Name: gold nanoparticle modified carbon fiber microelectrodes for enhanced neurochemical detection
Uploaded: 2019-05-13T14:45:00.000+00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection at JoVE.com

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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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. Gold modified carbon-fiber microelectrodes were shown to have significantly higher electroactive surface areas than bare electrodes in addition to faster electron transfer kinetics. They also had higher sensitivities and lower limits of detection than bare unmodified electrode materials. Furthermore, the electrodes showed a stability towards dopamine detection when tested in the flow cell for at least 4 h. There was a linear response with respect to peak oxidative current for dopamine detection with respect to both scan rate and concentration for the gold modified carbon fiber electrodes denoting adsorption control to the surface of the electrode.
Critical steps in the protocol include the pulling of the carbon-fiber microelectrodes with the vertical capillary puller and achieving interfacial adhesion between the glass capillary and carbon fiber using epoxy. Furthermore, the electrodeposition of gold onto the surface of the carbon fiber is quite challenging as to maintain a balance between having a thin uniform coating of gold on the surface of the electrode and over-depositing excess gold onto the surface of the electrode, which would hinder neurotransmitter detection through noise and signal overload. Modifications and troubleshooting the method include optimizing the method of electrodeposition with respect to both time and concentration. Different sources of gold (AuCl3, HAuCl4, and other gold hydrates) should be utilized to perform these experiments. Limitations of the method include the possibility of the electrodeposited gold overloading the signal of the potentiostat due to over-deposition. Furthermore, as a metal electrode material, gold modified electrodes could potentially oxidize water when scanning to higher potentials (over 1.45 V), which could interfere with analyte signal.
The method is a marked advancement in the field as gold nanoparticle modified microelectrodes significantly enhance neurotransmitter detection and have not been thoroughly examined for neurotransmitter detection using FSCV. Another method of enhancing electrochemical signals for CMFEs is through modification with carbon nanotubes24,25,26. Dipcoating electrodes into carbon nanotube suspensions often increases signal. However, the noise is also increased as the layer of deposited carbon nanotubes is heterogeneous. Gold nanoparticle deposition is a quick, reproducible, and effective method to create enhanced biomolecule sensors. Future method development will include the optimization of gold nanoparticle modification of carbon-fiber microelectrodes create thin, uniform layers of gold over the surface over the carbon fiber microelectrodes. Moreover, the study and optimization of the detection of other neurochemicals (norepinephrine, serotonin, histamine, adenosine, and others) will also be carried out. Lastly, these enhanced gold-modified microelectrodes will be used to perform in vivo measurements of neurotransmitters in rodent or fruit fly models. The enhancement of dopamine detection through gold nanoparticle modification allows for many possible applications and studies in the neurosciences such as studying Parkinson&#8217;s disease, drug abuse, and other disorders.

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

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 surface of the electrode. Lastly, a current vs. time plot is overlaid with a color plot. The current vs. time plot is a representation of dopamine oxidation. It is flat when there is no dopamine oxidation, and it rises vertically when dopamine is oxidized to dopamine-orthoquinone and reduced back down to dopamine as the analyte adsorbs, and subsequently, desorbs from the surface of the electrode. The color plot is a 3-dimensional plot of current. The yellow current is the background current (close to zero), while the green plot is the positive oxidation current (dopamine oxidation to dopamine orthoquinone), and the blue plot is the negative reduction current (dopamine orthoquinone reduction to dopamine).
SEM was utilized to image surface features of the bare and modified carbon electrodes. In Figure 2, we see unique difference in surface features amongst three different types of electrode materials. In Figure 2a, a bare carbon fiber microelectrode is shown. The fiber is approximately 7 &#956;m diameter with cylindrical ridges along the exterior. Figure 2b shows gold nanoparticles electrodeposited onto the surface of the carbon fiber for approximately 20 min with large sharp ridge of gold protruding from the surface of the carbon fiber. The presence of gold was further verified with EDS/EDX measurements. We then reduced the electrodeposition time to 5 min where we observed a thin uniform coating gold as shown in Figure 2c.
Comparison of Sensitivity and Electron Transfer
Figure 3a shows a comparison of sensitivity and electron transfer. As shown with the overlapping cyclic voltammograms, gold-modified carbon fiber microelectrodes have significantly higher peak oxidative currents (Figure 3b) and faster electron transfer kinetics (&#916;EP). Significance was measured with an unpaired t-test (P = .004 and .0016, respectively). Error bars are standard error of the mean.
Stability
The bare (Figure 4a) and gold nanoparticle modified (Figure 4b) CFMEs were placed in the flow cell for 4 h. Measurements were taken for the detection of 1 &#956;M dopamine every hour over 4 h. Both electrodes had a stable response with respect to dopamine. A stable response to dopamine (without water oxidation) is critically important for performing measurements in biological tissue. Error bars are standard error of the mean.
Scan Rate
The scan rate was varied from 100 V/s to 1,000 V/s. Both Bare (Figure 5a) and gold nanoparticle (Figure 5b) modified electrodes showed a linear response with respect to dopamine detection, therefore, indicating adsorption control to the surface of the bare and gold nanoparticle modified microelectrode. Error bars are standard error of the mean.
Concentration
The concentration was varied from 100 nM to 100 &#956;M dopamine for bare (Figure 6a) and gold nanoparticle modified (Figure 6b) carbon fiber microelectrodes. The linear range was from 100 nM to 10 &#956;M. After 10 &#956;M, we observe an asymptotic curve denoting that dopamine is supersaturated at the surface of the carbon fiber microelectrode. The linear response for the peak oxidation current of dopamine with respect to dopamine concentration denotes adsorption control to the surface of the electrode. The physiologically relevant concentrations of dopamine in the brain are within this range and vary between brain regions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59552/59552fig1v2.jpg" /> 
Figure 1. A schematic of dopamine oxidation. Overlay of carbon-fiber microelectrode oxidizing dopamine. Charge transfer is shown from the surface as dopamine is oxidized to dopamine-orthoquinone and back to dopamine as the triangle dopamine waveform is applied (-0.4 V to 1.3 V at 400 V/s). The current vs. time and color plots are shown denoting dopamine oxidation (green) and dopamine reduction (blue). <a href="https://www.jove.com/files/ftp_upload/59552/59552fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59552/59552fig2v2.jpg" /> 
Figure 2. SEM images of (a) bare carbon fiber microelectrode, (b) gold-nanoparticle modified carbon fiber microelectrodes with a 20 min electrode deposition time, and (c) gold-nanoparticle modified microelectrodes with a 5-min electrode deposition time. This provides proof of principle results that the size and thickness of gold nanoparticle coatings can be controlled by the electrodeposition time. <a href="https://www.jove.com/files/ftp_upload/59552/59552fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59552/59552fig3v3.jpg" /> 
Figure 3. Sensitivity comparison of bare and gold-nanoparticle modified electrodes. (A) Overlay of cyclic voltammograms of bare and gold nanoparticle modified microelectrodes. (B). Bar graph denoting differences in peak oxidative current of bare and gold nanoparticle modified microelectrodes. (C). Bar graph showing difference in &#916;EP between bare and gold nanoparticle modified microelectrodes. Error bars are standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/59552/59552fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59552/59552fig4v2.jpg" /> 
Figure 4. Stability experiment. (A) Bare and (B) gold nanoparticle-modified microelectrodes were placed in a flow cell for a total of at least 4 h. Their sensitivity towards 1 &#956;M dopamine was measured over 4 h. Both had a uniform response to dopamine over 4 h. Error bars are standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/59552/59552fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59552/59552fig5v2.jpg" /> 
Figure 5. Scan rate experiment. (A) Bare and (B) gold nanoparticle-modified microelectrodes were placed in a flow cell, and the scan rate was varied from 100 V/s to 1,000 V/s. Both bare and gold nanoparticle modified microelectrodes had a linear response with respect to scan rate, thus denoting adsorption control of dopamine to the surface of the bare and gold nanoparticle modified carbon fiber microelectrode. Error bars are standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/59552/59552fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59552/59552fig6v2.jpg" /> 
Figure 6. Concentration experiment. (A) Bare and (B) gold nanoparticle-modified microelectrodes were exposed to various concentrations of dopamine 100 nM &#8211; 100 &#956;M. Both bare and gold nanoparticle modified microelectrodes had a linear response with respect to dopamine up to 10 &#956;M, thus denoting adsorption control to the surface of the electrode. At concentrations higher than 10 &#956;M, we observe an asymptotic curve, which is indicative of dopamine saturation at the surface of the electrode by occupying all adsorption sites and resulting in more diffusion control. <a href="https://www.jove.com/files/ftp_upload/59552/59552fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

8.8K Views.  American University. In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

Video: Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers

Nitrogen-doped carbon nanotubes consist of many cup-shaped graphitic compartments termed as nitrogen-doped carbon nanotube cups (NCNCs). These as-synthesized graphitic nanocups from chemical vapor deposition (CVD) method were stacked in a head-to-tail fashion held only through noncovalent interactions. Individual NCNCs can be isolated out of their stacking structure through a series of chemical and physical separation processes. First, as-synthesized NCNCs were oxidized in a mixture of strong acids to introduce oxygen-containing defects on the graphitic walls. The oxidized NCNCs were then processed using high-intensity probe-tip sonication which effectively separated the stacked NCNCs into individual graphitic nanocups. Owing to their abundant oxygen and nitrogen surface functionalities, the resulted individual NCNCs are highly hydrophilic and can be effectively functionalized with gold nanoparticles (GNPs), which preferentially fit in the opening of the cups as cork stoppers. These graphitic nanocups corked with GNPs may find promising applications as nanoscale containers and drug carriers.

We discussed the synthesis of individual graphitic nanocups using a series of techniques including chemical vapor deposition, acid oxidation and probe-tip sonication. By citrate reduction of HAuCl4, the graphitic nanocups were effectively corked with gold nanoparticles due to the chemically reactive edges of the cups.

Nitrogen-doped carbon  nanotubes consist of many cup-shaped graphitic compartments termed as  nitrogen-doped carbon nanotube cups (NCNCs).  These ...

Engineering

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Bioengineering

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in nanoscience. However, electrochemically analyzing single nanoparticles is challenging, as the sensing nanointerface is uncontrollable. To address this challenge, we describe here the fabrication and characterization of a closed-type wireless nanopore electrode (WNE) that exhibits a highly controllable morphology and outstanding reproducibility. The facile fabrication of WNE enables the preparation of well-defined nanoelectrodes in a general chemistry laboratory without the use of a clean room and expensive equipment. One application of a 30 nm closed-type WNE in analysis of single gold nanoparticles in the mixture is also highlighted, which shows a high current resolution of 0.6 pA and high temporal resolution of 0.01 ms. Accompanied by their excellent morphology and small diameters, more potential applications of closed-type WNEs can be expanded from nanoparticle characterization to single molecule/ion detection and single-cell probing.

Here, we present a protocol for fabrication of a closed-type wireless nanopore electrode and subsequent electrochemical measurement of single nanoparticle collisions.

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in ...

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

Neuroscience

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

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

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

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

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

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Biology

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

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

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

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

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

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Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers

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Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

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

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