This study was supported in part by Merit Review Award IRX002611 (Capadona) and Research Career Scientist Award IK6RX003077 (Capadona) from the United States (US) Department of Veterans Affairs Rehabilitation Research and Development Service. Additionally, this work was also supported in part by the National Institute of Health, National Institute of Neurological Disorders and Stroke R01NS110823 (Capadona/Pancrazio), and the National Science Foundation Graduate Research Fellowship Program (Krebs).

All the coding files for 3D printing are provided in Supplementary Coding Files 1-16. The analysis provided in the Representative results is described using commercially acquired functional silicon planar electrode arrays (see Table of Materials).
1. Handling assembly for gas-phase deposition in a vacuum desiccator
NOTE: The assembled apparatus for handling and holding devices during gas-phase deposition is shown in <...

The described protocol was designed for the surface treatment of silicon planar microelectrode arrays. The 3D printed tools are customized to Michigan-style microelectrode arrays with low-profile connectors50. Non-functional probes were assembled by adhering a silicon probe to 3D printed tabs using a biocompatible adhesive. The 3D printed tabs were designed with similar dimensions to the connectors incorporated on the commercially available devices used. Files for the 3D printed tabs are available...

Neuroprosthetic devices aim to restore impaired or absent sensory and motor abilities in a wide range of patient populations, including those with spinal cord injury, Amyotrophic Lateral Sclerosis (ALS), cerebral palsy, and amputations1,2,3. Intracortical microelectrodes (IMEs) can establish a communication pathway between cortical neurons and the devices used to control neuroprosthetics. A distinct advantage of intracortical microelectrodes is their capability to record neural signals at the high spatial and temporal resolution, which is preferred for subsequent signal processing and control of brain-computer interfaces4,5. Unfortunately, the performance of intracortical microelectrodes dramatically reduces within months to a year following implantation2,6,7,8. The loss of signal quality and stability negatively affects the application of the technology.
A significant contributor to the observed performance decline is the biotic response to implantation-associated tissue damage and chronic neuroinflammation9,10,11. Implantation of IMEs inflicts damage on brain tissue, resulting in the release of signaling molecules that initiate cascades of reactionary cellular defense processes. Chronic interfacing exacerbates the foreign body response, leading to sustained neuroinflammation that damages tissue proximal to the device; often recognized as symptoms of neuroinflammation, scarring, and local neurodegeneration contributing to the decline of the recording of the signal quality12,13,14,15. Comprising a dense conglomerate of astrocytes with entrained activated microglia and macrophages, the scar that encapsulates the electrode creates an unfavorable local environment with reduced material transport and local accumulation of inflammatory factors16,15,16,17,18.
Many studies have described the brain&#39;s response to intracortical microelectrodes or approaches to mitigate the response7. Research and development into improving the tissue response have involved a range of strategies, including modifications to the overall structure, surface topology, materials, and coatings application. These efforts intend to minimize damage sustained from the implantation event, introduce a more favorable interface between the device and proximal cells, or reduce the tissue strain after devices are implanted7. Methods specifically targeting the chronic biologic response have led to several bioactive coatings that aim to stabilize the implantation site and chemically promote cell health. Examples include conductive polymers such as poly(ethylene dioxythiophene) (PEDOT)19,20, carbon nanotubes21, hydrogels22, and the addition of bioactive molecules and drugs to target specific cellular processes23,24,25. Our research group, in particular, have explored many mechanisms to promote a reduction of the inflammatory response to implanted microelectrodes including, but not limited to, minimizing the trauma associated with device implantation26, minimizing the device/tissue stiffness mismatch27,28,29,30,31,32,33, optimizing sterilization procedures34,35, reducing oxidative stress/damage28,36,37,38,39,40,41,42, exploring alternative electrode materials43, and mimicking the nano-architecture of the natural extracellular matrix44,45,46. Recent interest is the development of biomimetic surface coatings to mitigate the neuroinflammatory response at the microelectrode tissue interface directly39.
Modification of the interface offers the unique benefit of directly targeting the wound and the proximal tissue necessary for signal recording. A surface treatment that promotes healing without exacerbating the immune response can benefit the lifetime of quality recording and remove limitations in realizing the therapeutic and research potential of intracortical microelectrodes. The presented work details methods for applying surface treatments to microelectrode arrays that require extended reaction times while accommodating the fragility of the devices. The presented technique is intended to share surface modification methods to functional devices where the device cannot be handled throughout the treatment application. The tools are presented for handling non-functional dummy probes and functional silicon planar microelectrode arrays.
The presented approach to modify the electrode surface allows for the secure suspension of non-functional dummy probes or functional silicon planar electrode arrays for gas-phase deposition and reaction with aqueous solutions. Several 3D printed pieces are used to handle these fragile devices (Figure 1 and Figure 2). An example is provided of a procedure that utilizes both gas and solution phase steps for the surface modification with an antioxidative coating involving the immobilization of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP). MnTBAP is a synthetic metalloporphyrin possessing antioxidant properties with demonstrated mediation of inflammation47,48. The provided example on functional silicon planar electrode arrays validates an update to a previously reported protocol for non-functional devices40. The adaptation of a gas phase deposition technique from Munief et al. supports the protocol&#39;s compatibility with functional electrodes49. The gas-phase deposition is utilized to amine functionalize the surface in preparation for the aqueous reaction involving carbodiimide crosslinker chemistry to immobilize the active MnTBAP. The handling methodology developed here is provided as a platform that can be modified to accommodate other coatings and similar devices.
The protocol illustrates the approach using non-functional dummy probes comprising a silicon shank and 3D printed tab with similar dimensions to the functional silicon planar electrode arrays. The connector packaging of the device is considered analogous to the 3D printed tab of the non-functional dummy probe in the provided instruction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63500/63500fig01.jpg" /> 
Figure 1: 3D printed pieces for handling functional devices during the gas-phase deposition in a vacuum desiccator. (A) The structure&#39;s base includes holders for 1 cm x 1 cm sample silicon squares (top arrow) and holes for securing to desiccator plate (bottom arrow). (B) The plate is used to secure the suspension of devices. From here onward, each piece in this figure will be referred to as either piece 1A or 1B. Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/63500/63500fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63500/63500fig02.jpg" /> 
Figure 2: 3D printed pieces for handling functional devices for the surface reaction occurring in the aqueous solution. (A) Guide piece to be glued to the lid of the culture plate. (B) Benchtop pieces used to stabilize pieces (C) and (D) while assembling. (C) and (D) together secure the suspension of devices for placement in the well plate, and (E) further secures pieces (C) and (D) to the well plate lid. From here onward, individual pieces in each panel of this figure will be referred to as piece numbers corresponding to the panel number of this figure. Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/63500/63500fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>165344-1G</td>
			<td>Solid, stored desiccated at -20 &#176;C</td>
		</tr>
		<tr>
			<td>15 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>18 Pound Solid Nylon Cable/Zip Ties</td>
			<td>Cole-Parmer</td>
			<td>EW-06830-66</td>
			<td>Length 4 inches</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>4432-31-9</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>3-aminopropyltriethoxysilane (APTES)</td>
			<td>Sigma-Aldrich</td>
			<td>440140-100ML</td>
			<td>Liquid, container with Sure/Seal</td>
		</tr>
		<tr>
			<td>50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>01-213-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum weighing dishes</td>
			<td>Fisher Scientific</td>
			<td>08-732-102</td>
			<td>Diameter 66 mm</td>
		</tr>
		<tr>
			<td>Bel-Art Vacuum Desiccator</td>
			<td>Fisher Scientific</td>
			<td>08-594-15B</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar TC-Treated Multiple Well Plates</td>
			<td>Millipore Sigma</td>
			<td>CLS3527-100EA</td>
			<td>24-well plate, polystyrene</td>
		</tr>
		<tr>
			<td>Cyanoacrylate Adhesive</td>
			<td>LocTite</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Microscope</td>
			<td>Keyence</td>
			<td>VHX-S750E</td>
			<td></td>
		</tr>
		<tr>
			<td>Disco DAD3350 Dicing Saw</td>
			<td>Disco</td>
			<td>DAD3350</td>
			<td>Used to cut silicon wafer into 1 cm x 1 cm samples</td>
		</tr>
		<tr>
			<td>Double-Sided Polyimide Tape</td>
			<td>Kapton Tape</td>
			<td>PPTDE-1/4</td>
			<td>&#188;&#8221; x 36 yds.</td>
		</tr>
		<tr>
			<td>EP21LVMed &#8211; low viscosity, two component epoxy compound</td>
			<td>Masterbond</td>
			<td>EP21LVMed</td>
			<td>Meets USP Class VI certification, Passes ISO 10993-5 for cytotoxicity</td>
		</tr>
		<tr>
			<td>Epilog Fusion Pro 48 Laser Machine</td>
			<td>Epilog</td>
			<td>N/A</td>
			<td>CO2 laser</td>
		</tr>
		<tr>
			<td>Foam tape</td>
			<td>XFasten</td>
			<td>N/A</td>
			<td>1/8&#34; Thick</td>
		</tr>
		<tr>
			<td>Gamry Interface 1010E Potentiostat</td>
			<td>Gamry</td>
			<td>992-00129</td>
			<td></td>
		</tr>
		<tr>
			<td>High precision 45&#176; curved tapered very fine point tweezers/forceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab tape</td>
			<td>Fisher Scientific</td>
			<td>15-901-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP)</td>
			<td>EMD Millipore</td>
			<td>475870-25MG</td>
			<td>Solid, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>N-Hydroxysulfosuccinimide sodium salt, &#8805;98% (HPLC)</td>
			<td>Sigma-Aldrich</td>
			<td>56485-250MG</td>
			<td>Solid, stored desiccated at 4&#176;C</td>
		</tr>
		<tr>
			<td>Platinum clad niobium mesh anode</td>
			<td>Technic</td>
			<td>N/A</td>
			<td>Clad with 125&#956;&#8221; of platinum on one side, framed in titanium with (1) 1&#8221; x 6&#8221; titanium strap centered on one 6&#8221; dimension</td>
		</tr>
		<tr>
			<td>Silicon Planar Microelectrode Array, 16 Channel</td>
			<td>NeuroNexus</td>
			<td>A1x16-3mm-100-177-CM16LP</td>
			<td>Electrode site material is iridium, shank thickness is 15 &#956;m</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>University Wafer</td>
			<td>1575</td>
			<td>Diameter 100 mm, p-type, boron-doped, 100 oriented, resistivity 0.01-0.02 Ohm-cm, thickness 525 um, single side polished, prime grade</td>
		</tr>
		<tr>
			<td>Silver/silver Chloride reference electrode</td>
			<td>Gamry Instruments</td>
			<td>930-00015</td>
			<td></td>
		</tr>
		<tr>
			<td>Solidworks</td>
			<td></td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Phillips Flat Head Screws</td>
			<td>McMaster Carr</td>
			<td>96877A629</td>
			<td>#8-32, 1 1/2&#34;, fully threaded</td>
		</tr>
		<tr>
			<td>Type I deionized water</td>
			<td>ChemWorld</td>
			<td>CW-DI1-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultimaker 3 3D printer</td>
			<td>Ultimaker</td>
			<td>&#160;N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultimaker Cura</td>
			<td>Ultimaker</td>
			<td>N/A</td>
			<td>3D printing software</td>
		</tr>
		<tr>
			<td>Ultimaker NFC ABS Filament</td>
			<td>Dynamism, Inc.</td>
			<td>1621</td>
			<td>2.85 mm</td>
		</tr>
		<tr>
			<td>Ultimaker NFC PLA Filament</td>
			<td>Dynamism, Inc.</td>
			<td>1609</td>
			<td>2.85 mm</td>
		</tr>
		<tr>
			<td>Vacuum Gauge Vacuum Gauge</td>
			<td>Measureman Direct</td>
			<td>N/A</td>
			<td>Glycerin Filled, 2-1/2&#8221; Dial Size, &#188;&#8221;NPT, -30&#8221; Hg/-100kpa-0</td>
		</tr>
		<tr>
			<td>Wing nuts</td>
			<td>Everbilt</td>
			<td>934917</td>
			<td>#8-32, zinc plated</td>
		</tr>
	</tbody>
</table>

tools for surface treatment of silicon planar intracortical microelectrodes

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation durations. A substantial contributor to the observed decline is the damage to the neural tissue proximal to the implant and subsequent neuroinflammatory response. Efforts to improve device longevity include chemical modifications or coating applications to the device surface to improve the tissue response. Development of such surface treatments is typically completed using non-functional "dummy" probes that lack the electrical components required for the intended application. Translation to functional devices requires additional consideration given the fragility of intracortical microelectrode arrays. Handling tools greatly facilitate surface treatments to assembled devices, particularly for modifications that require long procedural times. The handling tools described here are used for surface treatments applied via gas-phase deposition and aqueous solution exposure. Characterization of the coating is performed using ellipsometry and x-ray photoelectron spectroscopy. A comparison of electrical impedance spectroscopy recordings before and after the coating procedure on functional devices confirmed device integrity following modification. The described tools can be readily adapted for alternative electrode devices and treatment methods that maintain chemical compatibility.

To demonstrate the use of the handling components, the described methodology was implemented to adapt the immobilization of an oxidant mediator to activated silicon. The application of this chemistry to IMEs to reduce oxidative stress was devised by Potter-Baker et al. and demonstrated on non-functional silicon dummy probes40. This surface treatment immobilizes the antioxidant, MnTBAP, to UV/ozone activated silicon surface via amine functionalization followed by carbodiimide crosslinking ...

Watch this Scientific Journal Video about Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes at JoVE.com

Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

The present protocol describes tools for handling silicon planar intracortical microelectrodes during treatments for surface modification via gas deposition and aqueous solution reactions. The assembly of the components used to handle the devices throughout the procedure is explained in detail.

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation ...

This protocol provides handling tools for the surface modification of intracortical microelectrode devices by gas-phase deposition and aqueous solution reaction. These handling methods maintain device integrity throughout extended reaction times. Methods for handling intracortical microelectrode devices are often not disclosed.This technique can benefit the research efforts to improve the performance of these devices by developing surface treatments and coatings. To begin, acquire intracortical microelectrode devices or nonfunctional probes for surface treatment. To facilitate material testing, acquire one-centimeter-squared samples of the substrate material for treatment alongside the devices.3D print or acquire pieces 1A and 1B. Attach a double-sided polyimide tape to piece 1A. Also, attach a 3.17-millimeter-thick foam strip with one-side adhesive to piece 1B.Then, adhere the connector packaging of the device to the tape on piece 1A. Join pieces 1A and 1B by aligning the holes and securing using stainless steel screws and wing nuts. Fasten the assembly to the vacuum desiccator tray using zip ties, utilizing the holes in the bottom of piece 1A.Place square material samples into the slits at the bottom of the frame. Place the solution in an appropriate receptacle in the vacuum desiccate opposite and in-line with the secured assembly. Put a vacuum gauge in the desiccator to record the exact pressure.Then, position the port of the desiccated lid near the secured assembly and in-line with the solution and complete the gas-phase deposition. First, cut rectangular holes of dimensions 19-by-10.5 millimeters into the lid of the well plate to suspend the electrode array of the device in solution. 3D print or acquire the guides.Align the rectangular holes in the guides to the holes in the lid, ensuring that the hole in the guide is unobstructed. Secure the guides to the lid using cyanoacrylate adhesive. Then, fill the desired solution in the wells where treatment will occur.To confirm surface treatment, submerge the square substrate samples in the reaction solution in a well of the plate. To assemble the probe device, tape piece 2B to a bench top. Place a double-sided polyimide tape to cover the base of piece 2C.Also, place a 3.17-millimeter foam tape with single-side adhesive to cover the base of piece 2D. Then, fit piece 2C into the groove of piece 2B. Adhere the connector packaging of the device onto the tape, oriented so the length of the device shank is suspended.Secure the device by sliding piece 2D into piece 2C. Hold the edges of the assembly and carefully lift to remove from piece 2B. Align the outward-facing semi-circles on pieces 2C and 2D with the corresponding guides on piece 2A to fit the assembly into the lid of the well plate.Secure assembly placement by press-fitting piece 2E over the guides. After suspending the devices in the well plate, transfer the assembly to a shaker and run at speeds under 100 rotations per minute. For reactions requiring multiple solutions or wash steps, carefully transfer the lid to a new well plate containing the desired solution in the appropriate well.After this step, tape piece 2B to a bench top. To remove devices from the well plate, remove piece 2E from the lid. Then, carefully remove the assembly by holding the device.Orient the assembly such that piece 2C faces the bench top and piece 2D faces upward. Align the shank of the device parallel to the bench top. Fit piece 2C of the assembly into piece 2B.Apply slight pressure on the tabs of piece 2C into the bench to separate piece 2D from piece 2C. Using forceps, remove the connector packaging of the device from the tape and transfer the device to a storage container. The surface treatment of Michigan-style microelectrode arrays in silicon square samples was demonstrated using this protocol.The gas-phase deposition method was applied for amine functionalization using APTES. Following this, carbodiimide cross-linking chemistry was used to immobilize manganese TBAP. After deposition ellipsometry measurements from the silicon samples produced a mean APTES layer thickness of 8.5 angstroms, compared to the theoretical monolayer thickness of 7 angstroms.X-ray photo-electron spectroscopy analysis showed an increase in the percentage atomic concentrations of nitrogen and carbon after the gas-phase APTES treatment, indicating chemical deposit. Similarly, manganese was detected following the solution-phase immobilization. Further, the Bode plot for electrical impedance spectroscopy analysis of the microelectrode arrays showed no statistical difference between the impedance magnitudes before and after the coating process.Thus, successful coating of the electrode array was performed using the coating processes. This protocol facilitates surface modification of intracortical microelectrodes by minimizing the risk of damage to the device. Others in the field may adapt the methodology to their devices and chemical procedures.

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Research

JoVE Journal

Bioengineering

2.3K vistas.  Case Western Reserve University. Este protocolo proporciona herramientas de manipulación para la modificación superficial de dispositivos microelectrodos intracorticales mediante deposición en fase gaseosa y reacción de solución acuosa. Estos métodos de manipulación mantienen la integridad del dispositivo a lo largo de tiempos de reacción prolongados. Los métodos para manejar dispositivos de microelectrodos intracorticales a menudo no se divulgan.Esta técnica puede beneficiar los esfuerzos de investigación ...