This work was sponsored by the Defense Advanced Research Projects Agency (DARPA) MTO under the auspices of Dr. Jack Judy and Dr. Doug Weber through the Space and Naval Warfare Systems Center, Pacific Grant/Contract No.N66001-12-C-4173. We would like to thank Thomas Eggers for his help in the fabrication process, and Ronald Triolo, Matthew Schiefer, Lee Fisher and Max Freeburg for their contribution in the development of the composite nerve cuff design.

1. Electrode Components Preparation
<ol>
	<li>Gather four electrode components that require precision cut (laser-cut was used, Please refer to the Materials List) prior to the manufacturing process. These components are (Figure 1): 
	Contacts array frame: This frame is made out of 125 &#181;m thick Polyether ether ketone (PEEK) sheet. It covers the entire width of the cuff and holds the middle contacts and has serpentine-shaped edges (Figure 1B). The middle contacts are wrapped in the guide channels; hence the exposed width of the contacts is limited by the width of the channels and the spacing is determined by the spacing between the channels. 
	The middle contacts strips: The middle contacts are formed by wrapping these strips around the contacts array frame (Figure 1B). Cut the strips out of the Platinum/10% Iridium sheet to the width of the guiding channels and add extra length to allow them to be fully folded around the frame. Spot-weld the contact&#39;s lead at 0&#186; angle with the strip&#39;s major axis. 
	The reference contacts: Four references are needed. The long dimension of these contacts is slightly shorter than the cuff width to fully contain them inside the cuff. Spot weld each reference contact to a lead at 90&#186; angle with the contact&#39;s major axis. 
	PEEK spacers: Spacers are used to create thinner region on the electrode to allow bending and closing (Figure 1C). All the spacers are made from PEEK (other material could be used) and cut to the length of the electrode. The width of the middle space is equal to the height of the electrode.</li>
</ol>
2. Contacts Array Preparation
<ol>
	<li>Clean the components made in step 1 by sonication in ethanol for 2 min at 40 KHz and room temperature, then 2 min in distilled deionized water under the same sonication parameters. Let dry.</li>
	<li>Visually inspect the contacts for any defects like laser-cut residuals or surface deformations.</li>
	<li>Position the contacts one by one under the microscope with the welding spot facing up. Hold the contact with tweezers at approximately 1/3 of length starting from the free end. Elevate the lead to a 45&#186; angle while holding the contact to make the first bend.</li>
	<li>Place the pre-bent contact underneath the array frame with the weld facing up. Hold the frame down with tweezers and elevate the lead to a 45&#186; angle to make a second bend. While Continuing holding the frame down, grab the free end of the contact with tweezers and bend at a 180&#186; angle (fold toward the middle line of the frame).</li>
	<li>Straighten and pull the contact toward operator and then bend at 180&#186; angle (fold to the middle line). The spot welding point should now be enclosed in between the two bent ends.</li>
	<li>Repeat steps 2.3 - 2.5 for the remaining contacts. Make as tight as possible. Alternate the contact leads on each side of the array frame.</li>
</ol>
3. Cuff Layout Guide
<ol>
	<li>Create a 2D diagram of the cuff in flat open position. 
	NOTE: Use any CAD software to produce a true-scale diagram. This diagram will determine the dimensions of the electrode and the placement site for the various electrode components.</li>
	<li>Print the 2D diagram on regular printing paper to scale using ordinary printing machine, and then cut out a 5 cm by 5 cm square piece with the drawing located in the center.</li>
	<li>Cut out 5 cm by 5 cm square piece of the thermal transparency sheet (T1) with a scalpel.</li>
	<li>Place the transparency piece T1 on top of the diagram paper, and then place both layers on the base plate with the diagram facing up. Tape them down to the base plate with adhesive tape.</li>
</ol>
4. Electrode Base Layer and Reference Contacts Placement
<ol>
	<li>Cut out 5 cm by 5 cm silicone sheet with a scalpel (S1), and then place it on the transparency layer. Start by dropping one corner then slowly lower the rest of the sheet to avoid trapping air bubbles in between T1 and S1 sheets (Figure 2A).</li>
	<li>Mix approximately 2 g of uncured silicone as directed on manufacturer data sheet. Rigorously stir the two parts together with sterilized wooden stirring stick. Place the mix in a vacuum chamber for 3 min. Cycle the vacuum to eliminate the bubbles as they rise to surface. Preheat the isotemp oven at 130 &#186;C. 
	Note: Latex gloves can inhibit the curing process of the silicone. Latex gloves also contain sulfur which can leave contaminants on the working surfaces. Using nitrile gloves instead is recommended.</li>
	<li>Using the dental pick tool, apply a thin line of uncured silicone along the middle of the spacer segments where they are located on the guiding diagram.</li>
	<li>Place the spacers onto the designated regions, and then press them down against the silicone sheet S1.</li>
	<li>Partially cure the silicone in the isotemp oven for 30 min, let it cool down for 10 min.</li>
	<li>Place the reference contacts onto designated areas. Ensure that the weld points are facing up and contact leads are routed towards the midline of the cuff to exit at the far end. After ensuring correct positioning, press the contacts down onto the silicone layer S1. Deposit uncured silicone into the through-holes.</li>
	<li>Tape down the leads and then fully cure the silicone at 130 &#186;C for 90 min, or overnight at room temperature (Figure 2B).</li>
</ol>
5. Center Contacts Array Placement
<ol>
	<li>Cut out 1.5 cm by 5 cm transparency piece with a scalpel (T2). Tape down the reference leads away from the middle region to prevent them from running underneath the contacts array during the next step.</li>
	<li>Place the contact arrays on the dedicated location with the leads side facing up. Deposit uncured silicone to tack the array in place.</li>
	<li>Place the piece from 5.1 (T2) across the midline of the electrode and over the arrays to hold them down, and then tape the ends while pressing down on the arrays. Manually align the array with the dedicated position. Tape down the leads outside the cuff&#39;s perimeter.</li>
	<li>Place the small fixture bar across the center of the electrode and over the transparency segment T2. Clamp it down to the base plate with moderate pressure to press the middle contacts against the base silicone layer S1.</li>
	<li>Fully cure the silicone for 90 min at 130 &#186;C, or overnight at RT.</li>
</ol>
6. Embedding the Electrode Components
<ol>
	<li>Remove the small fixture bar and gently remove the transparent sheet T2 to expose the middle contact arrays. Remove all the tapes that hold the leads for both references and middle contacts (Figure 2C).</li>
	<li>Cut a square piece of the transparency sheet with a scalpel to the same width of the electrode and 5 cm in length (T3), and then cut a square piece of silicone sheet to cover the entire electrode surface (S2).</li>
	<li>Lay the silicone sheet (S2) on top of the transparency piece (T3) and stretch it to remove any waves or irregularities and to eliminate air bubbles from being trapped in between.</li>
	<li>Cut four pieces of silicone tubing; 5 cm long each. Place them on the exit site of the leads as assigned on the guiding diagram. Leave a 2 mm space between the electrode edge and the tubes&#39; edges. While holding down each pair of tubes with tweezers, tape down the tubes starting at 1 mm away from the tube end. Repeat for the other pair.</li>
	<li>Arrange and leads of the middle contacts and the references into bundles, and then pass them through the corresponding tube near the exit sites. Repeat for the other three tubes. (Figure 2D).</li>
	<li>Deposit generous amount of uncured silicone over the entire electrode body. 
	NOTE: Avoid forming air bubbles during this step by either slowly pouring the uncured silicone from the vacuumed mixing container or injecting it with a syringe.</li>
	<li>Place the structure from 6.3 on top of the deposited uncured silicone with the silicone sheet S2 facing down. Align the transparency piece T3 with the electrode while keeping the silicone sheet S2 adhered to it.</li>
	<li>Tape down the transparency piece T3 and then apply pressure to channel out any trapped air bubbles. Place the large fixture bar across the center of the electrode and over the transparency segment T3. Then clamp it down to the base plate with moderate pressure. Fully cure the silicone for 90 min at 130 &#186;C, or overnight at RT.</li>
</ol>
7. Shielding Layer Placement (Recommended for Recording Cuffs)
<ol>
	<li>Remove the large fixture bar and delaminate the transparency piece (T3) with tweezers. Place the shielding sheet in the center of each face of the electrode and apply slight pressure to press them into the electrode. Deposit uncured silicone into the through-holes.</li>
	<li>Partially cure the silicone for 30 min at 130 &#186;C, and then let it cool completely to room temperature. Place adhesive tape over the outer ends of the electrode and over the closing flanges to prevent adding extra uncured silicone to these segments.</li>
	<li>Repeat steps 6.6 through 6.8.</li>
</ol>
8. Cutting out the Finished Electrode
<ol>
	<li>Peel off and cut the excess silicone on top of the adhesive tape added in step 7.2 using scalpel blade, then carefully remove the adhesive tape.</li>
	<li>Cut out windows through the silicone to expose the spacer segments through the S2 layer. Extract the embedded spacer segments with tweezers. This step will leave voids and form flexible single silicone sheet at these regions (originally S1).</li>
	<li>Peel off the excess silicone on top of the adhesive tapes that cover the silicone tubes, and then cut it with scalpel blade to level the tubes with the electrode body.</li>
	<li>Cut around the perimeter of the electrode down to base plate.</li>
	<li>Cut out a triangle between each tubes pair completely through the base plate, and on the outer side following the guiding diagram to shape the leads&#39; exit sites. Remove all the silicone material that was detached from the electrode body during last steps.</li>
</ol>
9. Exposing Contacts and Shielding Layers
<ol>
	<li>Cut out windows through the silicone layer S2 that covers the shielding layer. Glide the polypropylene suture filament in between the electrode base (layer S1) and the transparent layer T1 on the base plate to delaminate the finished cuff electrode.</li>
	<li>Flip the electrode such that the center contacts and the Silicone layer S1 are facing up, and then expose them by cutting out windows through the base silicone layer S1. Repeat for the outer reference contacts exposing 1 mm wide segments along the center of the contacts. Ensure that the stabilizing through-holes at the sides of the reference contacts are fully embedded inside the electrode&#39;s body.</li>
</ol>
10. Soldering a Connector to the Leads
<ol>
	<li>Deposit soldering substance onto the leads and onto the connecter pins separately, and then heat and fuse both parts together with soldering iron. 
	Note: The DFT lead wires consist of silver core surrounded by an outer layer made out of the Nickel-Cobalt base alloy MP35N. Depositing the solder substance onto these wires requires the use of specialty flux to allow adhering to the wire (please refer to the Materials List).</li>
</ol>

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The authors declare that they have no competing financial interests. The suppliers listed in this manuscript are provided for reference only.

The manufacturing method described in this article requires dexterous and fine movements in order to ensure the quality of the final cuff. The recording contacts must be placed precisely in the middle of the two reference electrodes. This placement has been shown to significantly reduce interferences from surrounding muscles electrical activity27. Any imbalance in the relative position of the contact during the fabrication can degrade the rejection of common mode interfering signals generated outside the cuff....

Interfacing with the peripheral nervous system (PNS) provides access to highly-processed neural command signals as they travel to different structures within the body. These signals are generated by axons confined within fascicles and surrounded by tightly-jointed perineurium cells. The magnitude of the measurable potentials resulting from the neural activities is affected by the impedance of the various layers within the nerve such as the highly resistive perineurium layer that surrounds the fascicles. Consequently, two interface approaches have been explored depending on the recording location with respect to the perineurium layer, namely intrafascicular and extrafascicular approaches. Intra-fascicular approaches place the electrodes inside the fascicles. Examples of these approaches are the Utah array17, the Longitudinal Intra-fascicular Electrode (LIFE)18, and the transverse intra-fascicular multichannel electrode (TIME)32. These techniques can record selectively from the nerve but have not been shown to reliably retain functionality for long periods of time in vivo, likely due to the size and the compliance of the electrode12.
Extra-fascicular approaches place the contacts around the nerve. The cuff electrodes used in these approaches do not compromise the perineurium nor the epineurium and have been shown to be both a safe and robust means of recording from the peripheral nervous system12. However, extra-fascicular approaches lack the ability to measure single unit activity&#160;&#8212; compared to intra-fascicular designs. Neuroprosthetic applications that utilize nerve cuff electrodes include activation of the lower extremity, the bladder, the diaphragm, treatment of chronic pain, block of neural conduction, sensory feedback, and recording electroneurograms1. Potential applications to utilize peripheral nerve interfacing include restoring movement to victims of paralysis with functional electrical stimulation, recording motor neuron activity from residual nerves to control powered limb prostheses in amputees, and interfacing with the autonomic nervous system to deliver bio-electronic medicines20.
A design implementation of the cuff electrode is the flat-interface nerve electrode (FINE)21. This design reshapes the nerve into a flat-cross section with larger circumference compared to a round shape. The advantages of this design are increased number of contacts that can be placed on the nerve, and the close proximity of the contacts with the rearranged internal fascicles for selective recording and stimulation. Furthermore, upper and lower extremity nerves in large animals and human can take various shapes and the reshaping generated by the FINE does not distort the natural geometry of the nerve. Recent trials have shown that FINE is capable of restoring sensation in the upper extremity16 and restoring movement in the lower extremity22 with functional electrical stimulation in humans.
The basic structure of a cuff electrode consists of placing several metal contacts on the surface of a nerve segment, and then insulating these contacts along with the nerve segment within a nonconductive cuff. To achieve this basic structure, several designs have been proposed in previous studies including:
(1) Metal contacts embedded into a Dacron mesh. The mesh is then wrapped around the nerve and the resulting cuff shape follows the nerve geometry4, 5.
(2) Split-cylinder designs which use pre-shaped rigid and non-conductive cylinders to fix the contacts around the nerve. The nerve segment that receives this cuff is reshaped into the cuff&#39;s internal geometry6-8.
(3) Self-coiling designs where the contacts are enclosed between two insulation layers. The internal layer is fused while stretched with an external un-stretched layer. With different natural resting lengths for the two bonded layers causes the final structure to form a flexible spiral that wraps itself around the nerve. The material used for these layers have typically been polyethylene9 polyimide10, and silicone rubber1.
(4) Uninsulated segments of the lead wires placed against the nerve to serve as the electrode contacts. These leads are either woven into silicone tubing11 or molded in silicone nested cylinders12. A similar principle was used to construct FINEs by arranging and fusing insulated wires to form an array, and then an opening through the insulation is made by stripping a small segment through the middle of these joined wires13. These designs assume a round nerve cross section and conform to this assumed nerve geometry.
(5) Flexible polyimide based electrodes33 with contacts formed by micromachining polyimide structure, and then integrating into stretched silicone sheets to form self-coiling cuffs. This design also assumes a round nerve cross section.
Cuff electrodes should be flexible and self-sizing in order to avoid stretching and compressing the nerve that can cause nerve damage3. Some of the known mechanisms by which cuff electrodes can induce these effects are the transmission of forces from adjacent muscles to the cuff and hence to the nerve, mismatch between the cuff&#39;s and nerve&#39;s mechanical properties, and the undue tension in the cuff&#39;s leads. These safety issues lead to specific set of design constraints on the mechanical flexibility, geometric configuration, and size1. These criteria are particularly challenging in the case of a high contact count FINE because the cuff must be at the same time stiff in the transverse direction to reshape the nerve and flexible in the longitudinal direction to prevent damage as well as accommodating multiple contacts. Self-sizing spiral designs can accommodate multiple contacts cuff14, but the resulting cuff is somewhat rigid. Flexible polyimide design can accommodate a high number of contacts but are prone to delamination. The wire array design13 produces a FINE with flat cross section, but in order to retain this geometry the wires are fused together along the length of the cuff producing stiff faces and sharp edges making then unsuitable for long term implants.
The fabrication technique described in this article produces a high contact density FINE with flexible structure that can be made by hand with consistently high precision. It uses a rigid polymer (Polyether ether ketone (PEEK)) to allow precise placement of the contacts. The PEEK segment maintains a flat cross section at the center of the electrode while remaining flexible in the longitudinal direction along the nerve. This design also minimizes the overall thickness and stiffness of the cuff since the electrode body does not have to be rigid in order to flatten the nerve or secure the contacts.

<table><tbody><tr><td>Platinum-Iridium foil</td><td>Alfa Aesar</td><td>41802</td><td>90% Platinum Iridium&amp;nbsp;</td></tr><tr><td>DFT wires</td><td>Fort Wayne Metals</td><td>35N LT-DFT-28%Ag</td><td></td></tr><tr><td>Lead connector</td><td>Omnetics Connector Corporation</td><td>MCS-27-SS</td><td></td></tr><tr><td>Silicone sheet</td><td>Speciality Silicon Fabricator</td><td>0.005&quot; x 12&quot; x 12&quot; Silicone Sheet</td><td>High durometer, vulcanized&amp;nbsp;</td></tr><tr><td>Polyether ether ketone (PEEK) sheet</td><td>Peek-Optima</td><td>0.005 sheet LT3 grade</td><td></td></tr><tr><td>polyester stabelizing mesh</td><td>Surgicalmesh</td><td>PETKM2002</td><td></td></tr><tr><td>Silicon tubing (0.04&quot; I.D. 0.085&quot; O.D.)</td><td>Silcon Medical/NewAge Industries.</td><td>2810458</td><td></td></tr><tr><td>Outer shielding layer</td><td>Alfa Aesar, A Johnson Matthey</td><td>MFCD00003436 (11391)</td><td>Gold foil, 0.004&quot; thick</td></tr><tr><td>Transparency sheet</td><td>APOLLO</td><td>APOCG7060</td><td></td></tr><tr><td>Ultrasonic bath cleaner</td><td>Terra Universal</td><td>2603-00A-220</td><td></td></tr><tr><td>Isotemp standard lab oven</td><td>Fisher Scientific</td><td>13247637G</td><td></td></tr><tr><td>Optical microscope</td><td>Fisher Scientific</td><td>15-000-101</td><td></td></tr><tr><td>Tweezers</td><td>Technik</td><td>18049USA (2A-SA)</td><td></td></tr><tr><td>Surgical blade handles</td><td>Aspen Surgical Products</td><td>371031</td><td></td></tr><tr><td>Base frame&amp;nbsp;</td><td>McMaster-Carr</td><td>9785K411</td><td></td></tr><tr><td>Support beam</td><td>McMaster-Carr</td><td>9524K359</td><td></td></tr><tr><td>Two parts silicone</td><td>Nusil</td><td>MED 4765</td><td></td></tr><tr><td>Soldering Flux</td><td>SRA Soldering Products</td><td>FLS71</td><td></td></tr><tr><td>Tape</td><td>3M Healthcare</td><td>1535-0 (SKUMMM15350H)</td><td>Paper, hypoallergenic surgical tape</td></tr><tr><td>Spot welding machine</td><td>Unitek</td><td>125 Power Supply with 101F Welding Head</td><td></td></tr><tr><td>Laser cutting platform</td><td>Universal Laser Systems</td><td>PLS6.150D</td><td>150 watts laser</td></tr></tbody></table>

fabrication of high contact-density, flat-interface nerve electrodes for recording and stimulation applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

Recording neural activity was performed with a customized pre-amplifier using super-&#946; input instrumentation amplifier (700 Hz - 7 kHz bandwidth and total gain of 2,000). An example of the fabricated FINE electrode with the presented protocol is shown in Figure 3. Implanting the FINE around the nerve is done by suturing the two free edges together. A demonstration of the cuff&#39;s flexibility (Figure 3B) indicates that the cuff flattens the nerve while retaining flexibility in the l...

Watch this Scientific Journal Video about Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications at JoVE.com

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

fabrication-high-contact-density-flat-interface-nerve-electrodes-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.7K Views.  Case Western Reserve University. This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Video: Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Microfluidic Channel-Based Soft Electrodes for Capacitive Pressure Sensing

Microfluidic Channel-Based Soft Electrodes and Their Application in Capacitive Pressure Sensing

Flexible and stretchable electrodes are essential components in soft artificial sensory systems. Despite recent advances in flexible electronics, most electrodes are either restricted by the patterning resolution or the capability of inkjet printing with high-viscosity super-elastic materials. In this paper, we present a simple strategy to fabricate microchannel-based stretchable composite electrodes, which can be achieved by scraping elastic conductive polymer composites (ECPCs) into lithographically embossed microfluidic channels. The ECPCs were prepared by a volatile solvent evaporation method, which achieves a uniform dispersion of carbon nanotubes (CNTs) in a polydimethylsiloxane (PDMS) matrix. Compared to conventional fabrication methods, the proposed technique can facilitate the rapid fabrication of well-defined stretchable electrodes with high-viscosity slurry. Since the electrodes in this work were made up of all-elastomeric materials, strong interlinks can be formed between the ECPCs-based electrodes and the PDMS-based substrate at the interfaces of the microchannel walls, which allows the electrodes to exhibit mechanical robustness under high tensile strains. In addition, the mechanical-electric response of the electrodes was also systematically studied. Finally, a soft pressure sensor was developed by combining a dielectric silicone foam and an interdigitated electrodes (IDE) layer, and this demonstrated great potential for pressure sensors in soft robotic tactile sensing applications.

Author Spotlight: Microfluidic Channel-Based Soft Electrodes and Their Application in Capacitive Pressure Sensing  

Flexible electrodes have a wide range of applications in soft robotics and wearable electronics. The present protocol demonstrates a new strategy to fabricate highly stretchable electrodes with high resolution via lithographically defined microfluidic channels, which paves the way for future high-performance soft pressure sensors.

Flexible and stretchable electrodes are essential components in soft artificial sensory systems. Despite recent advances in flexible electronics, most ...

Engineering

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 ...

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 ...

Bioengineering

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This paper presents a cleanroom "light" fabrication process of carbon fiber neural electrode arrays that can be learned quickly by an inexperienced cleanroom user. This carbon fiber electrode array fabrication process requires just one cleanroom tool, a Parylene C deposition machine, that can be learned quickly or outsourced to a commercial processing facility at marginal cost. This fabrication process also includes hand-populating printed circuit boards, insulation, and tip optimization. 
The three different tip optimizations explored here (Nd:YAG laser, blowtorch, and UV laser) result in a range of tip geometries and 1 kHz impedances, with blowtorched fibers resulting in the lowest impedance. While previous experiments have proven laser and blowtorch electrode efficacy, this paper also shows that UV laser-cut fibers can record neural signals in vivo. Existing carbon fiber arrays either do not have individuated electrodes in favor of bundles or require cleanroom fabricated guides for population and insulation. The proposed arrays use only tools that can be used at a benchtop for fiber population. This carbon fiber electrode array fabrication process allows for quick customization of bulk array fabrication at a reduced price compared to commercially available probes.

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. ...

 Fabricating Epimysial EMG Electrodes for Preclinical Rodent Models 

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to measure EMG signals in preclinical models. Although classical resources exist describing the principles of epimysial electrode fabrication, there is a sparsity of illustrative information translating electrode theory to practice. To remedy this, we provide an updated, easy-to-follow guide on fabricating and testing a low-cost epimysial electrode. 
Electrodes were made by folding and inserting two platinum-iridium foils into a precut silicone base to form the contact surfaces. Next, coated stainless steel wires were welded to each contact surface to form the electrode leads. Lastly, a silicone mixture was used to seal the electrode. Ex vivo testing was conducted to compare our custom-fabricated electrode to an industry standard electrode in a saline bath, where high levels of signal agreement (sine [intraclass correlation - ICC= 0.993], square [ICC = 0.995], triangle [ICC = 0.958]), and temporal-synchrony (sine [r = 0.987], square [r = 0.990], triangle [r= 0.931]) were found across all waveforms. Low levels of electrode impedance were also quantified via electrochemical impedance spectroscopy. 
An in vivo performance assessment was also conducted where the vastus lateralis muscle of a rat was surgically instrumented with the custom-fabricated electrode and signaling was acquired during uphill and downhill walking. As expected, peak EMG activity was significantly lower during downhill walking (0.008 &plusmn; 0.005 mV) than uphill (0.031 &plusmn; 0.180 mV, p = 0.005), supporting the validity of the device. The reliability and biocompatibility of the device were also supported by consistent signaling during level walking at 14 days and 56 days post implantation (0.01 &plusmn; 0.007 mV, 0.012 &plusmn; 0.007 mV respectively; p &gt; 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.

Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Summary

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Chapters in this video

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

Microfluidic Channel-Based Soft Electrodes and Their Application in Capacitive Pressure Sensing

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

Fabrication of Ti₃C₂ MXene Microelectrode Arrays for In Vivo Neural Recording

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing