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>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication&#8212;Carbon+Nanotube+Fiber+Microelectrodes+for+High+Temporal+Measurements+of+Dopamine.">Communication&#8212;Carbon Nanotube Fiber Microelectrodes for High Temporal Measurements of Dopamine.</a> Journal of The Electrochemical Society. 165, G3071-G3073 (2018).</li><li>Park, J., Takmakov, P., Wightman, R. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dopamine hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>H8502-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pine WaveNeuro Potentiostat</td>
			<td>Pine Instruments</td>
			<td>NEC-WN-BASIC</td>
			<td>This orders comes in bulk with all other accessories such as headstages, adapters, cords, and other electronics</td>
		</tr>
		<tr>
			<td>Pine Flow Cell and Micromanipulator</td>
			<td>Pine Instruments</td>
			<td>NEC-FLOW-1</td>
			<td>This is also another bulk order including the micromanipulator, flow cell, knobs, tubing, connectors, etc.</td>
		</tr>
		<tr>
			<td>Glass-Capillary</td>
			<td>A-M Systems</td>
			<td>602500</td>
			<td></td>
		</tr>
		<tr>
			<td>T-650 Carbon Fiber</td>
			<td>Goodfellow</td>
			<td>C 005711</td>
			<td></td>
		</tr>
		<tr>
			<td>Epon 828 Epoxy</td>
			<td>Miller-Stephenson</td>
			<td>EPON 828 TDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethelynetriamine</td>
			<td>Sigma Aldrich</td>
			<td>D93856-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold (III) chloride</td>
			<td>Sigma Aldrich</td>
			<td>254169</td>
			<td>Comes as either HAuCl4 or AuCl3</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Fisher</td>
			<td>S90528</td>
			<td></td>
		</tr>
		<tr>
			<td>Farraday Cage</td>
			<td>AMETEK TMC</td>
			<td>81-334-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>NEW ERA PUMP</td>
			<td>NE-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Pipettes and Tips</td>
			<td>Eppendorf</td>
			<td>2231000222</td>
			<td>This is also a bulk order containing multiple pipettes and tips</td>
		</tr>
		<tr>
			<td>10 -1,000 mL beakers</td>
			<td>VWR</td>
			<td>10536-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon fiber</td>
			<td>Goodfellow</td>
			<td>C 005711</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>JEOL</td>
			<td>JSM-IT100</td>
			<td></td>
		</tr>
	</tbody>
</table>

gold nanoparticle modified carbon fiber microelectrodes for enhanced neurochemical detection

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

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

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

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

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

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

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

Research

JoVE Journal

Chemistry

8.5K 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

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Extraction of Ramie Fiber in Alkali Hydrogen Peroxide System Supported by Controlled-release Alkali Source

This protocol demonstrates a method for ramie fiber extraction by scouring raw ramie in an alkali hydrogen peroxide system supported by a controlled-release alkali source. The fiber extracted from ramie is a type of textile material of great importance. In previous studies, ramie fiber was extracted in an alkali hydrogen peroxide system supported only by sodium hydroxide.However, due to the strong alkalinity of sodium hydroxide, the oxidation reaction speed of hydrogen peroxide was difficult to control and thus resulted in great damage to the treated fiber. In this protocol, a controlled-release alkali source, which is composed of sodium hydroxide and magnesium hydroxide, is used to provide an alkali condition and buffer the pH value of the alkali hydrogen peroxidesystem. The substitution rate of magnesium hydroxide can adjust the pH value of the hydrogen peroxide system and has great influence on the fiber properties. The pH value and oxidation-reduction potential (ORP) value, which represents the oxidation ability of alkali hydrogen peroxide system, were monitored using a pH meter and ORP meter, respectively. The residual hydrogen peroxide content in the alkali hydrogen peroxide system during the extraction process and the chemical oxygen demand (COD) value of wastewater after fiber extraction are tested by KMnO4 titration method. The yield of fiber is measured using a precision electronic balance, and residual gums of fiber are tested by a chemical analysis method. The polymerization degree (PD value) of fiber is tested by an intrinsic viscosity method using the Ubbelohde viscometer. The tensile property of fiber, including tenacity, elongation, and rupture, is measured using a fiber strength instrument. Fourier transform infrared spectroscopy and X-ray diffraction are used to characterize the functional groups and crystal property of fiber. This protocol proves that the controlled-release alkali source can improve the properties of the fiber extracted in an alkali hydrogen peroxide system.

Presented here is a protocol for extraction of ramie fiber in alkali hydrogen peroxide system supported by controlled-release alkali source.

This protocol demonstrates a method for ramie fiber extraction by scouring raw ramie in an alkali hydrogen peroxide system supported by a ...

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 ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced Raman spectroscopy (SERS) with a low detection limit of ~0.2 &#956;M for multiple characteristic peaks in the fingerprint region, using a modular spectrometer. This biosensing scheme is mediated by the host-guest complexation between a macrocycle, cucurbit[7]uril (CB7), and UA, and the subsequent formation of precise plasmonic nanojunctions within the self-assembled Au NP: CB7 nanoaggregates. A facile Au NP synthesis of desirable sizes for SERS substrates has also been performed based on the classical citrate-reduction approach with an option to be facilitated using a lab-built automated synthesizer. This protocol can be readily extended to multiplexed detection of biomarkers in body fluids for clinical applications.

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.