Name: Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection (Video) | JoVE Journal
Uploaded: 2019-05-13T14:45:00.000+00:00
Duration: 7 min 34 s
Description: Learn to modify carbon-fiber microelectrodes with gold nanoparticles for enhanced neurotransmitter detection. This quick, reproducible method improves sensitivity and temporal resolution for in vivo neurochemical studies.

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5 minutes

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>Create an electrode holder for the electrodes by cutting a piece of cardboard that is approximately 10 cm in length by 25 cm in width.</li>
	</ol>
	</li>
	<li>Pull the electrodes using a vertical capillary puller.</li>
	<li>Open the sliding door of the vertical capillary puller.</li>
	<li>Loosen and remove the metallic holder rod, by rotating the drill-chuck counterclockwise with enough space to insert the glass capillary.</li>
	<li>Insert the glass capillary into the electrode holder. Raise the glass capillary to the top of the vertical capillary manually by hand.</li>
	<li>Tighten the glass capillary with the drill-chucks clockwise without breaking or shattering the glass capillaries.</li>
	<li>Adjust the Heater 1, Heater 2, and Magnet settings to the manufacturer suggested levels to pull glass capillaries to a fine taper for electrode materials.</li>
	<li>Press the red start button to heat the coiled coil to pull the electrodes via pressure, gravity, and heating.</li>
	<li>Let the coiled coil cool from its red hot state. Cut the carbon fiber with scissors connecting the two pulled electrodes from top to bottom. Use the drill-chuck method to remove the glass capillary from the vertical capillary puller by twisting in the counter-clockwise direction.</li>
</ol>
2. Carbon-Fiber Microelectrode Preparation
<ol>
	<li>Under a stereoscope or microscope, cut the carbon fiber protruding from the surface of the glass capillary with surgical scissors or a sharp razor blade to approximately 100 &#8211;150 &#956;m in length.</li>
	<li>Prepare a solution of epoxy by mixing 10 g of epoxy with 0.2 mL of hardener in a 25 mL vial using a cotton swab.</li>
	<li>Dip only the tip of each electrode into the epoxy and hardener solution for approximately 15 s.</li>
	<li>Dip the aforementioned top of the carbon fiber microelectrode in acetone for approximately 3 s to wash away any excess epoxy from the barrel of the carbon fiber microelectrode.</li>
</ol>
3. Electrodeposition
<ol>
	<li>Place the working electrode (carbon-fiber microelectrode) in the solution of 0.5 mM HAuCl4 in addition to the reference electrode, silver-silver chloride (Ag/AgCl) using the micromanipulator.</li>
	<li>Connect the working electrode and the reference electrode to the potentiostat and headstage.</li>
	<li>Open the UNC HDCV Software. Change the settings on the software to apply the waveform. Enter the following waveform into the computer settings: scan from 0.2 V to &#8722;1.0 V in 0.1 M KCl solution containing 0.5 mM HAuCl4 at a scan rate of 50 mV/s for 10 cycles. Press the green arrow to apply the waveform. Then, press the start button to begin recording of the measurements.</li>
</ol>
4. Scanning electron microscopy
NOTE: Image bare and gold nanoparticle modified carbon fiber microelectrodes using scanning electron microscopy instrument (SEM). Load the sample onto black conductive tape and following the manufacturer described instructions.
<ol>
	<li>Turning on the instrument
	<ol>
		<li>Turn the key to START and release.</li>
		<li>Open the InTouchScope software by double clicking it.</li>
		<li>Release the key. It should land on the I symbol on its own.</li>
		<li>Wait for the EVAC button to stop blinking.</li>
		<li>Once the EVAC button stops blinking, press the VENT button.</li>
		<li>Wait for the VENT button to stop blinking.</li>
		<li>Ensure that the working distance (WD) is at 20 mm &#8211; 30 mm.</li>
		<li>While waiting, prepare the sample(s).</li>
	</ol>
	</li>
	<li>Scanning 
	<ol>
		<li>Once the VENT button stops blinking, load the sample(s) into the instrument.</li>
		<li>Ensure that the curved part of the sample-holder is pointed toward the instrument when loading sample(s).</li>
		<li>Press the EVAC button once the sample(s) is loaded.</li>
		<li>Adjust the working distance to 10 mm.</li>
		<li>Once the EVAC button stops blinking, turn on the computer.</li>
		<li>Click on the In Touch Scope software, located on the desktop. There are two In Touch Scope software, click on the one without the green and yellow circle.</li>
		<li>Once the software opens up, click on OBSERVE (top right of the screen), to turn on the beam. Ensure the EVAC button has stopped blinking before clicking on OBSERVE.</li>
		<li>Start analyzing the sample(s).</li>
		<li>Ensure that the voltage, working distance (WD) and probe current (PC) settings are to acceptable.</li>
		<li>Zoom out (~50X) for a higher setting and zoom in for a lower setting.</li>
		<li>Set the working distance at 10 mm.</li>
		<li>Before taking a picture of sample(s), ensure that the picture will be saved to the desired destination folder.</li>
		<li>To choose desired folder, click on the settings (top left of the screen).</li>
		<li>Export the pictures from the computer via a flash drive.</li>
	</ol>
	</li>
	<li>Turning off
	<ol>
		<li>Click on OBSERVE to turn off the beam.</li>
		<li>Press the VENT button and wait for it to stop blinking.</li>
		<li>While waiting for the VENT button to stop blinking, adjust the working distance back to 20 mm &#8211; 30 mm.</li>
		<li>Once the VENT button stops blinking, unload the sample(s) from the instrument.</li>
		<li>Press the EVAC button, and wait for it to stop blinking.</li>
		<li>Once the EVAC button stops blinking, exit out of the software and shut down the computer.</li>
		<li>Turn the key to the O symbol to completely turn off the instrument.</li>
	</ol>
	</li>
</ol>
5. Fast scan cyclic voltammetry testing
<ol>
	<li>Connect the carbon-fiber microelectrode to potentiostat and headstage along with the Ag/AgCl reference electrode.</li>
	<li>Using the micromanipulator, lower the carbon fiber microelectrode into the flow cell well by manually adjusting the X, Y, and Z measurement knobs.</li>
	<li>Prepare buffer solution in DI water (131.5 mM NaCl, 3.25 mM KCl, 1.2 mM CaCl2, 1.25 mM NaH2PO4, 1.2 mM MgCl2, and 2.0 mM Na2SO4 with the pH adjusted to 7.4).</li>
	<li>Fill the flow cell with phosphate buffered saline (PBS) buffer (pH = 7.4).</li>
	<li>Using a filled 60 mL buffer syringe, inject PBS buffer into the flow cell at approximately 1 mL/min.</li>
	<li>Place the electrode into the flow cell and apply the waveform by pressing the green button. Observe the oscilloscope and either cut the electrode or adjust the gain to prevent overloading. Allow for approximately 10 min of equilibration between each electrode run.</li>
	<li>Set the default waveform to the dopamine waveform. Scan from &#8211; 0.4 V to 1.3 V at 10 Hz and 400 V/s.</li>
	<li>Prepare stock solution of 10 mM dopamine, serotonin, norepinephrine, and others in perchloric acid. Dilute neurochemicals to final concentration of 1 &#956;M in buffer by pipetting 1 &#956;M&#160;of the dopamine stock solution in 10 mL of PBS buffer using a pipette.</li>
	<li>To begin measurements, press the record button. After 10 s, inject 0.2 mL of 1 &#956;M dopamine into the flow cell or any other concentration of neurotransmitter. Adjust the concentration, scan rate, waveform (holding potential or switching potential) accordingly. Set the total run time for 30 s.</li>
	<li>Analyze the run using the HDCV analysis software. Change the parameters as necessary.</li>
	<li>After the experiment is complete, clean the flow cell by injecting 3 mL of water and then air into the buffer and injection ports of the flow cell three times each.</li>
	<li>Turn off the waveform and the instrument.</li>
</ol>

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</ol>

The authors have nothing to disclose.

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><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

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...

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 ...

gold-nanoparticle-modified-carbon-fiber-microelectrodes-for-enhanced

Research

JoVE Journal

Chemistry

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Learn to modify carbon-fiber microelectrodes with gold nanoparticles for enhanced neurotransmitter detection. This quick, reproducible method improves sensitivity and temporal resolution for in vivo neurochemical studies.

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

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