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 Figure 3. Steps 1.1-1.8 describe the procedure required to place the devices into the apparatus for deposition (Figure 4A).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63500/63500fig03.jpg" /> 
Figure 3: Assembly of 3D printed pieces for handling functional devices during gas-phase deposition. The assembly is pictured without samples to be coated. Screws and wing nuts are used to fasten pieces 1A and 2B together. <a href="https://www.jove.com/files/ftp_upload/63500/63500fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63500/63500fig04.jpg" /> 
Figure 4: Image of assembly and placement of samples to be coated. This scheme describes the handling of functional devices during gas-phase deposition secured within a vacuum desiccator. (A) Double-sided polyimide tape placed on piece 1A and foam tape placed on 1B. (B) Devices secured onto tape. (C) Screws and wing nuts are used to fasten pieces 1B to 1A, and the assembly is attached to the desiccator tray using zip cable ties (red arrows). (D) 1 cm x 1 cm silicon square samples are placed into respective holders. (E) The aluminum weigh dish and pressure gauge are placed into the desiccator in the orientation shown. <a href="https://www.jove.com/files/ftp_upload/63500/63500fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>For surface treatment, acquire 1 cm x 1 cm square samples of the devices&#39; substrate material.
	<ol>
		<li>For silicon samples (selected for this protocol), cut the silicon wafer into 1 cm x 1 cm squares using a wafer dicing machine (see Table of Materials).</li>
	</ol>
	</li>
	<li>Print or acquire pieces 1A (Figure 1A, Supplementary Coding File 1, Supplementary Coding File 2) and 1B (Figure 1B, Supplementary Coding File 3, Supplementary Coding File 4).</li>
	<li>Attach double-sided polyimide tape to piece 1A and attach 1/8&#34; thick foam strip with one-side adhesive to piece 1B.</li>
	<li>Adhere the connector packaging of the device to the tape on piece 1A. 
	NOTE: The ideal orientation of the connector on the tape will leave the shank suspended over the edge, as shown in Figure 4B.</li>
	<li>Secure piece 1A and piece 1B together (Figure 4C). Align the holes and secure using stainless steel screws and wing nuts (see Table of Materials).</li>
	<li>Using zip ties, fasten the assembly to the vacuum desiccator tray using the holes in the bottom of piece 1A as shown in Figure 4C.</li>
	<li>If applicable, place square material samples into the slits at the bottom of the frame (Figure 4D). Here, 1 cm x 1 cm square silicon wafer diced samples are used as an example. 
	NOTE: The exact material will need to match the substrate of the treated device, which will vary depending on the device.</li>
	<li>Complete the gas-phase deposition by placing the solution into an appropriate receptacle within the vacuum desiccator opposite and in line with the secured assembly. 
	NOTE: Aluminum weigh dishes were used as receptacles for the (3-Aminopropyl)triethoxysilane (APTES) deposition, as an example here.
	<ol>
		<li>Place a vacuum gauge (see Table of Materials) within the desiccator to record the exact pressure. Position the port of the desiccator lid near the secured assembly and in line with the solution (Figure 4E). 
		NOTE: Further details regarding this method of gas-phase deposition are described in a previously published Reference49.</li>
	</ol>
	</li>
</ol>
2. Handling assembly for surface reaction via aqueous solution
NOTE: The components and assembled apparatus for handling and holding devices during aqueous phase deposition and surface treatment are illustrated in Figures 5-7. The following steps will detail the procedure required to place the devices into the apparatus for deposition and treatment.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63500/63500fig05.jpg" /> 
Figure 5: Assembly of 3D printed pieces for handling functional devices for the surface reaction occurring in aqueous solution. (A) Guide piece to be glued to the lid of the culture plate. (B) The benchtop piece was used to stabilize pieces (C) and (D) while assembling. (C) and (D) together secure the suspension of devices for placement in the well plate. (E) further secures pieces (C) and (D) to the well plate lid. Double-sided polyimide tape was placed on the lower portion of (C), and foam tape was placed on the lower portion of (D) (both boxed in red). <a href="https://www.jove.com/files/ftp_upload/63500/63500fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63500/63500fig06.jpg" /> 
Figure 6: Cell culture plate lid constructed with 6 guides (piece 2A). <a href="https://www.jove.com/files/ftp_upload/63500/63500fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63500/63500fig07.jpg" /> 
Figure 7: Sequence for securing and loading probes for solution reaction. The color of the parts was changed in this figure for clarity within the image. These are the same parts as Figure 5 and Figure 6. (A) Piece 2C is placed into piece 2B, and the device is secured to the taped portion of 2C. (B) Piece 2D fits into piece 2C to create an assembly that suspends the device shank. (C) The assembly of 2C, 2D, and the device is carefully positioned onto the lid of the well plate using the guide. (D) Piece 2E fits on top of the assembly to further secure the lid. <a href="https://www.jove.com/files/ftp_upload/63500/63500fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Construct a lid for the well plate to suspend the electrode array of the device in solution (Figure 6). This protocol describes the use of a 24-well plate.
	<ol>
		<li>Cut rectangular holes 19 mm x 10.5 mm into the lid of the well plate using a laser cutter or manually with a box cutter. Match the number of holes to the number of devices desired for treatment. 
		NOTE: For ease of assembly, it is recommended to treat six devices per well plate, or at minimum, place holes over non-adjacent wells (Figure 6).</li>
		<li>Print or acquire the appropriate number of guides (piece 2A (Figure 2A), Supplementary Coding File 5, Supplementary Coding File 6).</li>
		<li>Use cyanoacrylate adhesive to secure guides to the lid. Align the rectangular holes in the guides and lids while gluing to ensure the guide&#39;s rectangular hole is unobstructed, as shown in Figure 6.</li>
	</ol>
	</li>
	<li>Fill the well plate with the desired solution at locations where treatment will occur. For example purposes, the solution comprises EDC and Sulfo-NHS (see Table of Materials) in MES buffer. 
	NOTE: The volume of the solution will depend on the electrode device&#39;s dimensions. For Michigan-style microelectrode arrays (see Table of Materials) with low-profile connectors of 8.6 mm and shank length of 3 mm, there is ~9 mm clearance50. Using 2 mL of solution will allow for the shank of the device to be fully submerged while keeping the remainder of the device out of the reaction solution.
	<ol>
		<li>If substrate samples are being used to confirm surface treatment, place square material samples in a well of the plate and submerge them in the reaction solution.</li>
	</ol>
	</li>
	<li>Securely suspend the devices (see Table of Materials) in a well plate. The sequence is shown in Figure 7.
	<ol>
		<li>Tape piece 2B (Figure 2B, Supplementary Coding File 7, Supplementary Coding File 8) to a benchtop (Figure 7A).</li>
		<li>Place double-sided polyimide tape to cover the base of piece 2C (Figure 2C, Supplementary Coding File 9, Supplementary Coding File 10).</li>
		<li>Place 1/8&#34; foam tape with single-side adhesive to cover the base of piece 2D (Figure 2D, Supplementary Coding File 11, Supplementary Coding File 12).</li>
		<li>Fit piece 2C into the groove of piece 2B (Figure 7A).</li>
		<li>Adhere the connector packaging of the device onto the tape, oriented, so the length of the device shank is suspended (Figure 7B).</li>
		<li>Secure the device by sliding piece 2D (shown in orange in Figure 7) into piece 2C. This assembly effectively secures the device between the tool pieces (Figure 7B).</li>
		<li>Holding the edges of the assembly, carefully lift to remove from piece 2A.</li>
		<li>Fit the assembly into the lid by aligning the outward-facing semicircles on pieces 2C and 2D with the corresponding guides on piece 2A (shown in green in Figure 7C).</li>
		<li>Secure assembly placement by press-fitting piece 2E (Figure 2E) over the guides (shown in green in Figure 7D, Supplementary Coding File 13, Supplementary Coding File 14).</li>
		<li>For reactions that benefit from continuous mixing of the solution, agitate the well plate. Transfer the assembled well plate to a shaker table and run at speeds under 100 rpm.</li>
	</ol>
	</li>
	<li>If multiple solution-based reactions or wash steps are desired, carefully transfer the lid to a new well plate with desired solution(s) distributed to the appropriate wells. 
	NOTE: Step 2.4 is optional.</li>
	<li>Remove devices from the well plate.
	<ol>
		<li>Tape piece 2B to a benchtop.</li>
		<li>Remove piece 2E from the lid.</li>
		<li>Carefully remove the assembly holding the device from the well plate.</li>
		<li>Orient the assembly, so that piece 2C faces the benchtop and piece 2D faces upward. The shank of the device needs to be parallel to the benchtop. Fit piece 2C of the assembly into piece 2B as was completed previously (step 2.3.4) when fitting together the assembly.</li>
		<li>Separate piece 2D from piece 2C by carefully pulling them apart. Apply slight pressure on the tabs of piece 2C into the bench to provide stability for this task. 
		NOTE: The tabs of 2C are longer than that of 2D to facilitate this handling.</li>
		<li>Use forceps to hold onto the device&#39;s connector packaging to remove from the tape and transfer the device to the desired storage container.</li>
	</ol>
	</li>
</ol>

The contents do not represent the views of the US Department of Veterans Affairs, the National Institutes of Health, or the United States Government.

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><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 &amp;deg;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>&amp;frac14;&amp;rdquo; x 36 yds.</td></tr><tr><td>EP21LVMed &amp;ndash; 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>CO&lt;sub&gt;2&lt;/sub&gt; laser</td></tr><tr><td>Foam tape</td><td>XFasten</td><td>N/A</td><td>1/8&quot; 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&amp;deg; 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 &amp;deg;C</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Hydroxysulfosuccinimide sodium salt, &amp;ge;98% (HPLC)</td><td>Sigma-Aldrich</td><td>56485-250MG</td><td>Solid, stored desiccated at 4&amp;deg;C</td></tr><tr><td>Platinum clad niobium mesh anode</td><td>Technic</td><td>N/A</td><td>Clad with 125&amp;mu;&amp;rdquo; of platinum on one side, framed in titanium with (1) 1&amp;rdquo; x 6&amp;rdquo; titanium strap centered on one 6&amp;rdquo; 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 &amp;mu;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&quot;, 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>&amp;nbsp;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&amp;rdquo; Dial Size, &amp;frac14;&amp;rdquo;NPT, -30&amp;rdquo; 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 ...

tools-for-surface-treatment-silicon-planar-intracortical

Research

JoVE Journal

Bioengineering

2.4K Views.  Case Western Reserve University. 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.

Video: Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Neuroscience

A Novel Method for In Situ Electromechanical Characterization of Nanoscale Specimens

Electrically assisted deformation (EAD) is increasingly being used to improve the formability of metals during processes such as sheet metal rolling and forging. Adoption of this technique is proceeding despite disagreement concerning the underlying mechanism responsible for EAD. The experimental procedure described herein enables a more explicit study compared to previous EAD research by removing thermal effects, which are responsible for disagreement in interpreting previous EAD results. Furthermore, as the procedure described here enables EAD observation in situ and in real time in a transmission electron microscope (TEM), it is superior to existing post-mortem methods that observe EAD effects post-test. Test samples consist of a single crystal copper (SCC) foil having a free-standing tensile test section of nanoscale thickness, fabricated using a combination of laser and ion beam milling. The SCC is mounted to an etched silicon base that provides mechanical support and electrical isolation while serving as a heat sink. Using this geometry, even at high current density (~3,500 A/mm2), the test section experiences a negligible temperature increase (&#60;0.02 &#176;C), thus eliminating Joule heating effects. Monitoring material deformation and identifying the corresponding changes to microstructures, e.g. dislocations, are accomplished by acquiring and analyzing a series of TEM images. Our sample preparation and in situ experiment procedures are robust and versatile as they can be readily utilized to test materials with different microstructures, e.g., single and polycrystalline copper.

A Novel Method for In Situ Electromechanical Characterization of Nanoscale Specimens

Isolating electrical and thermal effects on electrically assisted deformation (EAD) is very difficult using macroscopic samples. Metallic sample micro- and nanostructures together with a custom test procedure have been developed to evaluate the impact of applied current on the formation without joule heating and evolution of dislocations on these samples.

Electrically assisted deformation (EAD) is increasingly being used to improve the formability of metals during processes such as sheet metal rolling and ...

Engineering

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single assembly step and prevents biofouling on silicon and gold surfaces. The SAM is used to coat microelectrodes patterned with standard, positive-tone lithography. Using the microtubule as an example, we apply a DC voltage to induce electrophoretic migration to the SAM-coated electrode in a reversible manner. A flow chamber is used for imaging the electrophoretic migration and microtubule patterning in situ using epifluorescence microscopy. This method is generally applicable to biomolecule patterning, as it employs electrophoresis to immobilize target molecules and thus does not require specific molecular interactions. Further, it avoids problems encountered when attempting to pattern the SAM molecules directly using lithographic techniques. The compatibility with electron beam lithography allows this method to be used to pattern biomolecules at the nanoscale.

We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

Biology

Metal-Assisted Electrochemical Nanoimprinting of Porous and Solid Silicon Wafers

Metal-assisted electrochemical imprinting (Mac-Imprint) is a combination of metal-assisted chemical etching (MACE) and nanoimprint lithography that is capable of direct patterning 3D micro- and nanoscale features in monocrystalline group IV (e.g., Si) and III-V (e.g., GaAs) semiconductors without the need of sacrificial templates and lithographical steps. During this process, a reusable stamp coated with a noble metal catalyst is brought in contact with a Si wafer in the presence of a hydrofluoric acid (HF) and hydrogen peroxide (H2O2) mixture, which leads to the selective etching of Si at the metal-semiconductor contact interface. In this protocol, we discuss the stamp and substrate preparation methods applied in two Mac-Imprint configurations: (1) Porous Si Mac-Imprint with a solid catalyst; and (2) Solid Si Mac-Imprint with a porous catalyst. This process is high throughput and is capable of centimeter-scale parallel patterning with sub-20 nm resolution. It also provides low defect density and large area patterning in a single operation and bypasses the need for dry etching such as deep reactive ion etching (DRIE).

A protocol for metal-assisted chemical imprinting of 3D microscale features with sub-20 nm shape accuracy into solid and porous silicon wafers is presented.

Metal-assisted electrochemical imprinting (Mac-Imprint) is a combination of metal-assisted chemical etching (MACE) and nanoimprint lithography that is ...

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

Summary

Explore More Videos

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

A Novel Method for In Situ Electromechanical Characterization of Nanoscale Specimens

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Metal-Assisted Electrochemical Nanoimprinting of Porous and Solid Silicon Wafers

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

Fabrication of Ti₃C₂ MXene Microelectrode Arrays for In Vivo Neural Recording

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain