This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1.光掩膜制造<ol><li> 20分钟，在120℃下浸在石英罐:清洁用食人鱼溶液4"的玻璃晶片（1比率的 H 2 SO 4 / H 2 O 2中2）。 </li><li>沉积一层铬/金（铬的Au / 1微米30纳米）层使用电子束蒸发器7（图1A），在玻璃晶片上。 <ol><li>放置晶片的电子束蒸发器。一旦真空度达到5×10 -6乇时，打开高压源（10千伏）。通过控制显示器面板的厚度。 </li><li>预清洁各材料进行30秒，使用电子束枪，保持闸板"关"（以避免在晶片上的沉积）。 </li></ol></li><li>生成的Cr / Au的光致抗蚀剂掩模层为玻璃深湿法腐蚀。 <ol><li>通过纺丝5 ml的30秒的溶液以3,000rpm使用旋喷涂机系统应用一个2微米厚的光致抗蚀剂。预烘烤的加热板上的光致抗蚀剂在100℃下1.5分钟。 </li><li>揭露和硬烘焙光致抗蚀剂在120℃下进行30分钟的热板上。关键是要产生一个疏水表面和光致抗蚀剂到金属层​​的附着力强。模式通过利用光致抗蚀剂Cr和Au蚀刻剂的铬/金图层蒙版8,9。 </li></ol></li><li>为保护的未图案化的玻璃表面，暂时粘结玻璃晶片以一个伪硅晶片。9 <ol><li>将玻璃晶片上的热板上在110℃和熔化在玻璃晶片的相对侧上的蜡（以这样一种方式，在晶片的整个表面覆盖有蜡）。 </li><li>放置一个虚设的硅晶片与所述玻璃晶片接触，然后按以除去过量的蜡。为了避免蜡的溢出，放置一个洁净室薄纸上热板。 </li></ol></li><li>执行使用优化氢氟酸透镜的各向同性蚀刻（49％体积/体积）和盐酸（37％体积/体积）溶液（以10的体积比:1）。用磁搅拌器为8.5分钟10存在的盐酸是在实现所产生的透镜的表面质量良好的关键。 <ol><li>确保蚀刻速度为7μm/分钟;使用200毫升的蚀刻液的总体积。在塑料容器中进行蚀刻，并采取安全预防措施该处理步骤。 </li><li>通过漂洗和进一步干燥在RT清洗在去离子（DI）水的晶片。 </li></ol></li><li>该过程完成后，从虚拟硅晶片分离玻璃晶片和用热板在100℃，15秒预热蜡。作为蜡熔化在该温度下，分离从虚拟硅晶片，玻璃晶片。 </li><li>除去残留的蜡，光致抗蚀剂和悬垂铬/金层在使用超声透镜的边缘1小时使用 N-甲基-2-吡咯烷酮作为在80℃下在一个超声容器中的溶剂。</li><li>创建制造在光掩模11上的微透镜的一个模具的PDMS复制品。 </li><li>复制品使用扫描电子显微镜和立体显微镜分别表征光掩模尺寸（长度和宽度）和微透镜的PDMS模具（深度和直径）12-14 </li></ol> 2. MN轴加工<ol><li>创建的2.5厘米的空腔×使用载玻片安装在玻璃的两侧0.9厘米。载玻片堆叠在任一侧的数量将决定被称为间隔件厚度（图1B）的空腔的高度。 </li><li>通过施加一薄层含有聚（乙二醇）二丙烯酸酯（PEGDA，MW = 258道尔顿）的预聚物溶液与0.5％w / w的2-羟基-2-甲基苯丙酮（HMP）到固定在玻璃载片的每一层载玻片随后建立与高强度紫外（UV）光进行2秒的照射。 </li><li>放置photomaSK（先前制备）朝向腔内部的铬/金涂覆的表面。确保该腔壁的边不模糊嵌入在光掩模的透镜。 </li><li>填与预聚物溶液的空腔，直到铬/金涂覆的表面是与没有任何可见的气泡的溶液接触。 </li><li>与照射1秒钟所需强度的高强度UV光的设置，在使用UV固化站的320-500纳米的紫外线过滤器范围内的紫外光源的距离为3.5cm。用准直适配器UV光探头。 </li><li>测量紫外光的使用辐射计所用的力度。 </li><li>以下UV曝光，卸下光掩膜与MnS的阵列。倾未聚合的过程中回到其再利用原始容器中的过量预聚物溶液。 </li><li>量化使用根据制造商的说明在立体显微镜的移动节点的长度和顶圆直径。 </li></ol><p类="jove_title"&gt; 3。 MN背衬层制造<ol><li>用钳子，将移动节点（先前制造）在一个24孔板的孔中附着于光掩模中所示的图1C。 </li><li>添加指定量 - 该预聚物溶液（300 400微升）到井直到针浸没到一所需高度。这个体积决定了所得背层的厚度。 </li><li>照射高强度UV光（15.1瓦/厘米2），10.5厘米远离UV源为1秒的持续时间的设置。 </li><li>从光罩用锋利的刀片在MN阵列中独立的衬里层。 </li><li>量化的MN的长度，顶端直径和底座直径与使用根据制造商的说明在立体显微镜背层。 </li></ol>

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The authors declare no conflict of Interest.

上述用于MN的阵列的制造的协议已被提交给制作的约1cm 2的MN的阵列。该阵列可通过创建一个大尺寸空腔，并通过使用一个更大的光掩模进行放大。增加的空腔大小可以通过增加两侧的隔离物之间的宽度来创建。虽然各步骤以制造在协议在MN阵列是重要的，最重要的步骤是:光掩模定位安装的，的预聚物溶液的填充，和照射。光掩模的定位应以这样的方式，该铬/金涂覆的表面面对所述腔体的内部与腔体壁的侧面掩盖嵌入在光掩模的透镜。当填充与预聚物溶液的模具，确保气泡不会截留其可以以其他方式导致变形和低强度MN阵列。气泡可以通过控制芯吸作用通过缓慢加入prepolyme能够防止R染料溶液，并确保没有存在于预聚物溶液气泡。设置为照射定位应以引导的方式进行，以保证均匀的紫外线照射。在暴露于UV光之前，将组合体对准并放置在基座划界内。 使用光刻法形成的移动节点由微透镜的存在下极大地影响了作为透镜导致聚合在融合路径，这导致了更清晰的MnS的生成，相比使用面状光掩模形成的圆筒状的MN。在平面光罩，UV光通过它与小偏差传递（几乎是直的）导致形成圆柱形的MN与不太清晰的提示。而在微透镜嵌入光掩模中，UV光通过镜片经历折射和融合，从而产生的锐顶的MN的形成。已被用作predicti透镜制造者公式已经模型由与微透镜的焦距相关近似MN的长度，得到长度比实际少三倍的预测。这种差异可能是由于微透镜，而没有允许光折射象常规凸透镜的扁平凸面。13 为MN几何形状的另一个因素是在紫外线照射强度。的6.44 瓦 /平方厘米的强度的选择，因为在此强度产生的针具有足够的机械强度用于皮肤应用。另一个发现相关的强度，与在UV强度的每个增量，MN的长度增加而增加。这可以归因于微透镜的平顶，这使得一些光射线的行进超出焦点。14,15。此外，聚合程度有它的限制，根据光的平方反比定律， 也就是说 ，光损失能量的距离从源增加。16 这里描述的方案有很短的时间周期内的无霉变制造的优点。但是，我们无法预测在多大程度上可以进一步采取批量制造。所述移动节点的数组是从一个低成本的生物相容的聚合物制成。它可以潜在地用作用于药物和化妆品应用的经皮药物递送装置。更有趣的是，它可以被用作递送系统和设备的组合，作为治疗化合物可以被包封（通过混合或增溶剂中的预聚物溶液）的MN的制造过程中。5,6,17所添加的化合物的溶解度里面的预聚物溶液，应考虑到作为MN的特性， 例如 ，MN强度，可能发生变化。17

透皮药物递送提供了药物施用一个有吸引力的替代方法，特别是对生物分子，这是几乎完全通过施用皮下注射。然而，皮肤，尤其是顶层（角质层），是一项艰巨的屏障防止外源性分子进入人体。近日，美国明尼苏达设备已成为实现工具，通过皮肤输送药物。在MN设备创建角质层内临时孔，以允许药物分子通过实现具有改善的患者顺应性和便利1-3所需的生理活性。 各种制造方法已被制造的聚合移动节点4。然而，它们通常涉及复杂和多步工艺需要长的时间和/或高温来制造MN的阵列。4要 ，单一步骤的无霉变过程使用简化制造过程的光掩模最近5,6被开发。然而，利用这种方法，制造的MN不得不钝的针尖，由于没有机制已经到位修改涉及光刻的紫外（UV）光的路径。 在这项研究中，已经提出了在光掩模嵌入式微透镜以定义的MN的几何形状。该协议制造光掩膜包括嵌入式微透镜，随后MN制造与使用光掩模尖头的报告。

<table><tbody><tr><td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td><td>SIGMA&amp;nbsp;</td><td>475629-500ML</td><td></td></tr><tr><td>2-hydroxy-2-methyl-propiophenone (HMP)</td><td>SIGMA&amp;nbsp;</td><td>405655-50ML</td><td></td></tr><tr><td>Bovine collagen type 1, FITC conjugate&amp;nbsp;</td><td>SIGMA&amp;nbsp;</td><td>C4361</td><td></td></tr><tr><td>UV curing station&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>OmniCure S2000-XL</td><td></td></tr><tr><td>Collimating Adaptor&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>EXFO 810-00042</td><td></td></tr><tr><td>24-well plate</td><td>Thermo Fisher Scientific, USA</td><td></td><td></td></tr><tr><td>Nikon SMZ 1500 stereomicroscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr><tr><td>Dillon GL-500 digital force gauge&amp;nbsp;</td><td>Dillon, USA</td><td></td><td></td></tr><tr><td>A-1R confocal microscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr></tbody></table>

polymeric microneedle array fabrication by photolithography

这个手稿通过光刻法描述的聚合物微针（MN）的阵列的制造。它涉及通过使用光掩模，包括嵌入式微透镜的简单的无霉变过程。嵌入式微透镜被发现影响MN几何（清晰度）。强大的MN阵列尖直径介于41.5微米±8.4微米和71.6微米±13.7微米，有两种不同长度（1336微米±193微米和957微米±171微米）制造。这些MN阵列可以通过皮肤提供交付小分子和大分子治疗药物的潜在应用。

所述移动节点的几何形状可以显著受光掩模特征和嵌入式微透镜。折射的程度影响的紫外线，这影响了在MN几何（图2A）的传输路径。每个微透镜被发现有一个直径350微米，130微米扁平凸面，和一个62.3微米深度（图2B-D）。利用毕达哥拉斯定理，第一表面的曲率半径被发现是272.89微米。焦距计算为509.28微米（考虑Ñ 玻璃 = 1.53627; N 空气 = 1.000;λ= 365纳米）通过透镜制造者公式12如下所述: 的1 / f =（N 1 / n×m个-1）*（1 / R 1 -1 / R 2） 其中n 1是透镜材料的折射率，n m是周围介质的折射率，R 1是拉德曲率第一表面的， 和 r 2的IUS是第二表面的曲率半径。 紫外线强度对在MN长度，清晰度和结构变形的效果，研究了改变从3.14的UV光的强度，以15.1瓦/厘米2以恒定的焦距和光源的距离。结果发现，平均MN长度显著升高 （P &lt;0.05），随着强度从3.14到9.58 瓦 /平方厘米（ 图3A）。进一步增加强度高达15.1瓦/厘米2不产生在长度显著变化。尖端直径（测量清晰度）和MN部结构被发现随增加强度（图3B）。具有规则形状，没有任何结构变形的移动节点在6.4瓦/厘米2进行观察。 背层制作，以使除去MN的在贴剂的形式并且，使光掩模可重复使用的。它也提供了强度至MN轴上。因此，背层体积（预聚物溶液的体积，以形成背层）的效果也进行了研究。未受影响的尖端直径，范围的长度（1336±193微米为300微升和957±171微米为400微升）的移动节点的UV曝光（图4）后进行观察。 <img alt="图1" src="/files/ftp_upload/52914/52914fig1highres.jpg" width="700" /> 透镜嵌入光掩模制造工艺的图1（A）的示意图。 （1）4"玻璃晶片。 （2），使用电子束蒸发器的Cr / Au层沉积。 （3）曝光铬/金/光致抗蚀剂掩模层的UV光与光掩模。图案（4）上使用铬/金蚀刻剂层形成。玻璃（5）的一个伪硅晶片​​上临时粘接。 （6） - （7-）使用HF /盐酸蚀刻剂随后超声湿法蚀刻（各向同性）的过程。 （8）合分离虚设硅晶片和除去光致抗蚀剂层的。 （B）的针的制造工艺的示意图。镀铬的光掩模（9×9阵列），被放置在含预聚物溶液的空腔，并暴露于UV。 （C）的背层的制造过程的示意图。光罩，附有微针，被放置在一个井充满了预聚物和暴露于紫外线。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52914/52914fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52914/52914fig2highres.jpg" width="450" /> 光掩模图2.表征。（A）紫外线照射光聚焦到一个conica<html>升路，生产圆锥移动节点。 （B）和（C）的微透镜的SEM图像。 （D）的一部分，从微透镜的拷贝，表示扁平凸面的PDMS模具复本阵列的，在立体显微镜下。 （E）的光掩模显示图案。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52914/52914fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52914/52914fig3highres.jpg" width="450" /> 紫外线参数对微针形状的（A）强度和微针长度 （B）垫片厚度的影响图3.影响。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52914/52914fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 帐篷"FO:保持together.within页="总是"&gt; <img alt="图4" src="/files/ftp_upload/52914/52914fig4highres.jpg" width="700" /> 用于背层制造不同的预聚物量图4.影响。（A - B）的图像在不同的音量，平均MN长度短（957微米）和长期（1336微米）移动节点。 （C - D）图片断裂力试验后对应（AB）。 （五）减少MN长度增加用于背层的制造量。跨用来制造背层（BL）的两个预聚合物卷（F）MN断裂力。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52914/52914fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

Polymeric Microneedle Array Fabrication by Photolithography

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

polymeric-microneedle-array-fabrication-by-photolithography

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.0K 次观看.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

视频: Polymeric Microneedle Array Fabrication by Photolithography

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

Micropunching为微型和亚微米模式聚合物基板上生成的光刻

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of ...

科研

工程学

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

生物工程

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 ...

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

暂停规定微米/亚微米尺度的纤维结构的3-D直写经全自动点胶系统

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

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 ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

使用激光切割模具实现简单的无光刻单细胞微模式

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

发育生物学

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

用光刻胶喷涂和柔性掩制备医用注射用针尖上的微细电极

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

神经科学

3D 打印在微型神经元细胞培养设备中的应用

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

作者聚焦:用于分析大脑功能的模块化神经元网络

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

微尺度神经元细胞培养装置的双光子聚合3D打印

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...