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 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 ...

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

Research

JoVE Journal

Bioengineering

2.3K 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

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

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 ...