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Protocol
Results
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Bioengineering

用于硅平面皮质内微电极表面处理的工具

Published: June 08, 2022
doi:
10.3791/63500
, , , , , , ,
1Department of Biomedical Engineering,Case Western Reserve University, 2Advanced Platform Technology Center, Rehabilitation Research and Development,Louis Stokes Cleveland Department of Veterans Affairs Medical Center, 3Department of Bioengineering,The University of Texas at Dallas

Summary

本方案描述了在处理过程中处理硅平面皮质内微电极的工具， 以便通过 气体沉积和水溶液反应进行表面改性。详细解释了在整个过程中用于处理设备的组件的组装。

Abstract

皮质内微电极具有巨大的治疗潜力。但是，他们面临的挑战是在适度的植入时间后显着降低性能。观察到的下降的一个重要因素是植入物近端神经组织的损伤和随后的神经炎症反应。提高设备寿命的努力包括化学修饰或将应用涂覆到设备表面以改善组织响应。这种表面处理的开发通常使用非功能性“假”探头完成，这些探头缺乏预期应用所需的电气元件。鉴于皮质内微电极阵列的脆弱性，转换为功能器件需要额外的考虑。处理工具极大地促进了对组装设备的表面处理，特别是对于需要较长程序时间的修改。此处描述的处理工具用于 通过 气相沉积和水溶液暴露进行的表面处理。涂层的表征使用椭圆偏振和X射线光电子能谱进行。在功能器件上涂覆过程之前和之后的电阻抗谱记录的比较确认了修改后的器件完整性。所描述的工具可以很容易地适应于保持化学相容性的替代电极设备和治疗方法。

Introduction

神经假体装置旨在恢复各种患者群体中受损或缺失的感觉和运动能力，包括脊髓损伤、肌萎缩性侧索硬化症 （ALS）、脑瘫和截肢1，2，3。皮质内微电极（IME）可以在皮质神经元和用于控制神经假体的设备之间建立通信途径。皮质内微电极的一个明显优点是它们能够以高空间和时间分辨率记录神经信号，这是随后的信号处理和控制脑机接口4，5的首选。不幸的是，皮质内微电极的性能在植入后的几个月内急剧下降到一年2，6，7，8。信号质量和稳定性的损失会对该技术的应用产生负面影响。

观察到的性能下降的一个重要因素是对植入相关组织损伤和慢性神经炎症的生物反应9，10，11。IME的植入会对脑组织造成损害，导致信号分子的释放，从而引发反应性细胞防御过程的级联。慢性接口会加剧异物反应，导致持续的神经炎症，损害设备近端的组织;通常被认为是神经炎症，瘢痕形成和局部神经变性的症状，导致信号质量记录下降12，13，14，15。包括具有夹带活化小胶质细胞和巨噬细胞的致密星形胶质细胞团，封装电极的疤痕创造了不利的局部环境，物质转运减少和炎症因子的局部积累16，15，16，17，18。

许多研究描述了大脑对皮质内微电极的反应或减轻反应的方法7。改善组织反应的研究和开发涉及一系列策略，包括对整体结构，表面拓扑，材料和涂层应用的修改。这些努力旨在最大限度地减少植入事件造成的损害，在装置和近端细胞之间引入更有利的界面，或在装置植入7后减少组织应变。专门针对慢性生物反应的方法已经产生了几种生物活性涂层，旨在稳定着床部位并化学促进细胞健康。实例包括导电聚合物如聚（乙烯-二氧噻吩）（PEDOT）19，20、碳纳米管21、水凝胶22，以及添加生物活性分子和药物以靶向特定的细胞过程23、24、25。特别是我们的研究小组，已经探索了许多促进对植入微电极的炎症反应减少的机制，包括但不限于最小化与设备植入相关的创伤26，最小化设备/组织刚度不匹配27，28，29，30，31，32，33，优化灭菌程序34，35，还原氧化应激/损伤28，36，37，38，39，40，41，42，探索替代电极材料43，并模仿天然细胞外基质的纳米结构44，45，46.最近的兴趣是仿生表面涂层的发展，以减轻微电极组织界面39处的神经炎症反应。

界面的修改提供了直接靶向伤口和信号记录所需的近端组织的独特好处。在不加剧免疫反应的情况下促进愈合的表面处理可以有益于质量记录的寿命，并消除实现皮质内微电极的治疗和研究潜力的限制。所介绍的工作详细介绍了将表面处理应用于微电极阵列的方法，这些方法需要延长反应时间，同时适应器件的脆弱性。所提出的技术旨在将表面改性方法共享给功能设备，其中设备在整个处理应用中无法处理。这些工具用于处理非功能性假探头和功能性硅平面微电极阵列。

所提出的修改电极表面的方法允许非功能性假探针或功能性硅平面电极阵列的安全悬浮，用于气相沉积和与水溶液的反应。几个3D打印件用于处理这些易碎的设备（图1 和 图2）。提供了一个程序示例，该程序利用气体和溶液相步骤进行表面改性，使用涉及Mn（III）四（4-苯甲酸）卟啉（MnTBAP）固定化的抗氧化涂层。MnTBAP是一种合成的金属卟啉，具有抗氧化特性，并已证明可以调解炎症47，48。提供的关于功能性硅平面电极阵列的示例验证了对先前报告的用于非功能性设备40的协议的更新。Munief等人 对气相沉积技术的适应性支持该协议与功能电极49的兼容性。气相沉积用于胺官能化表面，为涉及碳二亚胺交联剂化学的水反应做准备，以固定活性MnTBAP。这里开发的处理方法作为一个平台提供，可以对其进行修改以适应其他涂层和类似设备。

该协议说明了使用非功能性假探头的方法，这些探头包括硅柄和3D打印标签，其尺寸与功能性硅平面电极阵列相似。该设备的连接器包装被认为类似于所提供的说明中非功能性假探头的3D打印选项卡。

Figure 1
图 1：用于在真空干燥器中气相沉积期间处理功能器件的 3D 打印件 （A） 结构的底座包括用于 1 cm x 1 cm 样品硅方块的支架（顶部箭头）和用于固定到干燥器板的孔（底部箭头）。（B）板用于固定设备的悬挂。从这里开始，此图中的每个部分将称为部分1A或1B。比例尺 = 1 cm .请点击此处查看此图的放大图。

Figure 2
图2：用于处理水溶液中发生表面反应的功能器件的3D打印件。（B） 用于在组装时稳定 （C） 和 （D） 件的台式件。（C）和（D）一起固定装置的悬浮液以放置在孔板中，并且（E）进一步将（C）和（D）片固定在孔板盖上。从这里开始，此图的每个面板中的单个部件将称为与该图的面板编号相对应的部件号。比例尺 = 1 cm.请点击此处查看此图的放大图。

Protocol

3D打印的所有编码文件都在 补充编码文件1-16中提供。代表结果中提供的分析是使用商业获得的功能性硅平面电极阵列描述的（参见 材料表）。 1. 真空干燥器中气相沉积的处理组件 注：用于在气相沉积过程中处理和保持装置的组装设备如图 3所示。步骤1.1-1.8描述了将设备放入设备中进行沉积所需…

Representative Results

为了演示处理组件的使用，实施了所述方法以使氧化介质的固定化适应活化的硅。波特-贝克等人设计了将这种化学物质应用于IME以减少氧化应激，并在非功能性硅假探针40上进行了演示。这种表面处理通过胺官能化，然后 碳 二亚胺交联化学51，将抗氧化剂MnTBAP固定到UV/臭氧活化硅表面。胺官能化 通过 气相沉积完成，碳二亚胺交联化学 通过…

Discussion

所述方案设计用于硅平面微电极阵列的表面处理。3D打印工具定制为密歇根风格的微电极阵列，带有薄型连接器50。非功能性探针是通过使用生物相容性粘合剂将硅探针粘附到3D打印标签上来组装的。3D打印的标签设计与所使用的商用设备上的连接器具有相似的尺寸。3D 打印选项卡的文件可用作 补充编码文件 15、补充编码文件 16。丙烯腈丁二烯苯乙烯（ABS）长丝用于?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了美国退伍军人事务部康复研究与发展服务部的优异审查奖IRX002611（卡帕多纳）和研究职业科学家奖IK6RX003077（卡帕多纳）的部分支持。此外，这项工作还得到了美国国立卫生研究院，国家神经疾病和中风研究所R01NS110823（Capadona / Pancrazio）以及美国国家科学基金会研究生研究奖学金计划（克雷布斯）的部分支持。

Materials

1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDC) Sigma-Aldrich 165344-1G Solid, stored desiccated at -20 °C
15 mL Conical Centrifuge Tubes Fisher Scientific 14-959-70C
18 Pound Solid Nylon Cable/Zip Ties Cole-Parmer EW-06830-66 Length 4 inches
2-(N-Morpholino)ethanesulfonic acid (MES) Sigma-Aldrich 4432-31-9 Solid
3-aminopropyltriethoxysilane (APTES) Sigma-Aldrich 440140-100ML Liquid, container with Sure/Seal
50 mL Conical Centrifuge Tubes Fisher Scientific 14-959-49A
Aluminum foil Fisher Scientific 01-213-103
Aluminum weighing dishes Fisher Scientific 08-732-102 Diameter 66 mm
Bel-Art Vacuum Desiccator Fisher Scientific 08-594-15B
Corning Costar TC-Treated Multiple Well Plates Millipore Sigma CLS3527-100EA 24-well plate, polystyrene
Cyanoacrylate Adhesive LocTite N/A
Digital Microscope Keyence VHX-S750E
Disco DAD3350 Dicing Saw Disco DAD3350 Used to cut silicon wafer into 1 cm x 1 cm samples
Double-Sided Polyimide Tape Kapton Tape PPTDE-1/4 ¼” x 36 yds.
EP21LVMed – low viscosity, two component epoxy compound Masterbond EP21LVMed Meets USP Class VI certification, Passes ISO 10993-5 for cytotoxicity
Epilog Fusion Pro 48 Laser Machine Epilog N/A CO2 laser
Foam tape XFasten N/A 1/8" Thick
Gamry Interface 1010E Potentiostat Gamry 992-00129
High precision 45° curved tapered very fine point tweezers/forceps Fisher Scientific 12-000-131
Lab tape Fisher Scientific 15-901-10L
Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) EMD Millipore 475870-25MG Solid, stored at -20 °C
N-Hydroxysulfosuccinimide sodium salt, ≥98% (HPLC) Sigma-Aldrich 56485-250MG Solid, stored desiccated at 4°C
Platinum clad niobium mesh anode Technic N/A Clad with 125μ” of platinum on one side, framed in titanium with (1) 1” x 6” titanium strap centered on one 6” dimension
Silicon Planar Microelectrode Array, 16 Channel NeuroNexus A1x16-3mm-100-177-CM16LP Electrode site material is iridium, shank thickness is 15 μm
Silicon Wafer University Wafer 1575 Diameter 100 mm, p-type, boron-doped, 100 oriented, resistivity 0.01-0.02 Ohm-cm, thickness 525 um, single side polished, prime grade
Silver/silver Chloride reference electrode Gamry Instruments 930-00015
Solidworks N/A
Stainless Steel Phillips Flat Head Screws McMaster Carr 96877A629 #8-32, 1 1/2", fully threaded
Type I deionized water ChemWorld CW-DI1-20
Ultimaker 3 3D printer Ultimaker  N/A
Ultimaker Cura Ultimaker N/A 3D printing software
Ultimaker NFC ABS Filament Dynamism, Inc. 1621 2.85 mm
Ultimaker NFC PLA Filament Dynamism, Inc. 1609 2.85 mm
Vacuum Gauge Vacuum Gauge Measureman Direct N/A Glycerin Filled, 2-1/2” Dial Size, ¼”NPT, -30” Hg/-100kpa-0
Wing nuts Everbilt 934917 #8-32, zinc plated
DOWNLOAD MATERIALS LIST

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Keywords: Surface TreatmentSiliconIntracortical MicroelectrodesGas-phase DepositionAqueous Solution ReactionHandling ToolsDevice IntegritySubstrate Material3D PrintingPolyimide TapeFoam StripVacuum DesiccatorGas-phase DepositionWell PlateCyanoacrylate AdhesiveSurface ConfirmationProbe Device Assembly
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Krebs, O. K., Mittal, G., Ramani, S., Zhang, J., Shoffstall, A. J., Cogan, S. F., Pancrazio, J. J., Capadona, J. R. Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes. J. Vis. Exp. (184), e63500, doi:10.3791/63500 (2022).
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