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. 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

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 ...

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

Research

JoVE Journal

Chemistry

8.6K 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

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

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

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

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

Bioengineering

Fabrication of 3D Carbon Microelectromechanical Systems (C-MEMS)

A wide range of carbon sources are available in nature, with a variety of micro-/nanostructure configurations. Here, a novel technique to fabricate long and hollow glassy carbon microfibers derived from human hairs is introduced. The long and hollow carbon structures were made by the pyrolysis of human hair at 900 &#176;C in a N2 atmosphere. The morphology and chemical composition of natural and pyrolyzed human hairs were investigated using scanning electron microscopy (SEM) and electron-dispersive X-ray spectroscopy (EDX), respectively, to estimate the physical and chemical changes due to pyrolysis. Raman spectroscopy was used to confirm the glassy nature of the carbon microstructures. Pyrolyzed hair carbon was introduced to modify screen-printed carbon electrodes ; the modified electrodes were then applied to the electrochemical sensing of dopamine and ascorbic acid. Sensing performance of the modified sensors was improved as compared to the unmodified sensors. To obtain the desired carbon structure design, carbon micro-/nanoelectromechanical system (C-MEMS/C-NEMS) technology was developed. The most common C-MEMS/C-NEMS fabrication process consists of two steps: (i) the patterning of a carbon-rich base material, such as a photosensitive polymer, using photolithography; and (ii) carbonization through the pyrolysis of the patterned polymer in an oxygen-free environment. The C-MEMS/NEMS process has been widely used to develop microelectronic devices for various applications, including in micro-batteries, supercapacitors, glucose sensors, gas sensors, fuel cells, and triboelectric nanogenerators. Here, recent developments of a high-aspect ratio solid and hollow carbon microstructures with SU8 photoresists are discussed. The structural shrinkage during pyrolysis was investigated using confocal microscopy and SEM. Raman spectroscopy was used to confirm the crystallinity of the structure, and the atomic percentage of the elements present in the material before and after pyrolysis was measured using EDX.

Fabrication of 3D Carbon Microelectromechanical Systems C-MEMS

Long and hollow glassy carbon microfibers were fabricated based on the pyrolysis of a natural product, human hair. The two fabrication steps of carbon microelectromechanical and carbon nanoelectromechanical systems, or C-MEMS and C-NEMS, are: (i) photolithography of a carbon-rich polymer precursor and (ii) pyrolysis of the patterned polymer precursor.

A wide range of carbon sources are available in nature, with a variety of micro-/nanostructure configurations. Here, a novel technique to fabricate long ...

Engineering

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 ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

Neuroscience

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 ...

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, ...

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred &#181;m, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

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 ...

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 ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

Summary

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Chapters in this video

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

Fabrication of 3D Carbon Microelectromechanical Systems (C-MEMS)

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Fabrication of Ti₃C₂ MXene Microelectrode Arrays for In Vivo Neural Recording

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