This work was supported by the National Institutes of Health (grant no. R21-NS106434), the Citizens United for Research in Epilepsy Taking Flight Award, the Mirowski Family Foundation and Neil and Barbara Smit (F.V.); the National Science Foundation Graduate Research Fellowship Program (grant no. DGE-1845298 to N.D. and B.M.); the Army Research Office (Cooperative Agreement Number W911NF-18-2-0026 to K.M.); and by the U.S. Army via the Surface Science Initiative Program at the Edgewood Chemical Biological Center (PE 0601102A Project VR9 to Y.G. and K.M.). This work was carried out in part at the Singh Center for Nanotechnology, which is supported by the National Science Foundation National Nanotechnology Coordinated Infrastructure Program (NNCI-1542153).

All in vivo procedures conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania.
1. Synthesis of Ti3C2 MXene
NOTE: The reaction procedures described in this section are intended for use inside a chemical fume hood. Washing steps included in this procedure are intended to be used with balanced centrifuge tubes. All waste produced is considered hazardous waste and should be discarded appropriately following University guidelines.
CAUTION: Hydrofluoric acid (HF) is an extremely dangerous, highly corrosive acid. Consult the materials safety data sheets (MSDS) for the chemicals used to synthesize MXenes before use and implement and follow appropriate safety measures. Appropriate personal protective equipment (PPE) for handling HF includes a laboratory coat, acid resistant apron, close-toed shoes, long pants, goggles, full face shield, nitrile gloves, and HF resistant gloves made of butyl rubber or neoprene rubber.
<ol>
	<li>MAX phase synthesis
	<ol>
		<li>Synthesize Ti3AlC2 by ball milling TiC (2 &#181;m), Ti (44 &#181;m), and Al (44 &#181;m) powders at a molar ratio (TiC:Ti:Al) of 2:1:1 for 18 h using zirconia balls. Place the powders in an alumina crucible, heat to 1,380 &#176;C (5 &#176;C heating rate) and hold for 2 h under argon. After the powders have been cooled, mill the MAX block and sieve through a 200 mesh sieve (&#60;74 &#181;m particle size). 
		NOTE: The Ti3AlC2 MAX phase precursor used to synthesize MXenes has been shown to have direct implications on the resulting Ti3C2 MXene properties38. The Ti3C2 used to fabricate neural electrodes was selectively etched from MAX prepared following a previous procedure26.</li>
	</ol>
	</li>
	<li>Etching: Removal of the Al layer in Ti3AlC2 in an acidic etchant solution (Figure 1A)
	<ol>
		<li>Prepare the selective etching solution in a 125 mL plastic container by first adding 12 mL of deionized water (DI H2O) followed by the addition of 24 mL of hydrochloric acid (HCl). Wearing all appropriate HF etching PPE, add 4 mL of HF to the etchant container. Perform selective etching by slowly adding 2 g of Ti3AlC2 MAX phase to the reaction container and stirring with a Teflon magnetic bar for 24 h at 35 &#176;C at 400 rpm.</li>
	</ol>
	</li>
	<li>Washing: Bringing the material to neutral pH.
	<ol>
		<li>Fill two 175 mL centrifuge tubes with 100 mL of DI H2O. Split the etching reaction mixture into 175 mL centrifuge tubes and wash the material by repeated centrifugation at 3,500 rpm (2,550 x g) for 5 min. Decant the acidic supernatant into a plastic hazardous waste container. Repeat until the pH reaches 6.</li>
	</ol>
	</li>
	<li>Intercalation: Insertion of molecules between multilayer MXene particle to waken out-of-plane interactions (Figure 1B)
	<ol>
		<li>Add 2 g of lithium chloride (LiCl) to 100 mL of DI H2O and stir at 200 rpm until dissolved. Mix 100 mL of LiCl/H2O with the Ti3C2/Ti3AlC2 sediment and stir the reaction for 12 h at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Delamination: Exfoliation from bulk multilayer particle into single- to few- layer Ti3C2 MXene (Figure 1C)
	<ol>
		<li>Wash the intercalation reaction in 175 mL centrifuge tubes by centrifugation at 2,550 x g for 5 min. Decant the clear supernatant. Repeat until a dark supernatant is found.</li>
		<li>Continue to centrifuge for 1 h at 2,550 x g. Decant the dilute-green supernatant.</li>
		<li>Re-disperse the swollen sediment with 150 mL of DI H2O. Transfer supernatant to 50 mL centrifuge tubes and centrifuge at 2,550 x g for 10 min to separate remaining MAX (sediment) from MXene (supernatant). 
		NOTE: Re-dispersion of the sediment will become difficult and will require agitation or manual shaking.</li>
		<li>Collect supernatant as Ti3C2 MXene. Perform further size selection and optimization of the solution to isolate single- to few-layer flakes by collecting the supernatant following a centrifugation step at 2,550 x g for 1 h.</li>
	</ol>
	</li>
	<li>Solution storage: Packaging the MXene ink for long-term storage (Figure 1D)
	<ol>
		<li>Argon bubble the solutions for 30 min prior to packaging in an Argon sealed headspace vial (transfer via a syringe). Store solutions at high concentrations (&#62;5 mg/mL), away from sunlight, and at low temperatures (&#8804;5 &#176;C) to ensure longevity.</li>
	</ol>
	</li>
</ol>
2. Fabrication of Ti3C2 MXene Microelectrode Arrays
NOTE: The procedure described in this section is intended for use inside a standard university clean room facility, such as the Singh Center for Nanotechnology at the University of Pennsylvania. This facility, as well as similar facilities, are accessible to outside users as part of the National Nanotechnology Infrastructure Network (NNIN) supported by the National Science Foundation (NSF). In these facilities, many of the tools, equipment, and materials described in this section are provided along with access to the clean room facility and would not require separate purchase.
CAUTION: Many of the chemicals used in the fabrication of MXene electrodes are hazardous, including photoresists, RD6 developer, remover PG, aluminum etching solution, and buffered oxide etchant. Consult MSDS for these chemicals before use and implement and follow appropriate safety measures at all times. All chemicals should be handled in a fume hood.
<ol>
	<li>Deposit a 4 &#956;m thick bottom layer of parylene-C onto a clean Si wafer (see Figure 2A).</li>
	<li>Use the first photomask (mask-1) to define the metal interconnects of the devices, as well as a metal ring around the edge of the wafer to aid in later lift-off steps (Figure 2B).
	<ol>
		<li>Spin coat NR71-3000p onto the wafer at 3,000 rpm for 40 s. Soft bake the wafer on a hot plate for 14.5 min at 95 &#176;C.</li>
		<li>Load the wafer and mask-1 into a mask aligner. Position the wafer so that the ring on the photomask overlaps with all edges of the wafer.</li>
		<li>Expose with i-line (365 nm wavelength) at a dose of 90 mJ/cm2. Hard bake the wafer on a hot plate for 1 min at 115 &#176;C.</li>
		<li>Immerse the wafer in the RD6 developer for 2 min, continuously agitating the solution. Rinse thoroughly with DI H2O and blow dry with an N2 gun.</li>
		<li>Use an electron beam evaporator to deposit 10 nm Ti, followed by 100 nm Au onto the wafer. 
		NOTE: Typical deposition parameters are a base pressure of 5 x 10-7 Torr and a rate of 2 &#197;/s.</li>
		<li>Immerse the wafer in remover PG for ~10 min until the photoresist has dissolved and the excess metal has fully lifted off, leaving Ti/Au only in the desired interconnect traces and the ring around the edge of the wafer. Once lift-off appears complete, sonicate for 30 s to remove any remaining traces of unwanted metal. Rinse wafer first in clean remover PG solution, then thoroughly rinse in DI H2O and dry the wafer with an N2 gun.</li>
	</ol>
	</li>
	<li>Deposit the sacrificial parylene-C layer (Figure 2C).
	<ol>
		<li>Expose the wafer to O2 plasma for 30 s to render the underlying parylene-C layer hydrophilic. Spin coat 2% cleaning solution (e.g., Micro-90) in DI H2O onto the wafer at 1,000 rpm for 30 s. Allow wafer to air dry for at least 5 min. 
		NOTE: The dilute soap solution acts as an anti-adhesive, allowing the sacrificial parylene-C layer to be peeled up later in the process.</li>
		<li>Deposit 3 &#956;m of parylene-C onto the wafer.</li>
	</ol>
	</li>
	<li>Use the second photomask (mask-2) to define the MXene patterns and a ring around the edge of the wafer (Figure 2D).
	<ol>
		<li>Repeat steps 2.2.1&#8722;2.2.4, this time using mask-2 and carefully aligning the alignment marks between the wafer and photomask before exposure.</li>
		<li>Use O2 plasma reactive ion etching (RIE) to etch through the sacrificial parylene-C layer in the areas not covered by the photoresist to define the MXene electrodes and traces, which should partially overlap with the Ti/Au interconnects, as well as the ring around the edges of the wafer. Confirm complete etching of the sacrificial parylene-C layer by using a profilometer to measure the profile between the exposed Ti/Au interconnects and the bottom parylene-C layer. 
		NOTE: When etching is complete, the profile across the exposed metal surface will be smooth, while the bottom parylene-C layer will be rough and partially etched. This etch step should be completed in a planar etch RIE system, not a barrel asher, and etch times and parameters will be highly dependent on the RIE system.</li>
	</ol>
	</li>
	<li>Spin-coat the MXene solution onto the wafer (Figure 2E).
	<ol>
		<li>Pipette MXene solution onto each of the desired MXene patterns, then spin the wafer at 1,000 rpm for 40 s. Dry the wafer on a 120 &#176;C hot plate for 10 min to remove any residual water from the MXene film.</li>
	</ol>
	</li>
	<li>Use an electron beam evaporator to deposit 50 nm SiO2 onto the wafer, to act as a protective layer over the MXene patterns for subsequent processing steps. 
	NOTE: Typical deposition parameters are a base pressure of 5 x 10-7 Torr and a rate of 2 &#197;/s.</li>
	<li>Remove the sacrificial parylene-C layer to pattern the MXene and SiO2 layers (Figure 2F).
	<ol>
		<li>Apply a small drop of DI H2O to the edge of the wafer and use tweezers to peel up the sacrificial parylene-C layer, beginning where its edges are defined in the ring around the outside of the wafer. 
		NOTE: The water will combine with the soap residue beneath the sacrificial parylene-C layer to enable this lift-off.</li>
		<li>Rinse the wafer thoroughly in DI H2O to remove any remaining cleaning solution residue. Dry the wafer with an N2 gun, then place on a 120 &#176;C hot plate for 1 h to remove any residual water from the patterned MXene films.</li>
	</ol>
	</li>
	<li>Deposit the 4 &#956;m thick top layer of parylene-C (Figure 2G).</li>
	<li>Use the third photomask (mask-3) to define device outline and openings over electrodes and Au bonding pads (VIAs) (Figure 2H).
	<ol>
		<li>Repeat steps 2.2.1&#8722;2.2.4, this time using mask-3 and carefully aligning the alignment marks between the wafer and photomask before exposure.</li>
		<li>Use an electron beam evaporator to deposit 100 nm Al onto the wafer. 
		NOTE: Typical deposition parameters are a base pressure of 5 x 10-7 Torr and a rate of 2 &#197;/s.</li>
		<li>Immerse the wafer in remover PG for ~10 min until the metal has fully lifted off, leaving Al covering the devices with openings for the electrodes and bonding pads. When lift-off is complete, sonicate for 30 s to remove any remaining traces of unwanted metal. Rinse wafer first in clean remover PG solution, then thoroughly rinse in DI H2O and dry the wafer with an N2 gun.</li>
	</ol>
	</li>
	<li>Etch the parylene-C to pattern the device outline and openings over electrodes and Au bonding pads (VIAs) (Figure 2I). Use O2 plasma RIE to etch through the parylene-C layers surrounding the devices, and through the top parylene-C layer covering both the MXene electrode contacts and the Au bonding pads. 
	NOTE: Etching is complete when no parylene-C residue remains on the wafer between devices. The SiO2 layer covering the MXene will act as an etch-stop layer, preventing the O2 plasma from etching into or damaging the MXene electrode contacts.</li>
	<li>Etch the Al layer covering the devices using a wet chemical etch in Al etchant type A at 50 &#176;C either for 10 min, or for 1 min past when all visual traces of Al have disappeared, whichever comes first. Etch the SiO2 covering the MXene electrodes using a wet chemical etch in 6:1 buffered oxide etchant (BOE) for 30 s (Figure 2J). 
	NOTE: The MXene microelectrode arrays are now complete.</li>
	<li>Release the devices from the Si substrate wafer by placing a small drop of DI H2O at the edge of a device, and gently peeling up the device as water is wicked underneath it by capillary action (Figure 2K and Figure 3).</li>
</ol>
3. Adapter Construction and Interfacing
NOTE: At this point, the thin film microelectrode arrays must be interfaced with an adapter to connect to the electrophysiology recording system. The 128ch stimulation/recording controller with the RHS2000 16-ch stim/record headstage (Table of Materials) used in this protocol requires input via a connector compatible with the 18-pin connector A79039-001. This section uses a printed circuit board (PCB, Figure 4A) with a zero-insertion force (ZIF) connector for interfacing with the Au bonding pads on the microelectrode array and the connector A79040-001 for interfacing with the head-stage of the recording system. Depending on the data acquisition system, different connectors can be used on the PCB to enable interfacing with the electrophysiology headstage.
<ol>
	<li>Solder the Omnetics and ZIF connectors to the PCB by applying a thin film of solder paste to each of the contact pads on the PCB, placing the parts in their appropriate locations, and heating on a hot plate until the solder reflows to form connections (Figure 4B). 
	NOTE: Reflow soldering can be done very easily on a hot plate or in a toaster oven and does not require the use of a costly reflow oven.</li>
	<li>Apply two layers of polyimide tape (Table of Materials) to the back side of the Au bonding pad region of the MXene microelectrode array to give the device sufficient thickness to be secured in the ZIF connector. After applying the tape, trim any excess beyond the edges of the parylene-C device using a razor blade or precision scissors (Figure 4C).</li>
	<li>Either under an inspection scope or using magnifying glasses, align the MXene microelectrode array in the ZIF connector so that the Au bonding pads align with the pins inside the ZIF connector, then close the ZIF to form a secure connection (Figure 4D,E). 
	NOTE: The ZIF connector used here is an 18-channel connector, while the device used here has 16 channels. The extra uncontacted channels are easily identified as an open circuit by means of impedance testing during recording sessions.</li>
	<li>Test the electrochemical impedance of the MXene electrodes using a potentiostat to ensure successful fabrication and connection to the PCB adapter. 
	NOTE: Reasonable impedance values are given in the discussion section to aid in troubleshooting.</li>
</ol>
4. Acute Implantation and Neural Recording
NOTE: Surgeries on adult male Sprague Dawley rats are performed using sterile instruments and with aseptic technique. Respiratory rate, palpebral reflex, and pedal pinch reflex are checked every 10 min to monitor depth of anesthesia. Body temperature is maintained with a heating pad.
<ol>
	<li>Administer preemptive analgesia (subcutaneous injection of buprenorphine sustained release [SR], 1.2 mg/kg).</li>
	<li>Administer anesthesia (intraperitoneal injection of a mixture of 60 mg/kg ketamine and 0.25 mg/kg dexmedetomidine).</li>
	<li>Confirm proper level of anesthesia every 10 min throughout the experiment by checking for absence of palpebral and pedal pinch reflexes.</li>
	<li>Secure rat in stereotaxic frame, apply ocular lubricant to the eyes, and clean shaved scalp with 10% povidone-iodine.</li>
	<li>Expose the calvaria with single midline scalp incision and blunt dissection of underlying tissue.</li>
	<li>Place a 00-90 screw into the skull to serve as the ground for recordings.</li>
	<li>Using a dental drill with a small burr, make a craniotomy at the desired cortical recording site.</li>
	<li>Secure the array connector to a stereotaxic manipulator and position the device over the craniotomy. Gently lower until the entire array is in contact with the exposed cortex.</li>
	<li>Wrap the ground wire around the skull screw.</li>
	<li>Connect the recording system headstage to the array and begin recording spontaneous activity.</li>
</ol>

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R., Zhao, M. Q., Gogotsi, Y., Barsoum, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conductive+two-dimensional+titanium+carbide+&#8216;clay&#8217;+with+high+volumetric+capacitance.">Conductive two-dimensional titanium carbide &#8216;clay&#8217; with high volumetric capacitance.</a> Nature. 516 (7529), 78-81 (2014).</li><li>Lukatskaya, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high-rate+pseudocapacitive+energy+storage+in+two-dimensional+transition+metal+carbides.">Ultra-high-rate pseudocapacitive energy storage in two-dimensional transition metal carbides.</a> Nature Energy. 2, 17105 (2017).</li><li>Zhu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon-Based+Supercapacitors+Produced+by+Activation+of+Graphene.">Carbon-Based Supercapacitors Produced by Activation of Graphene.</a> Science. 332 (6037), 1537-1541 (2011).</li><li>Heon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+carbide-derived+carbon+films+with+high+volumetric+capacitance.">Continuous carbide-derived carbon films with high volumetric capacitance.</a> Energy &amp; Environmental Science. 4 (1), 135-138 (2011).</li><li>Yang, X., Cheng, C., Wang, Y., Qiu, L., Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid-mediated+dense+integration+of+graphene+materials+for+compact+capacitive+energy+storage.">Liquid-mediated dense integration of graphene materials for compact capacitive energy storage.</a> Science. 341 (6145), 534-537 (2013).</li><li>Zhang, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent,+Flexible,+and+Conductive+2D+Titanium+Carbide+(MXene)+Films+with+High+Volumetric+Capacitance.">Transparent, Flexible, and Conductive 2D Titanium Carbide (MXene) Films with High Volumetric Capacitance.</a> Advanced Materials. 29 (36), 1702678 (2017).</li><li>Han, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+Ultrathin+MXene-Based+Drug-Delivery+Nanoplatform+for+Synergistic+Photothermal+Ablation+and+Chemotherapy+of+Cancer.">2D Ultrathin MXene-Based Drug-Delivery Nanoplatform for Synergistic Photothermal Ablation and Chemotherapy of Cancer.</a> Advanced Healthcare Materials. 7 (9), 1701394 (2018).</li><li>Dai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+2D+Titanium+Carbide+(MXenes)+Composite+Nanosheets+for+pH-Responsive+MRI-Guided+Tumor+Hyperthermia.">Biocompatible 2D Titanium Carbide (MXenes) Composite Nanosheets for pH-Responsive MRI-Guided Tumor Hyperthermia.</a> Chemistry of Materials. 29 (20), 8637-8652 (2017).</li><li>Xu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+MXene-Micropattern-Based+Field-Effect+Transistor+for+Probing+Neural+Activity.">Ultrathin MXene-Micropattern-Based Field-Effect Transistor for Probing Neural Activity.</a> Advanced Materials. 28 (17), 3333-3339 (2016).</li><li>Driscoll, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Dimensional+Ti3C2+MXene+for+High-Resolution+Neural+Interfaces.">Two-Dimensional Ti3C2 MXene for High-Resolution Neural Interfaces.</a> ACS Nano. 12 (10), 10419-10429 (2018).</li><li>Sessolo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy-to-Fabricate+Conducting+Polymer+Microelectrode+Arrays.">Easy-to-Fabricate Conducting Polymer Microelectrode Arrays.</a> Advanced Materials. 25 (15), 2135-2139 (2013).</li><li>Shuck, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Ti3AlC2+MAX+Phase+on+Structure+and+Properties+of+Resultant+Ti3C2Tx+MXene.">Effect of Ti3AlC2 MAX Phase on Structure and Properties of Resultant Ti3C2Tx MXene.</a> ACS Applied Nano Materials. 2 (6), 3368-3376 (2019).</li><li>Hantanasirisakul, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Ti3C2Tx+MXene+Transparent+Thin+Films+with+Tunable+Optoelectronic+Properties.">Fabrication of Ti3C2Tx MXene Transparent Thin Films with Tunable Optoelectronic Properties.</a> Advanced Electronic Materials. 2 (6), 1600050 (2016).</li><li>Xu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+MXene-Micropattern-Based+Field-Effect+Transistor+for+Probing+Neural+Activity.">Ultrathin MXene-Micropattern-Based Field-Effect Transistor for Probing Neural Activity.</a> Advanced Materials. 28 (17), 3333-3339 (2016).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive-free+MXene+inks+and+direct+printing+of+micro-supercapacitors.">Additive-free MXene inks and direct printing of micro-supercapacitors.</a> Nature Communications. 10 (1), 1795 (2019).</li><li>Quain, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Writing+of+Additive-Free+MXene-in-Water+Ink+for+Electronics+and+Energy+Storage.">Direct Writing of Additive-Free MXene-in-Water Ink for Electronics and Energy Storage.</a> Advanced Materials Technologies. 4 (1), 1800256 (2019).</li><li>Salles, P., Quain, E., Kurra, N., Sarycheva, A., Gogotsi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Scalpel+Patterning+of+Solution+Processed+Thin+Films+for+Fabrication+of+Transparent+MXene+Microsupercapacitors.">Automated Scalpel Patterning of Solution Processed Thin Films for Fabrication of Transparent MXene Microsupercapacitors.</a> Small. 14 (44), 1802864 (2018).</li></ol>

The authors have nothing to disclose.

The MXene synthesis and delamination procedure described in this protocol (HF/HCl/LiCl) was built from the MILD etching approach which employed a LiF/HCl (in situ HF) etchant medium26. The MILD approach allows for large Ti3C2 flakes (several &#181;m in lateral size) to be spontaneously delaminated during washing once pH ~5&#8722;6 has been attained. Compared to etching with HF alone, this results in material with higher quality and improved material properties, such as electr...

Understanding the fundamental mechanisms underlying neural circuits, and how their dynamics are altered in disease or injury, is a critical goal for developing effective therapeutics for a broad range of neurological and neuromuscular disorders. Microelectrode technologies have been widely used to elucidate neural dynamics on fine spatial and temporal scales. However, obtaining stable recordings with high signal-to-noise ratio (SNR) from microscale electrodes has proven to be particularly challenging. As the dimensions of the electrodes are reduced to approach cellular scale, a corresponding rise in electrode impedance degrades signal quality1. Additionally, numerous studies have shown that rigid electrodes comprised of conventional silicon and metal electronic materials produce significant damage and inflammation in the neural tissue, which limits their usefulness for long-term recording2,3,4,5. Given these facts, there has been significant interest in developing microelectrodes with new materials which can reduce the electrode-tissue interface impedance and can be incorporated into soft and flexible form factors.
One commonly used method for reducing the electrode-tissue interface impedance is increasing the area over which ionic species in the extracellular fluid can interact with the electrode, or the &#34;effective surface area&#34; of the electrode. This can be achieved through nanopatterning6, surface roughening7, or electroplating with porous additives8,9. Nanomaterials have gained significant attention in this field because they offer intrinsically high specific surface areas and unique combinations of favorable electrical and mechanical properties10. For example, carbon nanotubes have been used as a coating to significantly reduce electrode impedance11,12,13, graphene oxide has been processed into soft, flexible free-standing probe electrodes14, and laser-pyrolyzed porous graphene has been utilized for flexible, low-impedance micro-electrocorticography (micro-ECoG) electrodes15. Despite their promise, a lack of scalable assembly methods has limited the widespread adoption of nanomaterials for neural interfacing electrodes. Carbon-based nanomaterials in particular are typically hydrophobic, and thus require the use of surfactants16, superacids17, or surface functionalization18 to form aqueous dispersions for solution-processing fabrication methods, while alternative methods of fabrication, such as chemical vapor deposition (CVD), typically require high temperatures which are incompatible with many polymeric substrates19,20,21,22.
Recently, a class of two-dimensional (2D) nanomaterials, known as MXenes, has been described which offers an exceptional combination of high conductivity, flexibility, volumetric capacitance, and inherent hydrophilicity, making them a promising class of nanomaterials for neural interfacing electrodes23. MXenes are a family of 2D transition metal carbides and nitrides which are most commonly produced by selectively etching the A element from layered precursors. These are typically MAX phases with the general formula Mn+1AXn, where M is an early transition metal, A is a group 12&#8722;16 element of the periodic table, X is carbon and/or nitrogen, and n = 1, 2, or 324. Two-dimensional MXene flakes have surface-terminating functional groups that can include hydroxyl (&#8722;OH), oxygen (&#8722;O) or fluorine (&#8722;F). These functional groups make MXenes inherently hydrophilic and enable flexible surface modification or functionalization. Of the large class of MXenes, Ti3C2 has been the most extensively studied and characterized25,26,27. Ti3C2 shows remarkably higher volumetric capacitance (1,500 F/cm3)28 than activated graphene (~60&#8722;100 F/cm3)29, carbide-derived carbons (180 F/cm3)30, and graphene gel films (~260 F/cm3)31. Furthermore, Ti3C2 shows extremely high electronic conductivity (~10,000 S/cm)32, and its biocompatibility has been demonstrated in several studies33,34,35,36. The high volumetric capacitance of Ti3C2 films is advantageous for biological sensing and stimulation applications, because electrodes that exhibit capacitive charge transfer can avoid potentially harmful hydrolysis reactions.
Our group has recently demonstrated flexible, thin-film Ti3C2 microelectrode arrays, prepared using solution processing methods, which are capable of recording both micro-electrocorticography (micro-ECoG) and intracortical neuronal spiking activity in vivo with high SNR36. These MXene electrodes showed significantly reduced impedance compared to size-matched gold (Au) electrodes, which can be attributed to the high conductivity of MXene and the high surface area of the electrodes. In this protocol, we describe the key steps for fabricating planar microelectrode arrays of Ti3C2 MXene on flexible parylene-C substrates and utilizing them in vivo for intraoperative micro-ECoG recording. This method takes advantage of the hydrophilic nature of MXene, which makes possible the use of solution processing methods that are simple and scalable while not requiring the use of surfactants or superacids to achieve stable aqueous suspensions. This ease of processability may enable cost-effective production of MXene biosensors at industrial scales, which has been a major limitation to the widespread adoption of devices based on other carbon nanomaterials. The key innovation in the electrode fabrication lies in the use of a sacrificial polymeric layer to micropattern the MXene after spin-coating, a method adapted from literature on solution-processed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) microelectrodes37, but which had not previously been described for patterning MXene. The exceptional electrical properties of Ti3C2, coupled with its processability and 2D morphology make it a very promising material for neural interfaces. In particular, Ti3C2 offers a route towards overcoming the fundamental trade-off between electrode geometric area and electrochemical interface impedance, a primary limiting factor for micro-scale electrode performance. Additionally, the fabrication procedure described in this protocol can be adapted to produce MXene electrode arrays of varying sizes and geometries for different recording paradigms, and can also easily be adapted to incorporate other conductive inks besides MXene.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>00-90 screw</td>
			<td>McMaster-Carr</td>
			<td>90910A630</td>
			<td>Skull screw around which ground wire is wrapped</td>
		</tr>
		<tr>
			<td>128ch stimulation/recording controller</td>
			<td>Intan Technologies</td>
			<td></td>
			<td>A component of the neural recording system.</td>
		</tr>
		<tr>
			<td>175 mL polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>REF: 352076</td>
			<td>Used for washing</td>
		</tr>
		<tr>
			<td>18 position 0.5 mm pitch ZIF connector</td>
			<td>Molex</td>
			<td>505110-1892</td>
			<td>Used to interface the flexible Parylene microelectrode array with the PCB adapter board.</td>
		</tr>
		<tr>
			<td>18 position dual row male nano-miniature (.025&#34;/.64mm) connector</td>
			<td>Omnetics Connector Corporation</td>
			<td>A79008-001</td>
			<td>Used to interface the PCB adapter board to the recording headstage.</td>
		</tr>
		<tr>
			<td>3ML Disposable Plastic Set Transfer Graduated Pipettes</td>
			<td>Rienar</td>
			<td>Rienar-3ML-20PCS</td>
			<td>Used for transferring etchant or MXene solutions</td>
		</tr>
		<tr>
			<td>50 mL polyproylene (PP) concial centrifuge tube</td>
			<td>Falcon</td>
			<td>REF: 352070</td>
			<td>Used for washing and size selection</td>
		</tr>
		<tr>
			<td>Al etchant Type A</td>
			<td>Transene</td>
			<td>060-0026000-QT</td>
			<td>For removing Al etch mask layer after final Parylene-C etch.</td>
		</tr>
		<tr>
			<td>Aluminum Powder, -325 Mesh, 99.5% (metals basis), particle size &#60; 44 &#181;m</td>
			<td>Alfa Aesar</td>
			<td>CAS: 7429-90-5</td>
			<td>Used for MAX synthesis</td>
		</tr>
		<tr>
			<td>AutoCAD software</td>
			<td>Autodesk Inc.</td>
			<td></td>
			<td>Design software for drawing photomasks. Free alternatives include DraftSight and LayoutEditor.</td>
		</tr>
		<tr>
			<td>Buffered Oxide Etchant 6:1</td>
			<td>JT Baker</td>
			<td>1178-03</td>
			<td>For removing SiO2 layer to expose MXene electrode contacts at the end of the fabrication procedure.</td>
		</tr>
		<tr>
			<td>Buprenorphine SR</td>
			<td>Wildlife Pharmaceuticals</td>
			<td></td>
			<td>Analgesia for rat surgery</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hermle</td>
			<td>Benchmark Z 446</td>
			<td>Used for washing and size selection</td>
		</tr>
		<tr>
			<td>Dexdomitor</td>
			<td>Midwest Veterinary Supply</td>
			<td>193.13250.3</td>
			<td>Anesthesia for rat surgery</td>
		</tr>
		<tr>
			<td>Drill burr</td>
			<td>Fine Science Tools</td>
			<td>19007-07</td>
			<td>Burrs for drill</td>
		</tr>
		<tr>
			<td>Electric drill</td>
			<td>Foredom</td>
			<td>K.1070</td>
			<td>Micromotor drill for craniotomies</td>
		</tr>
		<tr>
			<td>Electron beam evaporator</td>
			<td>Kurt J. Lesker Company</td>
			<td></td>
			<td>Used to evaporate Ti, Au, and SiO2 during fabrication. Most university clean rooms have this or a similar tool.</td>
		</tr>
		<tr>
			<td>Ground wire</td>
			<td>A-M Systems</td>
			<td>781500</td>
			<td>Bare silver wire</td>
		</tr>
		<tr>
			<td>Headspace Vial, glass</td>
			<td>Supelco</td>
			<td>REF: 27298</td>
			<td>Used for storing MXene solutions</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (12.1N)</td>
			<td>Fisher Scientific</td>
			<td>CAS: 7647-01-0</td>
			<td>Corrosive; etchant material</td>
		</tr>
		<tr>
			<td>Hydrofluoric Acid, (48-51% solution in H2O)</td>
			<td>Acros</td>
			<td>CAS: 7664-39-3</td>
			<td>Etchant material</td>
		</tr>
		<tr>
			<td>Jupiter II RIE system</td>
			<td>March Plasma Systems Inc.</td>
			<td></td>
			<td>Planar RIE etching system used to etch the Parylene-C using O2 plasma. Most university clean rooms have a comparable planar RIE etching system.</td>
		</tr>
		<tr>
			<td>Kapton standard polyimide tape, 1/4&#34;</td>
			<td>DuPont</td>
			<td></td>
			<td>Used to add thickness to the Au bonding pad region of the flexible Parylene microelectrode array for insertion into the ZIF connector.</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospital of the Univ. of Penn.</td>
			<td></td>
			<td>Anesthesia for rat surgery</td>
		</tr>
		<tr>
			<td>KLA P-7 Stylus Profilometer</td>
			<td>KLA Corporation</td>
			<td></td>
			<td>Used the measure 2D profiles to confirm complete etching through the sacrificial parylene-C layer in step 2.4.2. Most university clean rooms have this or a comparable stylus profilometer tool.</td>
		</tr>
		<tr>
			<td>Lithium chloride, 99% for analysis, anhydrous</td>
			<td>Acros</td>
			<td>CAS: 7447-41-8</td>
			<td>Hygroscopic; delamination material</td>
		</tr>
		<tr>
			<td>MA6 mask aligner</td>
			<td>Karl Suss Microtec AG</td>
			<td></td>
			<td>Used to align each photomask to the pattern on the wafer and expose the wafer to UV light. Most university clean rooms have this or a similar tool.</td>
		</tr>
		<tr>
			<td>Micro-90 cleaning solution</td>
			<td>International Products Corporation</td>
			<td>M-9050-12</td>
			<td>Used as the anti-adhesive layer to enable removal of the sacrificial Parylene-C layer to pattern the MXene</td>
		</tr>
		<tr>
			<td>NR71-3000p photoresist</td>
			<td>Futurrex Inc.</td>
			<td>NR71-3000p</td>
			<td>Negative photoresist used to define Ti/Au traces and MXene patterns in the devices.</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Midwest Veterinary Supply</td>
			<td>193.63200.3</td>
			<td>To prevent corneal drying during surgery</td>
		</tr>
		<tr>
			<td>Parylene deposition system</td>
			<td>Specialty Coating Systems</td>
			<td></td>
			<td>Used to evaporate thin conformal films of Parylene-C</td>
		</tr>
		<tr>
			<td>Parylene-C dimer</td>
			<td>Specialty Coating Systems</td>
			<td>980130-c-01lbe</td>
			<td>Flexible polymer used as bottom and top passivating layers for the flexible MXene devices</td>
		</tr>
		<tr>
			<td>Photomasks (chrome on soda lime glass)</td>
			<td>University of Pennsylvania</td>
			<td></td>
			<td>Our photomasks were produced in the University clean room using a Heidelberg DWL66+ laser writer system, however several vendors manufacture photomasks from provided design files.</td>
		</tr>
		<tr>
			<td>Povidone-iodine solution</td>
			<td>Medline</td>
			<td>MDS093901</td>
			<td>To help prevent infection around scalp incision</td>
		</tr>
		<tr>
			<td>Printed Circuit Board (PCB)</td>
			<td>Advanced Circuits</td>
			<td></td>
			<td>Used to interface between the MXene electrode array and the measurement electronics such as the potentiostat and the Intan recording system. Advanced Circuits and other vendors manufacture and assemble PCBs based on the provided design files.</td>
		</tr>
		<tr>
			<td>RD6 Developer</td>
			<td>Futurrex Inc.</td>
			<td>RD6 Developer</td>
			<td>Used to develop NR71-3000p negative photoresist following UV exposure</td>
		</tr>
		<tr>
			<td>Reference 600 potentiostat</td>
			<td>Gamry Instruments</td>
			<td></td>
			<td>Used to measure the electrodes&#39; impedance to assess quality of the devices</td>
		</tr>
		<tr>
			<td>Remover PG</td>
			<td>MicroChem Corp.</td>
			<td>G050200</td>
			<td>Used to remove NR71-3000p following metal deposition to perform lift-off patterning</td>
		</tr>
		<tr>
			<td>RHS2000 Stim SPI interface cable</td>
			<td>Intan Technologies</td>
			<td></td>
			<td>A component of the neural recording system.</td>
		</tr>
		<tr>
			<td>RHS2116 amplifier board</td>
			<td>Intan Technologies</td>
			<td></td>
			<td>A component of the neural recording system.</td>
		</tr>
		<tr>
			<td>Si wafers</td>
			<td>Wafer World</td>
			<td>2885</td>
			<td>Substrate for fabrication</td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Cost Effective Equipment</td>
			<td></td>
			<td>For coating wafers with resists and applying the Micro-90 and MXene layers. Most university clean rooms have spin coaters.</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Kopf Instruments</td>
			<td>Model 902</td>
			<td>For positioning the rat for neurosurgery</td>
		</tr>
		<tr>
			<td>Teflon-coated magnetic stir bar</td>
			<td>Corning</td>
			<td>REF: 1233W95</td>
			<td>Used to stir during etching and intercalation</td>
		</tr>
		<tr>
			<td>Titanium carbide, 99.5% (metals basis), particle size ~2 &#181;m</td>
			<td>Alfa Aesar</td>
			<td>CAS: 12070-08-5</td>
			<td>Used for MAX synthesis</td>
		</tr>
		<tr>
			<td>Titanium powder, -325 mesh, 99% (metals basis), particle size &#60; 44&#181;m</td>
			<td>Alfa Aesar</td>
			<td>CAS: 7440-32-6</td>
			<td>Used for MAX synthesis</td>
		</tr>
		<tr>
			<td>Ultrasonic bath sonicator</td>
			<td>Reynolds Tech</td>
			<td></td>
			<td>For removing metal and photoresist particles during lift-off processes to pattern metals.</td>
		</tr>
		<tr>
			<td>UV vis spectrophotometer</td>
			<td>ThermoScientific</td>
			<td>Evolution 201</td>
			<td>Used to determine concentration and observe absorption peak</td>
		</tr>
		<tr>
			<td>Zetasizer, Particle Size Analysis</td>
			<td>Malvern Panalytical</td>
			<td>Nano ZS</td>
			<td>Used to determine particle lateral size distibution</td>
		</tr>
	</tbody>
</table>

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.

Sample micro-ECoG data recorded on a MXene microelectrode array is shown in Figure 5. Following application of the electrode array onto the cortex, clear physiologic signals were immediately apparent on the recording electrodes, with approximately 1 mV amplitude ECoG signals appearing on all MXene electrodes. Power spectra of these signals confirmed the presence of two brain rhythms commonly observed in rats under ketamine-dexmedetomidine anesthesia: 1&#8722;2 Hz slow oscillations and &#947;...

Watch this Scientific Journal Video about Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording at JoVE.com

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.

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 ...

fabrication-ti3c2-mxene-microelectrode-arrays-for-vivo-neural

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JoVE Journal

Bioengineering

13.2K Views.  University of Pennsylvania. We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Video: Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...