We would like to thank American University, the Faculty Research Support Grant, NASA DC Space Grant, and NSF-MRI#1625977.

1. Construction of carbon-fiber microelectrodes
<ol>
	<li>Preparation of carbon fibers
	<ol>
		<li>To create carbon-fiber microelectrodes, first separate the carbon fibers (carbon fiber,7 &#956;m in diameter) one by one using hands, gloves, and spatula.</li>
		<li>Pull or yank one fiber from the twisted yarn.</li>
		<li>Aspirate an isolated carbon fiber into a glass capillary (single-barrel borosilicate capillary glass without microfilament, 1.2 mm outer diameter, 0.68 mm inner diameter).</li>
		<li>Cr...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication&#8212;Carbon+Nanotube+Fiber+Microelectrodes+for+High+Temporal+Measurements+of+Dopamine.">Communication&#8212;Carbon Nanotube Fiber Microelectrodes for High Temporal Measurements of Dopamine.</a> Journal of The Electrochemical Society. 165, G3071-G3073 (2018).</li><li>Park, J., Takmakov, P., Wightman, R. 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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=Voltammetric+detection+of+hydrogen+peroxide+at+carbon+fiber+microelectrodes.">Voltammetric detection of hydrogen peroxide at carbon fiber microelectrodes.</a> Analytical chemistry. 82, 5205-5210 (2010).</li><li>Heien, M. L., Johnson, M. A., Wightman, R. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+Nanoelectrodes+for+the+Electrochemical+Detection+of+Neurotransmitters.">Carbon Nanoelectrodes for the Electrochemical Detection of Neurotransmitters.</a> International Journal of Electrochemistry. , (2018).</li><li>Kim, J. 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=Dopamine+neurons+derived+from+embryonic+stem+cells+function+in+an+animal+model+of+Parkinson's+disease.">Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease.</a> Nature. 418, 50 (2002).</li><li>Zestos, A. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+Nanotube-Based+Microelectrodes+for+Enhanced+Neurochemical+Detection.">Carbon Nanotube-Based Microelectrodes for Enhanced Neurochemical Detection.</a> ECS Transactions. 80, 1497-1509 (2017).</li><li>Zachek, M. K., Hermans, A., Wightman, R. M., McCarty, G. 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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=Concurrent+zero-dimensional+and+one-dimensional+biomineralization+of+gold+from+a+solution+of+Au3++and+bovine+serum+albumin.">Concurrent zero-dimensional and one-dimensional biomineralization of gold from a solution of Au3+ and bovine serum albumin.</a> Science and technology of advanced materials. 14, 065004 (2013).</li><li>Xiao, N., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+sensitive+detection+of+neurotransmitters+at+microelectrodes+modified+with+self-assembled+SWCNT+forests.">Rapid, sensitive detection of neurotransmitters at microelectrodes modified with self-assembled SWCNT forests.</a> Analytical chemistry. 84, 7816-7822 (2012).</li><li>Zestos, A. G., Jacobs, C. B., Trikantzopoulos, E., Ross, A. E., Venton, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine+Carbon+Nanotube+Fiber+Electrodes+for+Enhanced+Detection+of+Neurotransmitters.">Polyethylenimine Carbon Nanotube Fiber Electrodes for Enhanced Detection of Neurotransmitters.</a> Analytical chemistry. 86, 8568-8575 (2014).</li><li>Yang, 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=Carbon+nanotubes+grown+on+metal+microelectrodes+for+the+detection+of+dopamine.">Carbon nanotubes grown on metal microelectrodes for the detection of dopamine.</a> Analytical chemistry. 88, 645-652 (2015).</li></ol>

In this study, we demonstrate a novel method to construct gold-nanoparticle modified carbon fiber microelectrodes for the detection of neurotransmitters such as dopamine using fast scan cyclic voltammetry. The method is an efficient, green, and relatively inexpensive approach to enhancing the sensitivity of biomolecule detection. The thickness of gold deposited onto the surface of the carbon fiber can be controlled by the time of electrodeposition and the concentration of gold present in the electrodeposition solution. G...

Carbon-fiber microelectrodes (CFMEs)1 are best used as biosensors to detect the oxidation of several crucial neurotransmitters2, including dopamine3, norepinephrine4, serotonin5, adenosine6, histamine7, and others8. The biocompatibility and size of carbon fibers make them optimal for implantation as there is mitigated tissue damage compared to larger standard electrodes.9 CFMEs are known to possess useful electrochemical properties and are capable of making quick measurements when used with fast electrochemical techniques, most commonly fast-scan cyclic voltammetry (FSCV)10,11. FSCV is a technique that scans the applied potential rapidly and provides a specific cyclic voltammogram for specific analytes12,13. The large charging current produced by fast scanning is stable on carbon fibers and can be background-subtracted to produce specific cyclic voltammograms.
Due to its optimal electrochemistry and neurobiological importance, dopamine has been widely studied. The catecholamine dopamine is an essential chemical messenger that plays a pivotal role in the control of movement, memory, cognition, and emotion within the nervous system. A surplus or deficiency of dopamine can cause numerous neurological and psychological interference; among these are Parkinson&#8217;s disease, schizophrenia, and addictive behavior. Today, Parkinson&#8217;s disease continues to be a prevalent disorder due to the degeneration of midbrain neurons involved in dopamine synthesis14. Parkinson&#8217;s disease symptoms include tremor, slowness of movement, stiffness, and problems in maintaining balance. On the other hand, stimulants such as cocaine15&#160;and amphetamine16,17&#160;promote the overflow of dopamine. Drug abuse eventually substitutes the regular flow of dopamine and conditions the brain to require a surplus of dopamine, which eventually leads to addictive behaviors.
In recent years, there has been an emphasis on improving electrode functionality in neurotransmitter detection18. The most widespread method of enhancing electrode sensitivity is by coating the fiber surface. Surprisingly, there has been limited research done on metal nanoparticle electrodeposition onto carbon-fibers19. Noble metal-nanoparticles such as gold, may be electrodeposited onto the fiber surface with other functional materials20. For example, increasing the electroactive surface area for neurotransmitter adsorption to occur. Electrodeposited metal nanoparticles form rapidly, can be purified, and adhere to the carbon-fiber. Electrochemistry continues to be significant for both the deposition of noble metal nanoparticles and surface enhancement of carbon-fibers, as it allows for the control of nucleation and growth of these nanoparticles. Finally, the increased catalytic and conductive characteristics, and improved mass transport are among other advantages of utilizing metal nanoparticles for electroanalysis.
The Advanced Laboratory sequence course of American University (Experimental Biological Chemistry I and II CHEM 471/671-472/672) is a combination of Analytical, Physical, and Biochemistry laboratories. The first semester is an overview of laboratory techniques. The second semester is a student-driven and led research project21. For these projects, students have previously examined the mechanism of biomolecule, protein, peptide, and amino acid-facilitated synthesis of gold nanoparticles22,23. More recent work has focused on the formation of gold nanoparticle (AuNP) production on electrode surfaces and the evaluation of AuNPs effects on the ability of CFMEs to detect neurotransmitters. In the present work, the laboratory has applied this technique to demonstrate that the sensitivity of CFMEs in detecting the dopamine-oxidation is enhanced through the electrodeposition of AuNP onto the fiber surface. Each bare-CFME is characterized by varying scan-rate, stability and dopamine-concentration when detecting dopamine-oxidative currents to measure dopamine oxidation on the surface of the CFME. Au3+ was then electroreduced to Au0 and concurrently electrodeposited onto the fiber surface as nanoparticles, followed by a series of characterization experiments. After a direct comparison, the AuNP-CFMEs were found to possess higher sensitivity of dopamine detection. The uniform coating of AuNP onto the fiber surface via electrodeposition renders a higher electroactive surface area; thus, increasing the adsorption of dopamine onto the modified electrode surface. This led to higher dopamine oxidative currents. The potential separation of the dopamine oxidation and reduction peaks (&#8710;Ep) of AuNP-CFMEs was also smaller, suggesting faster electron transfer kinetics. Future works of this study includes the in vivo testing of both the bare- and AuNP-CFMEs for the detection of dopamine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dopamine hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>H8502-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pine WaveNeuro Potentiostat</td>
			<td>Pine Instruments</td>
			<td>NEC-WN-BASIC</td>
			<td>This orders comes in bulk with all other accessories such as headstages, adapters, cords, and other electronics</td>
		</tr>
		<tr>
			<td>Pine Flow Cell and Micromanipulator</td>
			<td>Pine Instruments</td>
			<td>NEC-FLOW-1</td>
			<td>This is also another bulk order including the micromanipulator, flow cell, knobs, tubing, connectors, etc.</td>
		</tr>
		<tr>
			<td>Glass-Capillary</td>
			<td>A-M Systems</td>
			<td>602500</td>
			<td></td>
		</tr>
		<tr>
			<td>T-650 Carbon Fiber</td>
			<td>Goodfellow</td>
			<td>C 005711</td>
			<td></td>
		</tr>
		<tr>
			<td>Epon 828 Epoxy</td>
			<td>Miller-Stephenson</td>
			<td>EPON 828 TDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethelynetriamine</td>
			<td>Sigma Aldrich</td>
			<td>D93856-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold (III) chloride</td>
			<td>Sigma Aldrich</td>
			<td>254169</td>
			<td>Comes as either HAuCl4 or AuCl3</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Fisher</td>
			<td>S90528</td>
			<td></td>
		</tr>
		<tr>
			<td>Farraday Cage</td>
			<td>AMETEK TMC</td>
			<td>81-334-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>NEW ERA PUMP</td>
			<td>NE-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Pipettes and Tips</td>
			<td>Eppendorf</td>
			<td>2231000222</td>
			<td>This is also a bulk order containing multiple pipettes and tips</td>
		</tr>
		<tr>
			<td>10 -1,000 mL beakers</td>
			<td>VWR</td>
			<td>10536-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon fiber</td>
			<td>Goodfellow</td>
			<td>C 005711</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>JEOL</td>
			<td>JSM-IT100</td>
			<td></td>
		</tr>
	</tbody>
</table>

gold nanoparticle modified carbon fiber microelectrodes for enhanced neurochemical detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

For Figure 1, we show a schematic where FSCV testing is utilized to measure the concentration of neurotransmitters in vitro. Figure 1 displays the dopamine waveform applied. The triangle waveform scans from -0.4 V to 1.3 V at 400 V/s. In the second part of the figure to the left, it displays the oxidation of dopamine to dopamine-ortho-quinone (DOQ), a two electron transfer process occurs from the surface of the analyte to the sur...

Watch this Scientific Journal Video about Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection at JoVE.com

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

The method is significant because it enables neurochemical detection with high spatial and temporal resolution which can potentially enhance in vivo methods of neurochemical detection. The main advantage of this technique is that it is a quick, easy and reproducible method to enhance the sensitivity and temporal resolution of neurotransmitter detection. Demonstrating the procedure will be Sanuja Mohanaraj and Pauline Wonnenberg, graduate students from my laboratory.To begin, separate carbon fiber material into individual strands and pull a single seven-micrometer diameter carbon fiber from a strand. Connect a vacuum line to a borosilicate glass capillary and aspirate the carbon fiber into the capillary. Then cut a 10-centimeter by 25-centimeter piece of cardboard to serve as an electrode holder.Tape a paper towel around the cardboard as a support, then insert the capillary into the electrode holder and carefully secure it in a vertical capillary puller. Configure the capillary puller to pull the glass capillary to a fine taper for electrode materials and start it. Once pulling finishes and the heating coil has cooled, cut the carbon fiber connecting the tube-pulled electrodes.Carefully remove the microelectrodes from the capillary puller. Guided by a stereoscope or microscope, use a sharp blade or surgical scissors to trim the protruding carbon fiber on each electrode to about 100 to 150 micrometers in length. Next, in a 25-milliliter vial, use a cotton swab to mix 10 grams of epoxy with 0.2 milliliters of hardener.Fill another vial with acetone. For each electrode, immerse the carbon fiber tip in epoxy for 15 seconds and then dip it in acetone for three seconds to remove excess epoxy. After epoxying, the epoxied electrodes are then cured in the oven for three hours at 125 degrees Celsius.Next, use a micromanipulator to place a carbon fiber microelectrode in a silver-silver-chloride reference electrode in a 0.5 millimolar solution of chloroauric acid in 0.1 molar aqueous potassium chloride. Connect the electrodes to a potentiostat with a carbon fiber microelectrode as the working electrode. Scan the electrode from 0.2 volts to minus one volt at 50 millivolts per second for 10 cycles to perform electrode deposition.It is critical to optimize the parameters for depositing the gold coating. Too much coating will cause noise and signal overload while too little coating will not enhance neurochemical detection. Before the test, prepare a 10-millimolar stock solution of dopamine in perchloric acid and about a liter of pH 7.4 PBS-based buffer in deionized water.Pipette one microliter of the dopamine stock solution into 10 milliliters of buffer to make an approximately one micromolar dopamine solution. Then connect a carbon fiber microelectrode and a silver-silver chloride reference electrode to a potentiostat. Fix the carbon fiber electrode and the reference electrode in the head stage of the flow cell apparatus and use the micromanipulator to lower them into the flow cell.Draw 60 milliliters of the PBS buffer into a syringe. Fill the flow cell with buffer, and mount the syringe in a syringe pump. Start flowing buffer through the flow cell at a rate of one milliliter per minute.Then configure the potentiostat to scan for minus 0.4 volts to 1.3 volts at 10 hertz and 400 volts per second. Briefly apply the waveform to the microelectrode, observe the oscilloscope, and adjust the gain to prevent overloading. Let the microelectrode equilibrate for 10 minutes in the buffer.Then draw the diluted dopamine solution into a syringe and connect it to the injection port of the flow cell. Set the total run time on the potentiostat to 30 seconds. Start recording the measurements, wait 10 seconds, and then inject 0.2 milliliters of dopamine solution into the flow cell.When the run finishes, process the data with high definition cyclic voltammetry analysis software. Let the microelectrode re-equilibrate for 10 minutes before performing another test. When the tests are finished, clean the flow cell by injecting three milliliters of water and three milliliters of air into the buffer and injection ports three times.Coated carbon fibers were imaged with scanning electron microscopy. The thickness and particle size of the gold nanoparticle coatings can be controlled by the electrode deposition time. 20 minutes of electrode deposition yielded a thick gold coating with sharp ridges while five minutes yielded a thin uniform gold coating.Gold nanoparticle coated carbon-fiber microelectrodes had significantly higher peak oxidative currents and faster electron transfer kinetics than unmodified electrodes. The gold nanoparticle coating had no significant effect on the stability of the electrode responses as demonstrated here in a solution of dopamine. Both bare and gold nanoparticle coated electrodes responded linearly to scan rate changes with a much greater magnitude of change in the gold-coated electrodes.This indicated that dopamine absorption could be controlled via the scan rate. Both bare and gold nanoparticle-coated electrodes responded linearly between dopamine concentrations of 100 nanomolar to 10 micromolar. An asymptotic curve was observed at higher concentrations, indicating that dopamine is supersaturated at the electrode surface.The ability to detect neurochemical changes on a faster time scale and at higher sensitivities will help answer complex questions in neuroscience. This method also has uses in analytical chemistry, metabolomics, and environmental science. While it is easy to learn, visual demonstration is critical for learning to make, modify, and test the microelectrodes.Future directions for this method include adjusting the deposition of gold and other coatings to account for thickness, size, shape, and morphology to optimize the detection of specific neurotransmitters. Researchers should practice handling small fibers under a microscope before trying this technique. Also, neurotransmitter stock solution should be prepared in a fume hood because they use 1 molar perchloric acid.

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JoVE Journal

Chemistry

8.4K vistas.  American University. El método es significativo porque permite la detección neuroquímica con alta resolución espacial y temporal que potencialmente puede mejorar los métodos in vivo de detección neuroquímica. La principal ventaja de esta técnica es que es un método rápido, fácil y reproducible para mejorar la sensibilidad y resolución temporal de la detección de neurotransmisores. Demostrando el procedimiento serán Sanuja Mohanaraj y Pauline Wonnenberg, estudiantes graduados de mi laboratorio.