Name: open-source toolkit: benchtop carbon fiber microelectrode array for nerve recording
Uploaded: 2021-10-29T14:00:00
Description: Watch this Scientific Journal Video about Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording at JoVE.com

This work was financially supported by the National Institutes of Neurological Disorders and Stroke (UF1NS107659 and UF1NS115817) and the National Science Foundation (1707316). The authors acknowledge financial support from the University of Michigan College of Engineering and technical support from the Michigan Center for Materials Characterization and the Van Vlack Undergraduate Laboratory. The authors thank Dr. Khalil Najafi for the use of his Nd:YAG laser and the Lurie Nanofabrication Facility for the use of their Parylene C deposition machine. We would also like to thank Specialty Coating Systems (Indianapolis, IN) for their help in the commercial coating comparison study.

All animal procedures were approved by the University of Michigan Institutional Animal Care and Use Committee.
1. Choosing a carbon fiber array
<ol>
	<li>Choose a printed circuit board (PCB) from one of the three designs shown in Figure 1. 
	NOTE: For this protocol, Flex Arrays will be the focus.
	<ol>
		<li>Refer to PCB designs on the Chestek Lab website (https://chestekresearch.engin.umich.edu), free of charge and ready to be sent to and ...

<ol>
	<li>Szostak, K. M., Grand, L., Constandinou, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+interfaces+for+intracortical+recording:+Requirements,+fabrication+methods,+and+characteristics.">Neural interfaces for intracortical recording: Requirements, fabrication methods, and characteristics.</a> Frontiers in Neuroscience. 11, 665 (2017).</li><li>Cunningham, J. P., 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=A+closed-loop+human+simulator+for+investigating+the+role+of+feedback+control+in+brain-machine+interfaces.">A closed-loop human simulator for investigating the role of feedback control in brain-machine interfaces.</a> Journal of Neurophysiology. 105 (4), 1932-1949 (2011).</li><li>Yoshida, K., Bertram, M. J., Hunter Cox, T. G., Riso, R. R., Horch, K., Kipke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+recording+electrodes+and+techniques.">Peripheral nerve recording electrodes and techniques.</a> Neuroprosthetics: Theory and Practice. , 377-466 (2017).</li><li>Dweiri, Y. M., Stone, M. A., Tyler, D. J., McCallum, G. A., Durand, D. M. <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+high+contact-density,+flat-interface+nerve+electrodes+for+recording+and+stimulation+applications.">Fabrication of high contact-density, flat-interface nerve electrodes for recording and stimulation applications.</a> Journal of Visualized Experiments: JoVE. (116), e54388 (2016).</li><li>Kim, H., 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=Cuff+and+sieve+electrode+(CASE):+The+combination+of+neural+electrodes+for+bi-directional+peripheral+nerve+interfacing.">Cuff and sieve electrode (CASE): The combination of neural electrodes for bi-directional peripheral nerve interfacing.</a> Journal of Neuroscience Methods. 336, 108602 (2020).</li><li>Ciancio, A. L., 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=Control+of+prosthetic+hands+via+the+peripheral+nervous+system.">Control of prosthetic hands via the peripheral nervous system.</a> Frontiers in Neuroscience. 10, 116 (2016).</li><li>Jiman, A. A., 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=Multi-channel+intraneural+vagus+nerve+recordings+with+a+novel+high-density+carbon+fiber+microelectrode+array.">Multi-channel intraneural vagus nerve recordings with a novel high-density carbon fiber microelectrode array.</a> Scientific Reports. 10 (1), 15501 (2020).</li><li>Welle, E. 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=Sharpened+and+mechanically+robust+carbon+fiber+electrode+arrays+for+neural+interfacing.">Sharpened and mechanically robust carbon fiber electrode arrays for neural interfacing.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 29, 993-1003 (2021).</li><li>Moffitt, M. A., McIntyre, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+analysis+of+cortical+recording+with+silicon+microelectrodes.">Model-based analysis of cortical recording with silicon microelectrodes.</a> Clinical Neurophysiology. 116 (9), 2240-2250 (2005).</li><li>Nerve-cuff electrodes. Micro-Leads Neuro Available from: <a target="_blank" href="https://www.microleadsneuro.com/research-products/?jumpto=nerve-cuff">https://www.microleadsneuro.com/research-products/?jumpto=nerve-cuff</a> (2021)</li><li>Mortimer, J. T., 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=Perspectives+on+new+electrode+technology+for+stimulating+peripheral+nerves+with+implantable+motor+prostheses.">Perspectives on new electrode technology for stimulating peripheral nerves with implantable motor prostheses.</a> IEEE Transactions on Rehabilitation Engineering. 3 (2), 145-154 (1995).</li><li>Boretius, T., 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=A+transverse+intrafascicular+multichannel+electrode+(TIME)+to+interface+with+the+peripheral+nerve.">A transverse intrafascicular multichannel electrode (TIME) to interface with the peripheral nerve.</a> Biosensors &amp; Bioelectronics. 26 (1), 62-69 (2010).</li><li>Grill, W. M., Norman, S. E., Bellamkonda, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implanted+neural+interfaces+biochallenges+and+engineered+solutions.">Implanted neural interfaces biochallenges and engineered solutions.</a> Annual Review of Biomedical Engineering. 11, 1-24 (2009).</li><li>Larson, C. E., Meng, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+for+the+peripheral+nerve+interface+designer.">A review for the peripheral nerve interface designer.</a> Journal of Neuroscience Methods. 332, 108523 (2020).</li><li>Christensen, M. 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=The+foreign+body+response+to+the+Utah+Slant+Electrode+Array+in+the+cat+sciatic+nerve.">The foreign body response to the Utah Slant Electrode Array in the cat sciatic nerve.</a> Acta Biomaterialia. 10 (11), 4650-4660 (2014).</li><li>Patel, P. 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=Chronic+in+vivo+stability+assessment+of+carbon+fiber+microelectrode+arrays.">Chronic in vivo stability assessment of carbon fiber microelectrode arrays.</a> Journal of Neural Engineering. 13 (6), 066002 (2016).</li><li>Yoshida Kozai, T. D., 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=Ultrasmall+implantable+composite+microelectrodes+with+bioactive+surfaces+for+chronic+neural+interfaces.">Ultrasmall implantable composite microelectrodes with bioactive surfaces for chronic neural interfaces.</a> Nature Materials. 11 (12), 1065-1073 (2012).</li><li>Saito, 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=Application+of+carbon+fibers+to+biomaterials:+A+new+era+of+nano-level+control+of+carbon+fibers+after+30-years+of+development.">Application of carbon fibers to biomaterials: A new era of nano-level control of carbon fibers after 30-years of development.</a> Chemical Society Reviews. 40 (7), 3824-3834 (2011).</li><li>Welle, E. 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=Fabrication+and+characterization+of+a+carbon+fiber+peripheral+nerve+electrode+appropriate+for+chronic+recording.">Fabrication and characterization of a carbon fiber peripheral nerve electrode appropriate for chronic recording.</a> FASEB Journal. 34 (1), 1 (2020).</li><li>Guitchounts, G., Cox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=64-Channel+carbon+fiber+electrode+arrays+for+chronic+electrophysiology.">64-Channel carbon fiber electrode arrays for chronic electrophysiology.</a> Scientific Reports. 10 (1), 3830 (2020).</li><li>Patel, P. 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=High+density+carbon+fiber+arrays+for+chronic+electrophysiology,+fast+scan+cyclic+voltammetry,+and+correlative+anatomy.">High density carbon fiber arrays for chronic electrophysiology, fast scan cyclic voltammetry, and correlative anatomy.</a> Journal of Neural Engineering. 17 (5), 056029 (2020).</li><li>Massey, T. L., 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=Open-source+automated+system+for+assembling+a+high-density+microwire+neural+recording+array.">Open-source automated system for assembling a high-density microwire neural recording array.</a> 2016 International Conference on Manipulation, Automation and Robotics at Small Scales (MARSS). , 1-7 (2016).</li><li>Schwerdt, H. 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=Subcellular+probes+for+neurochemical+recording+from+multiple+brain+sites.">Subcellular probes for neurochemical recording from multiple brain sites.</a> Lab Chip. 17, 1104-1115 (2017).</li><li>Welle, E. 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=Ultra-small+carbon+fiber+electrode+recording+site+optimization+and+improved+in+vivo+chronic+recording+yield.">Ultra-small carbon fiber electrode recording site optimization and improved in vivo chronic recording yield.</a> Journal of Neural Engineering. 17 (2), 026037 (2020).</li><li>Guitchounts, G., Markowitz, J. E., Liberti, W. A., Gardner, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+carbon-fiber+electrode+array+for+long-term+neural+recording.">A carbon-fiber electrode array for long-term neural recording.</a> Journal of Neural Engineering. 10 (4), 046016 (2013).</li><li>Gillis, W. F., 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+fiber+on+polyimide+ultra-microelectrodes.">Carbon fiber on polyimide ultra-microelectrodes.</a> Journal of Neural Engineering. 15 (1), 016010 (2018).</li><li>Dong, T., Chen, L., Shih, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+sharpening+of+carbon+fiber+microelectrode+arrays+for+brain+recording.">Laser sharpening of carbon fiber microelectrode arrays for brain recording.</a> Journal of Micro and Nano-Manufacturing. 8 (4), 041013 (2020).</li><li>Massey, T. L., 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=A+high-density+carbon+fiber+neural+recording+array+technology.">A high-density carbon fiber neural recording array technology.</a> Journal of Neural Engineering. 16 (1), 016024 (2019).</li><li>Romeni, S., Valle, G., Mazzoni, A., Micera, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tutorial:+a+computational+framework+for+the+design+and+optimization+of+peripheral+neural+interfaces.">Tutorial: a computational framework for the design and optimization of peripheral neural interfaces.</a> Nature Protocols. 15 (10), 3129-3153 (2020).</li><li>Khani, H., Wipf, D. O. <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+tip-protected+polymer-coated+carbon-fiber+ultramicroelectrodes+and+pH+ultramicroelectrodes.">Fabrication of tip-protected polymer-coated carbon-fiber ultramicroelectrodes and pH ultramicroelectrodes.</a> Journal of The Electrochemical Society. 166 (8), 673-679 (2019).</li><li>El-Giar, E. E. D. M., Wipf, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+tip-protected+poly(oxyphenylene)+coated+carbon-fiber+ultramicroelectrodes.">Preparation of tip-protected poly(oxyphenylene) coated carbon-fiber ultramicroelectrodes.</a> Electroanalysis. 18 (23), 2281-2289 (2006).</li><li>Venkatraman, S., 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=In+vitro+and+in+vivo+evaluation+of+PEDOT+microelectrodes+for+neural+stimulation+and+recording.">In vitro and in vivo evaluation of PEDOT microelectrodes for neural stimulation and recording.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 19 (3), 307-316 (2011).</li><li>Petrossians, A., 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=Electrodeposition+and+Characterization+of+Thin-Film+Platinum-Iridium+Alloys+for+Biological+Interfaces.">Electrodeposition and Characterization of Thin-Film Platinum-Iridium Alloys for Biological Interfaces.</a> Journal of the Electrochemical Society. 158 (6), 269-276 (2011).</li><li>Lee, C. D., Hudak, E. M., Whalen, J. J., Petrossians, A., Weiland, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-impedance,+high+surface+area+Pt-Ir+electrodeposited+on+cochlear+implant+electrodes.">Low-impedance, high surface area Pt-Ir electrodeposited on cochlear implant electrodes.</a> Journal of The Electrochemical Society. 165 (12), 3015-3017 (2018).</li><li>Cassar, I. 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=Electrodeposited+platinum-iridium+coating+improves+in+vivo+recording+performance+of+chronically+implanted+microelectrode+arrays.">Electrodeposited platinum-iridium coating improves in vivo recording performance of chronically implanted microelectrode arrays.</a> Biomaterials. 205, 120-132 (2019).</li><li>Taylor, I. 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=Enhanced+dopamine+detection+sensitivity+by+PEDOT/graphene+oxide+coating+on+in+vivo+carbon+fiber+electrodes.">Enhanced dopamine detection sensitivity by PEDOT/graphene oxide coating on in vivo carbon fiber electrodes.</a> Biosensors and Bioelectronics. 89, 400-410 (2017).</li><li>Mohanaraj, S., 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=Gold+nanoparticle+modified+carbon+fiber+microelectrodes+for+enhanced+neurochemical+detection.">Gold nanoparticle modified carbon fiber microelectrodes for enhanced neurochemical detection.</a> Journal of Visualized Experiments: JoVE. (147), e59552 (2019).</li><li>Pusch, J., Wohlmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2+-+Carbon+fibers.">Chapter 2 - Carbon fibers.</a> Inorganic and composite fibers. Production, properties, and applications. , 31-51 (2019).</li><li>Budai, D., Hern&#225;di, I., M&#233;sz&#225;ros, B., Bali, Z. K., Gulya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+responses+of+carbon+fiber+microelectrodes+to+dopamine+in+vitro+and+in+vivo.">Electrochemical responses of carbon fiber microelectrodes to dopamine in vitro and in vivo.</a> Acta Biologica Szegediensis. 54 (2), 155-160 (2010).</li></ol>

Material substitutions 
While all materials used are summarized in the Table of Materials, very few of the materials are required to come from specific vendors. The Flex Array board must come from the listed vendor as they are the only company that can print the flexible board. The Flex Array connector must also be ordered from the vendor listed as it is a proprietary connector. Parylene C is highly recommended as the insulation material for the fibers as it provides a conformal coa...

Much of neuroscience research relies upon recording neural signals using electrophysiology (ePhys). These neural signals are crucial to understanding the functions of neural networks and novel medical treatments such as brain machine and peripheral nerve interfaces1,2,3,4,5,6. Research surrounding peripheral nerves requires custom-made or commercially available neural recording electrodes. Neural recording electrodes-unique tools with micron-scale dimensions and fragile materials-require a specialized set of skills and equipment to fabricate. A variety of specialized probes have been developed for specific end uses; however, this implies that experiments must be designed around currently available commercial probes, or a laboratory must invest in the development of a specialized probe, which is a lengthy process. Due to the wide variety of neural research in peripheral nerve, there is high demand for a versatile ePhys probe4,7,8. An ideal ePhys probe would feature a small recording site, low impedance9, and a financially realistic price point for implementation in a system3.
Current commercial electrodes tend to either be extraneural or cuff electrodes (Neural Cuff10, MicroProbes Nerve Cuff Electrode11), which sit outside the nerve, or intrafascicular, which penetrate the nerve and sit within the fascicle of interest. However, as cuff electrodes sit further away from the fibers, they pick up more noise from nearby muscles and other fascicles that may not be the target. These probes also tend to constrict the nerve, which can lead to biofouling-a build-up of glial cells and scar tissue-at the electrode interface while the tissue heals. Intrafascicular electrodes (such as LIFE12, TIME13, and Utah Arrays14) add the benefit of fascicle selectivity and have good signal-to-noise ratios, which is important in discriminating signals for machine interfacing. However, these probes do have issues with biocompatibility, with nerves becoming deformed over time3,15,16. When bought commercially, both these probes have static designs with no option for experiment-specific customization and are costly for newer laboratories.
In response to the high cost and biocompatibility issues presented by other probes, carbon fiber electrodes may offer an avenue for neuroscience laboratories to build their own probes without the need for specialized equipment. Carbon fibers are an alternative recording material with a small form factor that allows for low damage insertion. Carbon fibers provide better biocompatibility and considerably lower scar response than silicon17,18,19 without the intensive cleanroom processing5,13,14. Carbon fibers are flexible, durable, easily integrated with other biomaterials19, and can penetrate and record from nerve7,20. Despite the many advantages of carbon fibers, many laboratories find the manual fabrication of these arrays arduous. Some groups21 combine carbon fibers into bundles that collectively result in a larger (~200 &#181;m) diameter; however, to our knowledge, these bundles have not been verified in nerve. Others have fabricated individuated carbon fiber electrode arrays, although their methods require cleanroom-fabricated carbon fiber guides22,23,24 and equipment to populate their arrays17,23,24. To address this, we propose a method of fabricating a carbon fiber array that can be performed at the laboratory benchtop that allows for impromptu modifications. The resulting array maintains individuated electrode tips without specialized fiber populating tools. Additionally, multiple geometries are presented to match the needs of the research experiment. Building from previous work8,17,22,25, this paper provides detailed methodologies to build and modify several styles of arrays manually with minimal cleanroom training time needed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 prong clams</td>
			<td>05-769-6Q</td>
			<td>Fisher</td>
			<td>Qty: 2 
			Unit Cost (USD): 20</td>
		</tr>
		<tr>
			<td>3,4-ethylenedioxythiophene (25 g) 
			(PEDOT)</td>
			<td>96618</td>
			<td>Sigma-Aldrich</td>
			<td>Qty: 1 
			Unit Cost (USD): 102</td>
		</tr>
		<tr>
			<td>353ND-T Epoxy (8oz)++ 
			(ZIF and Wide Board Only)</td>
			<td>353ND-T/8OZ</td>
			<td>Epoxy Technology</td>
			<td>Qty: 1 
			Unit Cost (USD): 48</td>
		</tr>
		<tr>
			<td>Ag/AgCl (3M NaCl) Reference Electrode (pack of 3)</td>
			<td>50-854-570</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 100</td>
		</tr>
		<tr>
			<td>Autolab</td>
			<td>PGSTAT12</td>
			<td>Metrohm</td>
			<td></td>
		</tr>
		<tr>
			<td>Blowtorch</td>
			<td>1WG61</td>
			<td>Grainger</td>
			<td>Qty: 1 
			Unit Cost (USD): 36</td>
		</tr>
		<tr>
			<td>Carbon Fibers</td>
			<td>T-650/35 3K</td>
			<td>Cytec Thornel</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Carbon tape</td>
			<td>NC1784521</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 27</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicator</td>
			<td>WOD1002</td>
			<td>MediChoice</td>
			<td>Qty: 1 
			Unit Cost (USD): 0.57</td>
		</tr>
		<tr>
			<td>Delayed Set Epoxy++</td>
			<td>1FBG8</td>
			<td>Grainger</td>
			<td>Qty: 1 
			Unit Cost (USD): 3</td>
		</tr>
		<tr>
			<td>DI Water</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Qty: n/a 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Dumont Tweezers #5</td>
			<td>50-822-409</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 73</td>
		</tr>
		<tr>
			<td>Flex Array**</td>
			<td>n/a</td>
			<td>MicroConnex</td>
			<td>Qty: 1 
			Unit Cost (USD): 68</td>
		</tr>
		<tr>
			<td>Flux</td>
			<td>SMD291ST8CC</td>
			<td>DigiKey</td>
			<td>Qty: 1 
			Unit Cost (USD): 13</td>
		</tr>
		<tr>
			<td>Glass Capillaries (pack of 350)</td>
			<td>50-821-986</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 60</td>
		</tr>
		<tr>
			<td>Glass Dish</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Hirose Connector 
			(ZIF Only)</td>
			<td>H3859CT-ND</td>
			<td>DigiKey</td>
			<td>Qty: 2 
			Unit Cost (USD): 2</td>
		</tr>
		<tr>
			<td>Light-resistant Glass Bottle</td>
			<td>n/a</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Micropipette Heating Filiment</td>
			<td>FB315B</td>
			<td>Sutter Instrument Co</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>P-97</td>
			<td>Sutter Instrument Co</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Nitrile Gloves (pack of 200)</td>
			<td>19-041-171C</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 47</td>
		</tr>
		<tr>
			<td>Offline Sorter software</td>
			<td>n/a</td>
			<td>Plexon</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Omnetics Connector* 
			(Flex Array Only)</td>
			<td>A79025-001</td>
			<td>Omnetics Inc</td>
			<td>Qty: 1 
			Unit Cost (USD): 35</td>
		</tr>
		<tr>
			<td>Omnetics Connector* 
			(Flex Array Only)</td>
			<td>A79024-001</td>
			<td>Omnetics Inc</td>
			<td>Qty: 1 
			Unit Cost (USD): 35</td>
		</tr>
		<tr>
			<td>Omnetics to ZIF connector</td>
			<td>ZCA-OMN16</td>
			<td>Tucker-Davis Technologies</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Pin Terminal Connector 
			(Wide Board Only)</td>
			<td>ED11523-ND</td>
			<td>DigiKey</td>
			<td>Qty: 16 
			Unit Cost (USD): 10</td>
		</tr>
		<tr>
			<td>Probe storage box</td>
			<td>G2085</td>
			<td>Melmat</td>
			<td>Qty: 1 
			Unit Cost (USD): 2</td>
		</tr>
		<tr>
			<td>Razor Blade</td>
			<td>4A807</td>
			<td>Grainger</td>
			<td>Qty: 1 
			Unit Cost (USD): 2</td>
		</tr>
		<tr>
			<td>SEM post</td>
			<td>16327</td>
			<td>lnf</td>
			<td>Qty: 1 
			Unit Cost (USD): 3</td>
		</tr>
		<tr>
			<td>Silver Epoxy (1oz)++</td>
			<td>H20E/1OZ</td>
			<td>Epoxy Technology</td>
			<td>Qty: 1 
			Unit Cost (USD): 125</td>
		</tr>
		<tr>
			<td>Silver GND REF wires</td>
			<td>50-822-122</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 423.2</td>
		</tr>
		<tr>
			<td>Sodium p-toulenesulphonate(pTS)- 100g</td>
			<td>152536</td>
			<td>Sigma-Aldrich</td>
			<td>Qty: 1 
			Unit Cost (USD): 59</td>
		</tr>
		<tr>
			<td>Solder</td>
			<td>24-6337-9703</td>
			<td>DigiKey</td>
			<td>Qty: 1 
			Unit Cost (USD): 60</td>
		</tr>
		<tr>
			<td>Soldering Iron Tip</td>
			<td>T0054449899N-ND</td>
			<td>Digikey</td>
			<td>Qty: 1 
			Unit Cost (USD): 13</td>
		</tr>
		<tr>
			<td>Soldering Station</td>
			<td>WD1002N-ND</td>
			<td>Digikey</td>
			<td>Qty: 1 
			Unit Cost (USD): 374</td>
		</tr>
		<tr>
			<td>SpotCure-B UV LED Cure System</td>
			<td>n/a</td>
			<td>FusionNet LLC</td>
			<td>Qty: 1 
			Unit Cost (USD): 895</td>
		</tr>
		<tr>
			<td>Stainless steel rod</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Stir Plate</td>
			<td>n/a</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): n/a</td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>08-953-1B</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 100</td>
		</tr>
		<tr>
			<td>TDT Shroud 
			(ZIF Only)</td>
			<td>Z3_ZC16SHRD_RSN</td>
			<td>TDT</td>
			<td>Qty: 1 
			Unit Cost (USD): 3.5</td>
		</tr>
		<tr>
			<td>Teflon Tweezers</td>
			<td>50-380-043</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 47</td>
		</tr>
		<tr>
			<td>UV &#38; Visible Light Safety Glassees</td>
			<td>92522</td>
			<td>Loctite</td>
			<td>Qty: 1 
			Unit Cost (USD): 45</td>
		</tr>
		<tr>
			<td>UV Epoxy (8oz)++ 
			(Flex Array Only)</td>
			<td>OG142-87/8OZ</td>
			<td>Epoxy Technology</td>
			<td>Qty: 1 
			Unit Cost (USD): 83</td>
		</tr>
		<tr>
			<td>UV Laser</td>
			<td>n/a</td>
			<td>WER</td>
			<td>Qty: 1 
			Unit Cost (USD): 30</td>
		</tr>
		<tr>
			<td>Weigh boat 
			(pack of 500)</td>
			<td>08-732-112</td>
			<td>Fisher</td>
			<td>Qty: 1 
			Unit Cost (USD): 58</td>
		</tr>
		<tr>
			<td>Wide Board+</td>
			<td>n/a</td>
			<td>Advanced Circuits</td>
			<td>Qty: 1 
			Unit Cost (USD): 3</td>
		</tr>
		<tr>
			<td>ZIF Active Headstage</td>
			<td>ZC16</td>
			<td>Tucker-Davis Technologies</td>
			<td>Qty: 1 
			Unit Cost (USD): 925</td>
		</tr>
		<tr>
			<td>ZIF Passive Headstage</td>
			<td>ZC16-P</td>
			<td>Tucker-Davis Technologies</td>
			<td>Qty: 1 
			Unit Cost (USD): 625</td>
		</tr>
		<tr>
			<td>ZIF*</td>
			<td>n/a</td>
			<td>Coast to Coast Circuits</td>
			<td>Qty: 1 
			Unit Cost (USD): 9</td>
		</tr>
	</tbody>
</table>

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.

Tip validation: SEM images 
Previous work20 showed that scissor cutting resulted in unreliable impedances as Parylene C folded across the recording site. Scissor cutting is used here only to cut fibers to the desired length before processing with an additional finish cutting method. SEM images of the tips were used to determine the exposed carbon length and tip geometry (Figure 8).
Scissor and Nd:YAG laser-cut fibers w...

Watch this Scientific Journal Video about Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording at JoVE.com

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

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 ...

Carbon fiber electrodes, are smaller than neurons and do minimal damage. In addition, they can be manufactured with minimal specialized equipment on the bench top. The main advantage of this technique is in its simplicity.It allows users to build and customize neural arrays, with no cleanroom experience. Manipulating the carbon fibers and placing the silver epoxy is tricky and critical. These two parts take a lot of practice and fine motor control.The first day is always the worst, but with practice, after about a week, you will build a functional neural array. To begin, set a soldering iron to 315 degrees Celsius. Apply flux to all soldering pads.Form small mounds of solder on the back pads of the flex array, then solder the pins on either side of the connector. Once secure, gently push the soldering iron tip, between the front pins, to solder the remaining connections in the back. Apply an additional flux layer, if soldering takes a long time, then solder the front row of pins to the board.Clean away excess flux with 100%isopropyl alcohol, and a short bristle brush. Next, slowly push the laid set epoxy with a syringe, placed bevel side down on the pins, to encapsulate the soldered connection. Lay a small line of the epoxy across the board's backside, and pull it onto the connector's edges to secure it.Make capillaries with a glass puller and filament. Cut a pulled glass capillary, so that its tip fits between the traces of the array. Then, scoop approximately a one to one ratio of silver epoxy into a plastic dish, with the wooden ends of two cotton tipped applicators and mix it.Discard the applicators after mixing. Cut two to four millimeters, from the end of a carbon fiber bundle, onto a printer paper with a razor blade. Pull a laminated paper, gently, over the top of the bundle to separate the fibers and the bundle.Next, take a little epoxy onto the pulled capillary's end. And gently apply it between every other trace, on the end of the board, filling the gap. Place one carbon fiber in each epoxy trace, with teflon coated tweezers.Then, adjust the carbon fibers with a clean pulled capillary, to make perpendicular to the end of the flex array board and bury them beneath the epoxy. Place the arrays on a wooden block, with fibered ends overhanging the edge of the block. Bake the wooden block and arrays at 140 degrees Celsius for 20 minutes, to cure the silver epoxy and lock the fibers into place.If silver epoxy shorts two or more of the fibers together, it can be removed using a clean glass capillary, and gently scraping it off the board. Store the finished boards in a box with a raised platform, to suspend the fibered ends of the board, to prevent fiber breakage. Apply a tiny droplet of UV epoxy on the exposed traces with a clean capillary, and continue adding droplets, until the traces are completely covered.Cure the UV epoxy under a UV pen for two minutes and repeat it for the other side of the board. Cut the fibers to one millimeter with a stereoscope reticle and surgical scissors. To check electrical connections, set the potentiostat to zero volts, for five seconds, and stabilize the recorded signal.Run a one kilohertz impedance scan for each fiber with a potentiostat. Record the measurements via the potentiostat associated software. Then dip the fibers in deionized water in a beaker, three times, to rinse them.Gently scrape away Parylene C from the ground, and reference wires on the board with tweezers. Next, cut two five centimeter lengths of insulated silver wire with a razor blade. De-insulate two to three millimeters of wire, from one end and around 10 millimeters from the opposite end.Next, heat the soldering iron to 315 degrees Celsius, and apply a small flux to the wires. Insert two to three millimeters of one wire, into each electrophysiology wire on the board, and apply solder to the top of the wires. After allowing the probe to cool, flip it over to apply a little solder to the backside of the wire.Snip off any exposed wire sticking out of the back solder mound. Place the arrays in the storage box, bending the wires back, away from the fiber and secure the wires on the adhesive tape, to prevent potential fiber wire interactions. SEM images of the tips were used to determine the exposed carbon length and tip geometry.Scissor cut fibers have inconsistent tip geometries, with Parylene C folding over the end. The NDYAG laser cut fibers remain consistent, in the recording site area, shape and impedance. Blow torched fibers lead to the largest electrode size, and shape variability and a sharpened tip.On average, 140 micrometers of carbon were re-exposed. UV laser cut fibers were similar to blow torched fibers, showing 120 micro meters of carbon, exposed from the tip. The resulting impedances were within range, for electrophysiological recording.NDYAG laser cut fibers had the smallest surface area but the highest impedances. Followed by blow torched and UV laser cut fibers. However, in all cases, the PEDOT:pTS coated fibers, fell under the 110 kilo ohm threshold.Acute recordings from four UV laser treatment fibers, of two millimeter length, simultaneously implanted in rat motor cortex, showed three units across all fibers, suggesting that the treatment of the fibers with the inexpensive UV laser, is similar to other cutting methods. When attempting this protocol, give yourself a large, clean space when populating carbon fibers. It is easy to accidentally sweep all of your fibers off the table, because there's not enough maneuvering room.These arrays are now suitable for signal unit recordings from the brain. These building techniques allowed Dr.Barnes'group, at the University of Michigan, to report and Dr.Chiel's group at Case Western Reserve University, to report intracellular

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Research

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Bioengineering

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

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 ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...