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Introduction
Protocol
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Bioengineering

聚焦 Ion 光束平版印刷到蚀刻纳米架构到微电极

Published: January 19, 2020
doi:
10.3791/60004
, , , ,
1Department of Electrical Engineering and Computer Science,University of Michigan, 2Veteran Affairs Ann Arbor Healthcare System, 3Department of Neurology, School of Medicine,University of Michigan, 4Department of Biomedical Engineering,University of Michigan, 5Michigan Center for Materials Characterization,University of Michigan, 6Carl Zeiss SMT, Inc.

Summary

我们已经表明，将纳米结构蚀刻到皮内微电极装置中可以减少炎症反应，并有可能改善电生理记录。本文描述的方法概述了将纳米架构蚀刻到非功能和功能性单柄硅皮内微电极表面的方法。

Abstract

随着电子和制造技术的进步，皮内微电极得到了重大改进，使得具有更高分辨率和扩展能力的精密微电极的生产成为可能。制造技术的进步支持了仿生电极的发展，它旨在无缝地集成到脑气囊中，减少电极插入后观察到的神经炎反应，并提高质量和电生理记录的寿命。在这里，我们描述了一个协议，以采用一种最近被归类为纳米架构的仿生方法。该协议使用聚焦电子束光刻（FIB），将特定的纳米结构特征蚀刻到非功能和功能性单柄皮内微电极的表面。将纳米结构蚀刻到电极表面表明植入装置的生物相容性和功能可能有所改善。使用 FIB 的好处之一是在制造设备上蚀刻的能力，而不是在设备制造过程中蚀刻，从而在制造后为修改大量医疗设备提供了无限的可能性。本文介绍的协议可以针对各种材料类型、纳米架构特征和设备类型进行优化。增强植入式医疗设备的表面可以提高设备的性能并集成到组织中。

Introduction

皮内微电极（IME）是侵入性电极，提供一种在外部设备与大脑皮层1、2内神经元群之间直接连接的方法。这项技术是记录神经作用潜力的宝贵工具，以提高科学家探索神经元功能的能力，促进对神经系统疾病的理解，并开发潜在的疗法。皮内微电极，用作脑机器接口（BMI）系统的一部分，能够记录单个或小群神经元的运动电位，以检测可用于产生功能输出的运动意图3。事实上，BMI系统已经成功地用于假肢和治疗目的，如获得感觉运动节律控制，以操作电脑光标的患者肌萎缩性侧索硬化症（ALS）4和脊髓损伤5和恢复运动的人患有慢性四肢瘫痪6。

不幸的是，由于几种故障模式，包括机械、生物和材料因素7，8，ImME往往无法随时间持续地记录。植入电极后发生的神经炎反应被认为是导致电极故障9，10，11，12，13，14的一个相当大的挑战。神经炎反应是在IME的初始插入期间启动的，它切断血脑屏障，破坏局部脑气肿，破坏胶质和神经元网络15，16。这种急性反应的特点是活化胶质细胞（微胶质细胞/巨噬细胞和星形细胞），在植入部位17、18、19、20周围释放亲炎和神经毒性分子。胶质细胞的慢性活化导致异物反应，其特征是形成胶质疤痕，将电极从健康的脑组织7、9、12、13、17、21、22中分离。最终，由于电极和神经元之间的物理屏障以及神经元的退化和死亡23、24、25，阻碍了电极记录神经元作用电位的能力。

皮内微电极的早期失效为下一代电极的发展带来了大量研究，重点是生物仿生策略26、27、28、29、30。特别感兴趣的协议，这里描述的，是使用纳米结构作为一类仿生表面改变的IME31。现已证实，模仿自然体环境结构的表面具有更好的生物相容性反应32，33，34，35，36。因此，令人信服的假设是，脑组织的粗糙结构与皮内微电极平滑结构之间的不连续性可能导致神经炎和慢性异物对植入的IIMEs的反应（完整审查请参阅Kim等人31）。我们之前已经表明，与神经炎症37、38的体外和前体模型相比，利用与大脑细胞外基质结构相似的纳米结构特征可以减少从纳米结构基板上培养的细胞中的星形细胞炎标记物。此外，我们还表明，聚焦电光束（FIB）光刻技术直接蚀刻到硅探针上，与平滑对照组26相比，通过纳米架构探针植入的动物的神经元活力和亲炎基因表达能力显著增强。因此，本文介绍的协议的目的是描述FIB光刻在人造皮内微电极器件上蚀刻纳米结构的用法。该协议旨在利用自动化和手动工艺，将纳米结构尺寸特征蚀刻到皮内微电极刀柄的硅表面。这些方法简单、可重复，当然可以针对各种设备材料和所需的特征尺寸进行优化。

Protocol

注意：在佩戴适当的个人防护装备（如实验室外套和手套）时，执行以下步骤。 1. 安装用于聚焦式电子束 （FIB） 平版印刷的非功能硅探头 注：有关描述使用1000个探头制造SOI晶圆的完整过程，请参阅Ereifej等人39。 从包含 1，000 个探头的绝缘体 （SOI） 晶圆上的硅片中分离出一条 2-3 个硅探头。请勿制作包含三个以上硅探头的条带…

Representative Results

FIB 在单柄内皮探头表面蚀刻纳米结构利用本文描述的方法，在皮内探针按照既定的协议39蚀刻了特定的纳米结构。这些方法中描述的纳米架构设计的尺寸和形状是从以前的体外结果中实现的，这些结果描绘了与本文中描述的纳米架构设计进行培养时胶质细胞反应的降低。本文描述的方法利用聚焦的铀子束（FIB）将纳米?…

Discussion

此处概述的制造方案利用聚焦的电子束光刻法，将纳米结构有效且可重复地蚀刻到非功能和功能单柄硅微电极的表面。聚焦电子束（FIB）光刻允许使用细聚焦的电束50，51选择性消融基板表面。FIB是一种直接写入技术，可以产生各种具有纳米尺度分辨率和高纵横比50、52、53的特性。为…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了美国（美国）退伍军人事务部康复研究和发展服务奖的支持：#RX001664-01A1（CDA-1，Ereifej）和#RX002628-01A1（CDA-2，Ereifej）。其内容并不代表美国退伍军人事务部或美国政府的观点。作者要感谢FEI公司（现为热鱼科学的一部分）的工作人员协助和使用仪器，这有助于开发本研究中使用的脚本。

Materials

16-Channel ZIF-Clip Headstage Tucker Davis Technologies ZC16 The headstage and headstage holder may need to be changed, depending on the electrode used. https://www.tdt.com/zif-clip-digital-headstages.html
1-meter cable, ALL spring wrapped Thomas Scientific 1213F04 Any non treated petri dish will suffice. https://www.thomassci.com/Laboratory-Supplies/Cell-Culture-Dishes/_/Non-Treated-Petri-Dishes?q=petri%20dish%20cell%20culture
32-Channel ZIF-Clip Headstage Holder Tucker Davis Technologies Z-ROD32 The headstage and headstage holder may need to be changed, depending on the electrode used. https://www.tdt.com/zif-clip-digital-headstages.html
Acetone, Thinner/Extender/Cleaner, 30ml Ted Pella 16023 https://www.tedpella.com/SEMmisc_html/SEMpaint.htm#anchor16062
Baby-Mixter Hemostat Fine Science Tools 13013-14 Any curved hemostat will suffice. https://www.finescience.com/en-US/Products/Forceps-Hemostats/Hemostats/Baby-Mixter-Hemostat
Carbon Conductive Tape, Double Coated Ted Pella 16084-7 The protocol suggested three options for mounting the functional electrode to the aluminum stub (copper or carbon conductive tape or a low profile clip. We utilized the carbon conductive tape in our study. https://www.tedpella.com/semmisc_html/semadhes.htm
Corning Costar Not Treated Multiple Well Plates – 6 well Sigma Aldrich CLS3736-100EA Any non-treated 6 well plate will suffice. https://www.sigmaaldrich.com/catalog/substance/
Dumont #5 Fine Forceps Fine Science Tools 11251-30 Either this fine forceps or the vacuum pump will suffice. https://www.finescience.com/en-US/Products/Forceps-Hemostats/Dumont-Forceps/Dumont-5-Forceps/11251-30
Ethanol, 190 proof (95%), USP, Decon Labs Fisher Scientific 22-032-600 Any 95% ethanol will suffice. https://www.fishersci.com/shop/products/ethanol-190-proof-95-usp-decon-labs-10/22032600
Falcon Cell Strainer Fisher Scientific 08-771-1 https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711
FEI, Tescan, Zeiss (also for Philips, Leo, Cambridge, Leica, CamScan), aluminum, grooved edge, Ø32mm Ted Pella 16148 Depending on the SEM machine used, you may need a different size stub. https://www.tedpella.com/SEM_html/SEMpinmount.htm#_16180
Fisherbrand Aluminum Foil, Standard-gauge roll Fisher Scientific 01-213-101 Any aluminum foil will suffice. https://www.fishersci.com/shop/products/fisherbrand-aluminum-foil-7/p-306250
Fisherbrand Low- and Tall-Form PTFE Evaporating Dishes Fisher Scientific 02-617-149 Any Teflon plate will suffice, this is used to dry the probes after washing on a surface they will not stick onto. https://www.fishersci.com/shop/products/fisherbrand-low-tall-form-ptfe-evaporating-dishes-12/p-88552
Michigan-style silicon functional electrode NeuroNexus A1x16-3mm-100-177 http://neuronexus.com/electrode-array/a1x16-3mm-100-177/
Model 1772 Universal holder KOPF Model 1772 Other stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-1772-universal-holder/
Model 900-U Small Animal Stereotaxic Instrument KOPF Model 900-U Other stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-900-small-animal-stereotaxic-instrument1/
Model 960 Electrode Manipulator with AP Slide Assembly KOPF Model 960 Other stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-1772-universal-holder/
Parafilm M 10cm x 76.2m (4" x 250') Ted Pella 807-5 https://www.tedpella.com/grids_html/807-2.htm
PELCO Vacuum Pick-Up System, 220V Ted Pella 520-1-220 Either this vacuum pump or the fine forceps will suffice. http://www.tedpella.com/grids_html/Vacuum-Pick-Up-Systems.htm#anchor-520
PELCO Conductive Silver Paint Ted Pella 16062 https://www.tedpella.com/SEMmisc_html/SEMpaint.htm#anchor16062
SEM FIB FEI Helios 650 Nanolab Thermo Fisher Scientific Helios G2 650 This is the specific focused ion beam and scanning electron microscope used in the protocol. The Nanobuilder software is what it comes with. If a different FIB instrument is used, it may not be completely compatible with the protocol, specifically the steps requiring the Nanobuilder software. https://www.fei.com/products/dualbeam/helios-nanolab/
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Keywords: Focused Ion Beam LithographyFIBNano-architecturesMicroelectrodesBiocompatibilitySiliconMetalsPolymersNon-planar SurfacesPost-processingScanning Electron MicroscopeSEMSilver PaintStage CoordinatesNitrile GlovesNavigation CameraMicroscope User Interface
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Mahajan, S., Sharkins, J. A., Hunter, A. H., Avishai, A., Ereifej, E. S. Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes. J. Vis. Exp. (155), e60004, doi:10.3791/60004 (2020).
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