用于Vivo神经记录的 Ti3C2 MXene 微电极阵列的制造
1Department of Bioengineering,University of Pennsylvania, 2Center for Neuroengineering and Therapeutics,University of Pennsylvania, 3Corporal Michael J. Crescenz Veterans Affairs Medical Center, 4Department of Materials Science and Engineering,Drexel University, 5A.J. Drexel Nanomaterials Institute,Drexel University, 6Department of Neurosurgery,University of Pennsylvania, 7Department of Neurology,University of Pennsylvania, 8Department of Physical Medicine and Rehabilitation
Summary
在这里，我们描述了一种制造Ti3C2 MXene微电极阵列并利用它们进行体内神经记录的方法。
Abstract
植入式微电极技术已广泛用于阐明微尺度的神经动力学，从而更深入地了解脑病和损伤的神经基础。由于电极与单个电池的尺度小型化，接口阻抗的相应上升会限制记录信号的质量。此外，传统的电极材料是僵硬的，导致电极和周围脑组织之间的机械不匹配，导致炎症反应，最终导致设备性能下降。为了应对这些挑战，我们开发了一种基于 Ti3C2 MXene 的柔性微电极的制造工艺，这是一种最近发现的纳米材料，具有非常高的体积电容、导电性、表面功能和水分散物的可加工性。Ti3C2 MXene 微电极的柔性阵列具有极低的阻抗，因为 Ti3C2 MXene 薄膜的电导率高，特异性表面积高，并且已证明它们对记录神经元活动非常敏感。在该协议中，我们描述了一种将Ti3C2 MXene微阵列微阵列微阵列到柔性聚合物基板上的新方法，并概述了它们用于体内微电理学记录。该方法可轻松扩展，以创建任意尺寸或几何形状的 MXene 电极阵列，用于生物电子领域的一系列其他应用，并且除 Ti3C2 MXene 外，还可与其他导电油墨一起使用。该协议支持从基于解决方案的导电油墨中简单和可扩展地制造微电极，并特别允许利用亲水性 Ti3C2 MXene 的独特特性来克服长期以来阻碍高保真神经微电极广泛采用碳基纳米材料的许多障碍。
Introduction
了解神经回路的基本机制，以及它们在疾病或损伤中如何改变其动力学，是开发针对各种神经和神经肌肉疾病的有效疗法的关键目标。微电极技术已广泛用于阐明精细空间和时间尺度上的神经动力学。然而，从微尺度电极获得高信噪比（SNR）的稳定记录已证明特别具有挑战性。随着电极尺寸减小到接近蜂窝规模，电极阻抗的相应增加会降低信号质量1。此外，许多研究表明，由传统硅和金属电子材料组成的硬电极在神经组织中产生严重的损伤和炎症，这限制了它们对长期记录2、3、4、5的有用性。鉴于这些事实，人们一直对开发具有新材料的微电极非常感兴趣，这种材料可以减少电极组织界面阻抗，并可以融入软和灵活的外形中。
减少电极组织接口阻抗的一个常用方法是增加细胞外流体中的离子物种与电极相互作用的面积，或电极的”有效表面积”。这可以通过纳米图案6，表面粗加工7，或电镀与多孔添加剂8，9。纳米材料在这一领域得到了很大的关注，因为它们提供了内在高的特异性面积和独特的有利的电气和机械特性组合10。例如，碳纳米管已被用作涂层，以显著减少电极阻抗11，12，13，氧化石墨烯氧化物已加工成柔软，灵活的独立探头电极14，和激光热解多孔石墨烯已用于柔性，低阻抗微电化学（微-ECoG）电极15。尽管它们有希望，但缺乏可扩展的装配方法限制了纳米材料在神经接口电极中的广泛应用。碳基纳米材料尤其具有疏水性，因此需要使用表面活性剂16、超酸17或表面功能化18，形成用于溶液加工制造方法的水分散体，而化学气相沉积（CVD）等替代制造方法通常需要与许多聚合物基材19、20、21不相容的高温， 22.
最近，一类二维（2D）纳米材料，称为MXenes，已被描述，它提供了高导电性，灵活性，体积电容和固有的亲水性的特殊组合，使其成为一个有前途的一类纳米材料的神经接口电极23。MXenes 是一个 2D 过渡金属碳化物和硝化物系列，最常见的是选择性地从分层前体蚀刻 A 元素。这些通常是 MAX 相位与一般公式 Mn_1AXn，其中 M 是早期过渡金属，A 是周期表的组 12-16 元素，X 是碳和/或氮，n = 1、2 或 324。二维 MXene 片具有表面终止功能组，可包括羟基 （+OH）、氧 （+O） 或氟 （+F）。这些功能组使 MXenes 具有固有的亲水性，并实现灵活的表面改性或功能化。在大类MXenes中，Ti3C2的研究范围最广，特征为25、26、27。Ti3C2显示的体积电容 （1，500 F/cm3）28比活性石墨烯 （+60+100 F/cm3）29，硬质合金衍生碳素 （180 F/cm3）30，和石墨烯凝胶薄膜 （+260 F/cm3）31。此外，Ti3C2表现出极高的电子电导率 （+10，000 S/cm）32，其生物相容性已在若干研究中证明33、34、35、36。Ti3C2薄膜的高容积电容有利于生物传感和刺激应用，因为具有电容电荷转移的电极可以避免潜在的有害水解反应。
我们小组最近展示了灵活的薄膜Ti3C2微电极阵列，使用溶液处理方法制备，能够记录微电图（微-ECoG）和皮内神经尖峰活性，体内SNR36。与尺寸匹配的金 （Au） 电极相比，这些 MXene 电极的阻抗显著减少，这可归因于 MXene 的高导电性和电极的高表面积。在该协议中，我们描述了在柔性丙烯-C基板上制造Ti3C2 MXene的平面微电极阵列并将其用于术中微ECoG记录的关键步骤。此方法利用了 MXene 的亲水性，这使得可以使用简单且可扩展的解决方案处理方法，同时无需使用表面活性剂或超酸剂来实现稳定的水悬浮液。这种易于加工性可使 MXene 生物传感器在工业规模上具有成本效益，这是广泛采用基于其他碳纳米材料的设备的主要限制。电极制造的关键创新在于使用牺牲性聚合物层对自旋涂层后的MXene进行微模式，该方法根据溶液加工的聚（3，4-乙烯二氧硫磷）:p丙（苯乙烯磺酸）（PEDOT：PSS）微电极37进行了文献改编，但之前没有为MXene的图案描述。Ti3C2具有卓越的电气特性，加上其可加工性和二维形态学，使其成为神经接口非常有前途的材料。特别是，Ti3C2为克服电极几何面积和电化学接口阻抗之间的基本权衡提供了一条途径，电化学接口阻抗是微尺度电极性能的主要限制因素。此外，该协议中描述的制造过程可以调整以生产不同尺寸和几何形状的 MXene 电极阵列，以适应不同的记录模式，并且还可以轻松地进行调整，以合并除 MXene 之外的其他导电油墨。
Protocol
所有体内程序均符合美国国家卫生研究院（NIH）《实验室动物护理和使用指南》，并经宾夕法尼亚大学动物护理和使用委员会（IACUC）批准。 1. Ti3C2 MXene 合成 注：本节中描述的反应程序适用于化学烟气罩内。本步骤中包含的清洗步骤旨在与平衡的离心管一起使用。所有产生的废物均被视为危险废物，应按照大学准则适当丢弃。 <p …
Representative Results
在MXene微电极阵列上记录的微ECoG数据样本如图5所示。在将电极阵列应用于皮层后，清晰的生理信号立即在记录电极上显现出来，所有 MXene 电极上都出现了大约 1 mV 振幅 ECoG 信号。这些信号的功率谱证实了在氯胺酮-脱模素麻醉下大鼠中常见的两种大脑节律的存在：1⁄2 Hz缓慢振荡和40-70 Hz的β振荡。 此外，在慢振荡的”下降”状态下，观察到一个特征宽带功率衰减，在慢振荡的”…
Discussion
本议定书中描述的MXene合成和脱毛程序（HF/HCl/LiCl）采用MILD蚀刻方法，采用LiF/HCl（原位HF）蚀刻介质26。MILD 方法允许一旦达到 pH +5+6 即可在洗涤过程中自发地解压大 Ti3C2片（横向大小为几 μm）。与仅使用高频蚀刻相比，这会产生更高质量的材料，提高材料性能，如电子导电性和化学稳定性。HF/HCl/LiCl 方法利用 MILD 合成改进，同时额外分离每个步骤（蚀刻、间接?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院的支持（赠款号为No.R21-NS106434），公民癫痫研究联合组织飞行奖，米罗夫斯基家庭基金会和尼尔和芭芭拉·斯密特（F.V.）;国家科学基金会研究生研究奖学金计划（赠款号DGE-1845298 到 N.D. 和 B.M.;陆军研究办公室（合作协议号W911NF-18-2-0026至K.M.）;由美国陆军通过埃奇伍德化学生物中心的表面科学倡议计划（PE 0601102A项目VR9到Y.G.和K.M.）。这项工作部分在辛格纳米技术中心进行，该中心得到国家科学基金会国家纳米技术协调基础设施项目（NNCI-1542153）的支持。
Materials
| 00-90 screw | McMaster-Carr | 90910A630 | Skull screw around which ground wire is wrapped |
| 128ch stimulation/recording controller | Intan Technologies | A component of the neural recording system. | |
| 175 mL polypropylene (PP) conical centrifuge tubes | Falcon | REF: 352076 | Used for washing |
| 18 position 0.5 mm pitch ZIF connector | Molex | 505110-1892 | Used to interface the flexible Parylene microelectrode array with the PCB adapter board. |
| 18 position dual row male nano-miniature (.025"/.64mm) connector | Omnetics Connector Corporation | A79008-001 | Used to interface the PCB adapter board to the recording headstage. |
| 3ML Disposable Plastic Set Transfer Graduated Pipettes | Rienar | Rienar-3ML-20PCS | Used for transferring etchant or MXene solutions |
| 50 mL polyproylene (PP) concial centrifuge tube | Falcon | REF: 352070 | Used for washing and size selection |
| Al etchant Type A | Transene | 060-0026000-QT | For removing Al etch mask layer after final Parylene-C etch. |
| Aluminum Powder, -325 Mesh, 99.5% (metals basis), particle size < 44 µm | Alfa Aesar | CAS: 7429-90-5 | Used for MAX synthesis |
| AutoCAD software | Autodesk Inc. | Design software for drawing photomasks. Free alternatives include DraftSight and LayoutEditor. | |
| Buffered Oxide Etchant 6:1 | JT Baker | 1178-03 | For removing SiO2 layer to expose MXene electrode contacts at the end of the fabrication procedure. |
| Buprenorphine SR | Wildlife Pharmaceuticals | Analgesia for rat surgery | |
| Centrifuge | Hermle | Benchmark Z 446 | Used for washing and size selection |
| Dexdomitor | Midwest Veterinary Supply | 193.13250.3 | Anesthesia for rat surgery |
| Drill burr | Fine Science Tools | 19007-07 | Burrs for drill |
| Electric drill | Foredom | K.1070 | Micromotor drill for craniotomies |
| Electron beam evaporator | Kurt J. Lesker Company | Used to evaporate Ti, Au, and SiO2 during fabrication. Most university clean rooms have this or a similar tool. | |
| Ground wire | A-M Systems | 781500 | Bare silver wire |
| Headspace Vial, glass | Supelco | REF: 27298 | Used for storing MXene solutions |
| Hydrochloric acid (12.1N) | Fisher Scientific | CAS: 7647-01-0 | Corrosive; etchant material |
| Hydrofluoric Acid, (48-51% solution in H2O) | Acros | CAS: 7664-39-3 | Etchant material |
| Jupiter II RIE system | March Plasma Systems Inc. | Planar RIE etching system used to etch the Parylene-C using O2 plasma. Most university clean rooms have a comparable planar RIE etching system. | |
| Kapton standard polyimide tape, 1/4" | DuPont | Used to add thickness to the Au bonding pad region of the flexible Parylene microelectrode array for insertion into the ZIF connector. | |
| Ketamine | Hospital of the Univ. of Penn. | Anesthesia for rat surgery | |
| KLA P-7 Stylus Profilometer | KLA Corporation | Used the measure 2D profiles to confirm complete etching through the sacrificial parylene-C layer in step 2.4.2. Most university clean rooms have this or a comparable stylus profilometer tool. | |
| Lithium chloride, 99% for analysis, anhydrous | Acros | CAS: 7447-41-8 | Hygroscopic; delamination material |
| MA6 mask aligner | Karl Suss Microtec AG | Used to align each photomask to the pattern on the wafer and expose the wafer to UV light. Most university clean rooms have this or a similar tool. | |
| Micro-90 cleaning solution | International Products Corporation | M-9050-12 | Used as the anti-adhesive layer to enable removal of the sacrificial Parylene-C layer to pattern the MXene |
| NR71-3000p photoresist | Futurrex Inc. | NR71-3000p | Negative photoresist used to define Ti/Au traces and MXene patterns in the devices. |
| Ophthalmic ointment | Midwest Veterinary Supply | 193.63200.3 | To prevent corneal drying during surgery |
| Parylene deposition system | Specialty Coating Systems | Used to evaporate thin conformal films of Parylene-C | |
| Parylene-C dimer | Specialty Coating Systems | 980130-c-01lbe | Flexible polymer used as bottom and top passivating layers for the flexible MXene devices |
| Photomasks (chrome on soda lime glass) | University of Pennsylvania | Our photomasks were produced in the University clean room using a Heidelberg DWL66+ laser writer system, however several vendors manufacture photomasks from provided design files. | |
| Povidone-iodine solution | Medline | MDS093901 | To help prevent infection around scalp incision |
| Printed Circuit Board (PCB) | Advanced Circuits | Used to interface between the MXene electrode array and the measurement electronics such as the potentiostat and the Intan recording system. Advanced Circuits and other vendors manufacture and assemble PCBs based on the provided design files. | |
| RD6 Developer | Futurrex Inc. | RD6 Developer | Used to develop NR71-3000p negative photoresist following UV exposure |
| Reference 600 potentiostat | Gamry Instruments | Used to measure the electrodes' impedance to assess quality of the devices | |
| Remover PG | MicroChem Corp. | G050200 | Used to remove NR71-3000p following metal deposition to perform lift-off patterning |
| RHS2000 Stim SPI interface cable | Intan Technologies | A component of the neural recording system. | |
| RHS2116 amplifier board | Intan Technologies | A component of the neural recording system. | |
| Si wafers | Wafer World | 2885 | Substrate for fabrication |
| Spin Coater | Cost Effective Equipment | For coating wafers with resists and applying the Micro-90 and MXene layers. Most university clean rooms have spin coaters. | |
| Stereotaxic frame | Kopf Instruments | Model 902 | For positioning the rat for neurosurgery |
| Teflon-coated magnetic stir bar | Corning | REF: 1233W95 | Used to stir during etching and intercalation |
| Titanium carbide, 99.5% (metals basis), particle size ~2 µm | Alfa Aesar | CAS: 12070-08-5 | Used for MAX synthesis |
| Titanium powder, -325 mesh, 99% (metals basis), particle size < 44µm | Alfa Aesar | CAS: 7440-32-6 | Used for MAX synthesis |
| Ultrasonic bath sonicator | Reynolds Tech | For removing metal and photoresist particles during lift-off processes to pattern metals. | |
| UV vis spectrophotometer | ThermoScientific | Evolution 201 | Used to determine concentration and observe absorption peak |
| Zetasizer, Particle Size Analysis | Malvern Panalytical | Nano ZS | Used to determine particle lateral size distibution |
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Driscoll, N., Maleski, K., Richardson, A. G., Murphy, B., Anasori, B., Lucas, T. H., Gogotsi, Y., Vitale, F. Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording. J. Vis. Exp. (156), e60741, doi:10.3791/60741 (2020).