路易斯，密苏里州购买的单体和porogenic溶剂。 </li><li>该芯片是与紫外线紫外线交联剂照射在200兆焦耳/厘米2为1.1分引发聚合，从通道中删除多余的单体与15μL甲醇使用真空抽吸冲洗。 </li><li> （NanoPorts TM连接，厄普丘奇科学，橡树港，WA）流体连接，然后附着在微流体通道的引进和集水井。 </li><li>多孔的一块，然后再与甲醇洗涤使用注射泵流量的100mL/hour至少30分钟。 </li></ol>北海裂解一枝独秀制作塑料微流体通道内制备的CNT（碳纳米管）裂解庞然大物是一个两步的过程。首先，制造微表面改性（嫁接），在同样的方式在塑料设备的SPE柱。 后接枝过程中，形成微孔聚合物一枝独秀内的多壁碳纳米管浸渍微。 多孔的一块准备在原地如下： <ol><li>多壁碳纳米管悬浮在环己醇在2.27M的浓度。这再悬浮超声在50％幅度为30分钟和一个sonifier细胞干扰物（50％占空比Sonifier的S - 250丹伯里，必能信超声，CT）的研发，制造的。超声提供了碳纳米管的有效和稳定的0.5M的悬浮液。 Nanolabs（布赖顿，马萨诸塞州）均购自碳纳米管。 </li><li>除了现在的环己醇的解决方案+使用碳纳米管是表面改性通道都充满了类似的巨石前体的混合物。一枝独秀的混合物组成的布玛（15％WT），EDMA（10％WT），1 - 十二醇（52.5％WT），环己醇+碳纳米管（22.5％WT）。此解决方案添加光引发剂DMPAP（1.13％的单体重量）。单体，porogenic溶剂和DMPAPA均购自Sigma - Aldrich公司，圣路易斯，密苏里州。 </li><li>该芯片是在120兆焦耳/ 厘米 2照射紫外线紫外线交联剂为0.9分钟，引发聚合。该设备是180度翻转的crossliker，另一个在120兆焦耳/平方厘米0.9分钟照射。从通道中删除多余的单体与50μL甲醇使用真空抽吸冲洗。 </li><li> （NanoPorts TM连接，厄普丘奇科学，橡树港，WA）流体连接，然后附着在微流体通道的引进和集水井。 </li></ol>北海裂解一枝独秀使用的细菌样品制备一种细菌样品的样品制备涉及到在600nm处的光密度测量，以确保该样本是不是非常集中，以免堵塞通道。 <ol><li>在与媒体的细菌600nm处的OD读数，这是一个分光光度计（Eppendorf公司分光光度计，科学的Eppendorf公司，韦斯特伯里，纽约州）。稀释样品，直到OD值下降的范围内0.18-0.25答： </li><li>在5分钟6500转离心样品，倒出上清液。 </li></ol>裂解<ol><li> GuSCN悬浮细菌* + .01％SDS + 4％，蛋白酶K（0.8mg/ml）。涡简要（媒体细菌1毫升应悬浮1毫升GuSCN）。蛋白酶K被添加到解决方案援助变性，附着细菌的DNA和蛋白质抑制DNAases和RNAases。蛋白酶K的一个额外的好处是，它也打破细胞壁的蛋白质，这是可取的，因为革兰氏阳性菌和革兰氏阴性菌细胞壁中含有大量的蛋白质（虽然革兰氏阴性菌通常有更多的蛋白质艾滋病）。此外，离液剂缓冲区艾滋病的蛋白酶K与变性的蛋白质，而洗涤剂破坏细胞壁。蛋白酶K和胍硫氰酸盐（GuSCN）均购自Qiagen公司（瓦伦西亚，加利福​​尼亚州）。 SDS（​​十二烷基硫酸钠）购自皮尔斯（罗克福德，IL）。 </li><li> PEEK管连接到一个注射器，立即装入样品。使系统在没有空气吸入注射器。连接管nanoport和渠道。 </li></ol>一个KDS100注射泵（Holliston，马KD科学，制造）用于实验和使用的流量为450μL/小时的所有步骤。流450ul/hr样品裂解通道。 <ol><li>收集样品，并继续通过SPE提取DNA（DNA提取协议）。注意：这种细菌样本不会需要遵循载荷步2，自暂停GuSCN *. </li></ol> DNA的提取协议提取过程包括3个步骤： <ol><li>负载。 </li><li>华盛顿州</li><li>流出。 </li></ol> KDS100注射泵（Holliston，马KD科学，制造）用于实验和使用的流量为300μL/小时的所有步骤。 负载<ol><li>条件通道离液剂缓冲区（GuSCN *，硫氰酸胍）在实验开始前1-2分钟。 </li><li>在离液剂缓冲液按1:1的比例混合样品。 </li><li> 3分钟的通道流过样品的混合物。 </li></ol>洗<ol><li>洗2分钟与70％乙醇的通道。 </li><li>洗净与100％乙醇2分钟的渠道。 </li></ol>洗脱<ol><li> 3分钟的微孔水洗脱提取的DNA。收集15μL纯化，DNA PCR准备。 </li></ol> * GuSCN从Qiagen公司试剂盒（缓冲区RLT）。

<ol>
	<li>Bhattacharyya, A., Klapperich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+chemical+analysis+of+plasma+and+ultraviolet-ozone+surface+treatments+for+thermal+bonding+of+polymeric+microfluidic+devices.">Mechanical and chemical analysis of plasma and ultraviolet-ozone surface treatments for thermal bonding of polymeric microfluidic devices.</a> Lab Chip. 7, 876-882 (2007).</li></ol>

The authors have nothing to disclose.

fabrication of the thermoplastic microfluidic channels

在我们的实验室，我们已经成功地分离出核酸，直接从人体的血液，尿液和大便使用聚合物/纳米粒子复合材料的微观溶解和固相萃取柱微升和submicroliter卷。回收的样本PCRable无需任何额外的清理，集中，小体积样品。在这里，我们将演示如何制造热塑性微流体芯片采用热压成型和热封。然后，我们将演示如何使用通过密封芯片执导的原位光接枝表面和聚合，形成的复合材料的固相柱。我们展示嫁接和碳纳米管/聚合物复合列细菌细胞裂解聚合。然后，我们显示芯片采用硅/聚合物复合材料柱从样品核酸的固相萃取的裂解过程。所附的协议包含如何使裂解和固相萃取柱的详细说明。

Watch this Scientific Journal Video about Fabrication of the Thermoplastic Microfluidic Channels at JoVE.com

Fabrication of the Thermoplastic Microfluidic Channels

在这里我们将演示如何制造热塑性微流体芯片采用热压成型和热封。然后，我们将演示如何使用通过密封芯片执导的原位光接枝表面和聚合，形成的复合材料的固相柱。

制备热塑性微流体通道

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

fabrication-of-the-thermoplastic-microfluidic-channels

Research

JoVE Journal

Biology

13.3K 次观看.  Boston University. 在这里我们将演示如何制造热塑性微流体芯片采用热压成型和热封。然后，我们将演示如何使用通过密封芯片执导的原位光接枝表面和聚合，形成的复合材料的固相柱。

视频: Fabrication of the Thermoplastic Microfluidic Channels

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

科研

生物学

PDMS Device Fabrication and Surface Modification

PDMS的设备制造和表面改性

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

PDMS微流体装置的廉价制造的非等离子键合

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

微流控一次性诊断的应用

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

安培电极的制备

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...