This work was supported by an award from the NIGMS (GM111835).

1. Constructing a coating of nanoporous gold with hierarchical bimodal architecture on gold wires - Alloying
<ol>
	<li>Assemble an electrochemical cell in a 5 mL beaker. Use a Teflon-based lid with three holes to contain the three-electrode setup. 
	NOTE: Teflon is a popular material for making lids, as it does not react with other chemicals.</li>
	<li>Place a platinum wire counter-electrode, an Ag/AgCl (saturated KCl) reference electrode, and a gold wire with a diameter of 0.2 mm and a length of 5.0 mm functioning as a working electrode (see Table of Materials) conveniently in each hole of the lid. Maintain a distance of 0.7 cm between the working electrode and the counter electrode. 
	NOTE: The length of the wire is 1 cm, and after clipping the alligator clip, the exposed part going into the solution is 0.5 cm. The remaining portion is covered using polytetrafluoroethylene tape (see Table of Materials). This length is precisely measured using a scale each time the experiment is performed. The gold wire is cleaned in several steps before being used in the study; It is first immersed in concentrated nitric acid, then rinsed and dipped in piranha solution, and finally immersed in sodium borohydride. These strong reagents aid in eliminating any impurities that might be adhering to the wire.</li>
	<li>Prepare a 50 mM solution each of K[Ag(CN)2] and K[Au(CN)2] (see Table of Materials) in water. Add 0.5 mL of K[Au(CN)2] solution and 4.5 mL of K[Ag(CN)2] salt solution in the 5 mL beaker. 
	CAUTION: Be careful when handling and disposing of cyanide salts. Wear protective gloves, appropriate clothing, respiratory protection, and eye protection while handling it, and work in a fume hood. Cyanide salt solutions must be collected after the experiment is over and placed in a separate, clearly marked waste container. When in contact with acids, hazardous fumes are released. To preserve the ionic mobility, 0.25 M Na2CO3 is added to the stock 40 mM solutions of each of the two salts.</li>
	<li>Mix the solution thoroughly by inserting a magnetic stirrer bar into the electrochemical cell, at a constant stirring speed of 300 rpm. 
	NOTE: Verify that the stirrer bar is clean. Aqua regia can be used to clean it if there are contaminants.</li>
	<li>Circulate argon gas through the solution using a 1/32 inch (in) inner diameter, 5/32 in outer diameter, and 1/16 in wall thickness silicone tubing (see Table of Materials), going inside the electrochemical cell to take out any dissolved oxygen in the electrolyte solution. 
	NOTE: The leak in the setup is avoided by making it tight enough.</li>
	<li>Connect the potentiostat using alligator clips that are clipped to the appropriate electrodes once the electrochemical cell is assembled completely. 
	NOTE: It is best to label the clips with the name of the electrode they will be attached to in order to avoid confusion.</li>
	<li>Use the software (PowerSuite; see Table of Materials) to perform electrodeposition utilizing chronoamperometry after turning on the potentiostat. Configure the software using the desired parameters. For 600 s, the potential is timed with a fixed value of -1.0 V15. 
	NOTE: Let the booting process finish on its own before specifying the parameters. Supplementary File 1 shows screenshots for the use of this specific software.</li>
	<li>Press run and choose external cell to complete the alloy deposition on the working electrode. A thick white coating is seen at the end of the process. 
	&#8203;NOTE: Ensure the connections are secure and the solution is stirring steadily. If everything works as it should, the current oscillates between 300 and 400 &#181;A.</li>
</ol>
2. Constructing a coating of nanoporous gold with hierarchical bimodal architecture on gold wires - Dealloying
NOTE: This phase of the protocol is based on the partial dealloying of the alloyed wires.
<ol>
	<li>Configure the electrochemical cell once more, as in the prior process (step 1.2), in which three electrodes are kept 0.7 cm from each other in an electrolyte solution. Use 4 mL of 1 N nitric acid as the electrolyte solution for partial dealloying. 
	NOTE: Because the electrolyte is diluted, it does not completely remove the less noble element, which here is silver.</li>
	<li>Leave the stirrer bar running to evenly circulate the solution at a constant speed of 300 rpm.</li>
	<li>Once the electrochemical cell is set up, attach the potentiostat using alligator clips that are clipped to the correct electrodes.</li>
	<li>Employ the chronoamperometry software once more, but this time choose a potential of 0.6 V for 600 s.</li>
	<li>Press run, then select external cell to finish dealloying the deposited alloy on the working electrode. 
	NOTE: The color of the wire changes to greyish-black after this step.</li>
</ol>
3. Constructing a coating of nanoporous gold with hierarchical bimodal architecture on gold wires - Annealing
<ol>
	<li>Keep the dealloyed wires in a glass vial inside the furnace.</li>
	<li>Maintain the temperature in the furnace at 600 &#176;C for 3 h. 
	NOTE: The temperature and duration were chosen based on optimization studies, which showed that lower temperatures did not coarsen the pores, while higher temperatures and a longer time caused cracks in the structure.</li>
	<li>Remove the vial after the procedure is finished and the furnace is turned off. Wait till the vial cools to room temperature. 
	&#8203;NOTE: The hot vial is removed from the furnace using a set of tongs.</li>
</ol>
4. Constructing a coating of nanoporous gold with hierarchical bimodal architecture on gold wires - Dealloying
<ol>
	<li>Immerse the partially dealloyed-annealed wires in 4 mL of concentrated nitric acid. 
	NOTE: When transferring the concentrated acid into the glass vial containing the wires, ensure a glass pipette is used.</li>
	<li>Leave the partially dealloyed-annealed wires in the glass vial containing the strong nitric acid in the fume hood overnight. 
	NOTE: The dealloying period was kept at 24 h to allow for full dealloying.</li>
	<li>Create the hb-NPG-coated wires the following day (Figure 1). Rinse these thoroughly with deionized water, followed by an ethanol rinse, before being used in subsequent studies. After drying, use the wires in experiments. A fresh batch of clean gold wires is used each time to prepare hb-NPG. 
	&#8203;NOTE: Every step in the synthesis is crucial to arrive at the bimodal structure. If any of the steps are skipped, only a unimodal pore structure will result.</li>
</ol>
5. hb-NPG characterization
<ol>
	<li>Scanning electron microscope (SEM)-sample preparation
	<ol>
		<li>Ensure to use a clean aluminum stub as the base for sample preparation for SEM imaging. 
		NOTE: The stubs are cleaned with dilute nitric acid, rinsed with ethanol, dried, and kept in a paraffin film-covered container before being used again.</li>
		<li>Place a newly cut piece of carbon tape over the aluminum stub&#39;s flat base.</li>
		<li>With the aid of tweezers, cut little pieces of hb-NPG-coated wire and adhere them to the carbon tape. 
		NOTE: Ensure that the wire is gently gripped with tweezers to prevent the coating from coming off.</li>
		<li>Arrange the cut pieces horizontally to reveal their morphology under a SEM (see Table of Materials); pieces arranged vertically can reveal the thickness of the material deposited on the gold wire (Figure 2). 
		NOTE: If the electrode has any moisture, the chamber will become contaminated, resulting in hazy images. Therefore, place the electrode in a vacuum overnight before performing SEM imaging.</li>
	</ol>
	</li>
	<li>SEM chamber set up
	<ol>
		<li>Vent the chamber using the option &#34;vent&#34; from the software &#34;XT microscope&#34; to easily open the chamber door. 
		NOTE: The &#34;XT microscope&#34; software comes with the SEM system. The chamber must first be entirely vented before the door can be smoothly opened, which typically takes 3-5 min. In adiition, the pressure of the nitrogen gas cylinder must be under 5 psi. Supplementary File 1 shows screenshots for the use of this specific software.</li>
		<li>With the aid of a special tweezer&#39;s curved front, firmly grasp the circular aluminum stub, insert the clean sample that was previously placed on the stub into the chamber, and place it on the sample stage. 
		NOTE: Ensure that the stage is clean; if there are any spills, then them off with acetone and a lint-free wipe (see Table of Materials).</li>
		<li>Select the &#34;pump&#34; option after placing the sample inside the chamber to produce a vacuum-like environment. 
		NOTE: It takes approximately 3 min to pump the chamber. Before turning on the beam, the user needs to wait for the pumping to finish.</li>
		<li>Using the navigation camera, click on the real-time image to observe the sample&#39;s precise location.</li>
	</ol>
	</li>
	<li>Imaging the sample
	<ol>
		<li>Turn on the beam initially to capture an image of the sample. 
		NOTE: Before turning on the beam, ensure the system is vacuumed.</li>
		<li>To bring the sample image from the navigation camera&#39;s collected image to the center of the screen and sharpen the focus, double-click on it.</li>
		<li>Ensure that the images are produced utilizing imaging with the compatible software and the Everhart-Thornley detector (ETD). Optimizing the spot size, scan rate, and potential is necessary. 
		NOTE: Starting with lower potential and spot size before moving up is always promising.</li>
		<li>Adjust the stage distance from the beam source to 10 mm. 
		NOTE: To precisely measure the distance between the stage and the source; the system must be focused and connected to the Z axis.</li>
		<li>To learn more about the architecture that is present at a lower scale, increase the magnification after obtaining a focused image at the lower magnification (Figure 3). 
		NOTE: Every level of the magnification increase requires focus.</li>
	</ol>
	</li>
	<li>SEM and energy dispersive spectroscopy (EDS) for elemental composition of the sample
	<ol>
		<li>By choosing the color SEM option in the software, determine the elemental makeup of the material (Figure 4). It automatically assigns diverse colors to the sample&#39;s various elements. However, it is also possible to choose colors and elements manually. 
		NOTE: It is crucial to examine the elemental composition analysis chart and the atomic percentages of the elements to rule out choices that have an atomic percentage of 0.</li>
		<li>By inserting the EDS detector, subject the sample under study to energy-dispersive X-ray spectroscopy. Use 15 kV potential and a spot size of 12 for collecting the EDS data. 
		NOTE: After pressing the insert button, the screen displays the detector inserted in real time.</li>
		<li>Check that the program enables the creation of tiny boxes to select discrete regions on the sample image screen, to gather information for the elemental composition. 
		&#8203;NOTE: For accurate statistics, there must be between 50 and 100,000 counts. The panel displays the overall status of the data collected. At the conclusion, a report is produced.</li>
	</ol>
	</li>
</ol>
6. Cyclic voltammetry (CV) for electrochemically active surface area
<ol>
	<li>Perform the gold oxide stripping method, which involves manually setting up an electrochemical cell with the reference electrode, counter electrode, and working electrode immersed in an electrolyte. 
	NOTE: For the present study, Pt wire serves as the counter electrode, hb-NPG is the working electrode, and Ag/AgCl serves as the reference electrode. Sulphuric acid, 0.5 N, serves as the electrolyte. Cleaning the reference and counter electrodes in diluted nitric acid before usage is recommended. Moreover, avoid reusing the electrolyte.</li>
	<li>Once the connections are made to the potentiostat, select the CV option from the program and configure the parameters. Fix the potential range from -0.2-1.6 V (Figure 5) and back to -0.2 V (vs. Ag/AgCl) along with a scan rate of 100 mV/s. 
	NOTE: No stirring is required.</li>
	<li>Click on the run button to carry out the cyclic scan. Ensure the reverse scan reveals a noticeable peak. Using the reported conversion factor of 400 &#181;C cm-2, integrate the charge under the peak coming from the reduction of the gold oxide monolayer to determine the electrochemical surface area of the hb-NPG wire. 
	NOTE: The tangent must be drawn correctly to integrate the charge under the peak properly.</li>
	<li>Conduct an identical set of experiments on chemically dealloyed wire and after combining chemical and electrochemical dealloying on the Au:Ag (10:90) alloyed wire, to comprehend the crucial significance of dual dealloying procedures.</li>
</ol>
7. Solution depletion technique to study protein loading
<ol>
	<li>To study the real-time loading of protein molecules onto the hb-NPG surface, employ an ultraviolet-visible (UV-vis) spectrophotometer (see Table of Materials). 
	NOTE: In this study, protein solutions of fetuin, bovine serum albumin (BSA), and horseradish peroxidase (HRP) (see Table of Materials) were prepared separately at concentrations of 1 mg/mL, 0.5 mg/mL, and 1 mg/mL, respectively, in a 0.01 M (pH = 7.4) phosphate buffered saline (PBS) buffer. It is crucial to select a buffer and pH that maintains protein stability.</li>
	<li>Perform baseline correction using the buffer only. Choose the software&#39;s parameters, including wavelength, time, and rate, after the correction has been made. Then, place 500 &#181;L of the protein solution inside the cuvette.</li>
	<li>Ensure that the real-time protein monitoring starts after adding hb-NPG into the solution. Monitor the change in absorbance after every minute for 120 min at 280 nm after pressing the start button (Figure 6).</li>
</ol>

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The authors have nothing to disclose.

Using a multistep procedure involving alloying, partial dealloying, thermal treatment, and acid etching, fabricating hierarchically NPG with dual-sized pores and a higher active electrochemical surface area is demonstrated.
In alloying, the standard potential of metal precursors influences how reactive they are during electrodeposition. Au and Ag ions from liquid solutions are reduced during electrodeposition16,17.
<p class="jove_c...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65065">Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold</a>. The Authors section was updated from:
Palak Sondhi1 
Dharmendra Neupane2 
Jay K. Bhattarai3 
Hafsah Ali1 
Alexei V. Demchenko4 
Keith J. Stine1 
1Department of Chemistry and Biochemistry, University of Missouri-Saint Louis 
2Food and Drug Administration 
3Mallinckrodt Pharmaceuticals Company 
4Department of Chemistry, Saint Louis University
to:
Palak Sondhi1 
Dharmendra Neupane1 
Jay K. Bhattarai2 
Hafsah Ali1 
Alexei V. Demchenko3 
Keith J. Stine1 
1Department of Chemistry and Biochemistry, University of Missouri-Saint Louis 
2Mallinckrodt Pharmaceuticals Company 
3Department of Chemistry, Saint Louis University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65065">Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold</a>. The Authors section was updated.

Erratum: Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold

Often seen in nature, hierarchical porous architectures have been imitated at the nanoscale to alter the physical characteristics of materials for improved performance1. Interconnected structural elements of various scales of length are a characteristic of the hierarchical architecture of porous materials2. Dealloyed nanoporous metals typically have unimodal pore size distributions; hence, multiple techniques have been devised to produce hierarchically bimodal porous structures with two separate pore size ranges3. The two fundamental objectives of the material design approach, namely the large specific surface area for functionalization and rapid transport pathways, which are distinct and inherently in conflict with one another, are fulfilled by functional materials possessing structural hierarchy4,5. 
Performance of the electrochemical sensor is determined by the electrode morphology, since the nanomatrix's pore size is crucial for molecular transport and capture. Small pores have been found to aid in target identification in complicated samples, whereas bigger pores enhance the target molecule's accessibility, increasing the sensor's detection range6. The template-based fabrication, electroplating, bottom-up synthetic chemistry, thin film sputtering deposition7, complex flexible matrices based on polydimethylsiloxane support8, alloying of various metals followed by selective etching of the less noble metal, and electrodeposition are some of the methods that are frequently used to introduce nanostructures into the electrode. One of the best methods for creating porous structures is the dealloying procedure. Due to the disparity in dissolution rates, the sacrificial metal, which is the less noble metal, significantly influences the final morphology of the electrode. An interconnected network of pores and ligaments results from the effective process of creating nanoporous gold (NPG) structures, in which the less noble component selectively dissolves out of the starting alloy, and the remaining atoms reorganize and consolidate9. 
The method of dealloying/plating/re-dealloying used by Ding and Erlebacher to make these nanostructures involved first subjecting the precursor alloy composed of gold and silver to chemical dealloying using nitric acid, followed by heating at a higher temperature with a single pore size distribution to create the upper hierarchical level, and removing the remaining silver using a second dealloying to produce the lower hierarchical level. This method was applicable to thin films10. Using ternary alloys, which are comprised of two comparatively more reactive noble metals that are eroded away one at a time, was advised by Biener et al; Cu and Ag were initially removed from the Cu-Ag-Au material, leaving behind bimodally structured, low-density NPG samples11. Long-range ordered structures are not produced by the procedures outlined utilizing ternary alloys. Bigger pores were produced by extracting away one of the phases of the master alloy of Al-Au employed by Zhang et al., which produced the bimodal structure with a minimal degree of order12. An ordered hierarchical structure has reportedly been created by controlling several length scales, through the use of processing pathways that include disassembling bulk materials and putting basic components together into larger structures. In this case, a hierarchical NPG structure was made via direct ink writing (DIW), alloying, and dealloying13. 
Here, a two-step dealloying method for fabricating a hierarchical bimodal nanoporous gold (hb-NPG) structure employing various Au-Ag alloy compositions is presented. The amount of reactive element below which dealloying stops is, in theory, the parting limit. The surface diffusion kinetics is slightly impacted by the parting limit or dealloying threshold, which is typically between 50 and 60 atomic percentage for electrolytic dissolution of the more reactive component from a binary alloy. A large atomic fraction of Ag in the Au:Ag alloy is necessary for the successful synthesis of hb-NPG, since both the electrochemical and chemical dealloying processes cannot be successfully completed at low concentrations near the parting limit14.
The benefit of this method is that the structure and pore size can be tightly controlled. Each step in the protocol is crucial for fine-tuning the typical porosity length scale and the typical distance between ligaments15. To regulate the rate of ion interfacial diffusion and dissolution, the applied voltage is carefully calibrated. To prevent cracking during dealloying, the Ag dissolution rate is controlled.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Argon gas compressed</td>
			<td>Fisher Scientific Compay</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich (St.Louis, MO)</td>
			<td>A9418</td>
			<td>&#62; 98% purity</td>
		</tr>
		<tr>
			<td>Counter electrode (Platinum wire)</td>
			<td>Alfa Aesar</td>
			<td>43288-BU</td>
			<td>0.5 mm diameter</td>
		</tr>
		<tr>
			<td>Digital Lab furnace</td>
			<td>Barnstead Thermolyne 47,900</td>
			<td>F47915</td>
			<td>used for annealing at high temperatures</td>
		</tr>
		<tr>
			<td>Digital Potentiostat/galvanostat</td>
			<td>EG&#38;G Princeton Applied Research</td>
			<td>273A</td>
			<td>PowerPULSE software</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich (St.Louis, MO)</td>
			<td>CAS-64-17-5</td>
			<td>HPLC/spectrophotometric grade</td>
		</tr>
		<tr>
			<td>Fetuin from fetal calf serum</td>
			<td>Sigma-Aldrich (St.Louis, MO)</td>
			<td>F2379</td>
			<td>lyophilized powder</td>
		</tr>
		<tr>
			<td>Gold wire roll</td>
			<td>Electron Microscopy Sciences (Fort Washington, PA)</td>
			<td>73100</td>
			<td>0.2 mm diameter, 10 ft, 99.9%</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Chemical</td>
			<td>A144C-212</td>
			<td>36.5-38%</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>CAS-7732-18-5</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>KIMTECH Science brand, Kimberly-Clark professional</td>
			<td>34120</td>
			<td>4.4 x 8.2 in</td>
		</tr>
		<tr>
			<td>Nitric acid</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>A2008-212</td>
			<td>trace metal grade</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis PM996</td>
			<td>13-374-10</td>
			<td>4 IN. x 125 FT.</td>
		</tr>
		<tr>
			<td>Peroxidase from horseradish (HRP)</td>
			<td>Sigma-Aldrich (St.Louis, MO)</td>
			<td>9003-99-0</td>
			<td></td>
		</tr>
		<tr>
			<td>PharMed silicone tubing</td>
			<td>Norton</td>
			<td>AY242606&#160;</td>
			<td>1/32&#34; Inner Diameter, 5/32&#34; Outer Diameter, 1/16&#34; Wall Thickness, 25&#39; Length</td>
		</tr>
		<tr>
			<td>Potassium dicyanoargentate</td>
			<td>Sigma-Aldrich (St.Louis, MO)</td>
			<td>379166</td>
			<td>99.96%, 10 G</td>
		</tr>
		<tr>
			<td>Potassium dicyanoaurate</td>
			<td>Sigma-Aldrich (St.Louis, MO)</td>
			<td>389867</td>
			<td>99.98%, 1 G</td>
		</tr>
		<tr>
			<td>PowerSuite software</td>
			<td>EG&#38;G Princeton Applied Research</td>
			<td></td>
			<td>comes with the instrument</td>
		</tr>
		<tr>
			<td>PTFE tape</td>
			<td>Fisherbrand</td>
			<td>15-078-261</td>
			<td>1&#34; wide 600&#34; long</td>
		</tr>
		<tr>
			<td>Reference electrode (Ag/AgCl)</td>
			<td>Princeton Applied Research&#160;</td>
			<td>K0265</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscopy (SEM) Apreo 2C</td>
			<td>ThermoFisher scientific</td>
			<td>APREO 2 SEM</td>
			<td>equipped with Color SEM technology</td>
		</tr>
		<tr>
			<td>Simplicity UV system</td>
			<td>Millipore corporation, Boston, MA, USA</td>
			<td>SIMSV00WW</td>
			<td>for generating Milli-Q water(18.2 M&#937; cm at 25 &#176;C)&#160;</td>
		</tr>
		<tr>
			<td>Sodium Borohydride</td>
			<td>Sigma-Aldrich (St.Louis, MO)</td>
			<td>213462</td>
			<td>100 G</td>
		</tr>
		<tr>
			<td>Sodium Carbonate</td>
			<td>Sigma-Aldrich (St.Louis, MO)</td>
			<td>452882</td>
			<td>enzyme grade, &#62;99%, 100 G</td>
		</tr>
		<tr>
			<td>Stir bar</td>
			<td>Fisherbrand</td>
			<td>14-512-153</td>
			<td>5 x 2 mm</td>
		</tr>
		<tr>
			<td>Sulphuric acid</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>A300C-212</td>
			<td>certified ACS plus</td>
		</tr>
		<tr>
			<td>Supracil quartz cuvette</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>14-385-902C</td>
			<td>10 mm light path, volume capacity 1 mL</td>
		</tr>
		<tr>
			<td>UV-Visible Spectrophotometer</td>
			<td>Varian Cary 50</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

versatile technique to produce a hierarchical design in nanoporous gold

The potential to generate variable pore sizes, simplistic surface modification, and a breadth of commercial uses in the fields of biosensors, actuators, drug loading and release, and the development of catalysts have unquestionably accelerated the usage of nanoporous gold (NPG)-based nanomaterials in research and development. This article describes the process of the generation of hierarchical bimodal nanoporous gold (hb-NPG) by employing a step-wise procedure involving electrochemical alloying, chemical dealloying techniques, and annealing to create both macro- and mesopores. This is done to improve the utility of NPG by creating a bicontinuous solid/void morphology. The area available for surface modification is enhanced by smaller pores, while molecular transport benefits from the network of larger pores. The bimodal architecture, which is the result of a series of fabrication steps, is visualized using scanning electron microscopy (SEM) as a network of pores that are less than 100 nm in size and connected by ligaments to larger pores that are several hundred nanometers in size. The electrochemically active surface area of the hb-NPG is assessed using cyclic voltammetry (CV), with a focus on the critical roles that both dealloying and annealing play in creating the necessary structure. The adsorption of different proteins is measured by solution depletion technique, revealing the better performance of hb-NPG in terms of protein loading. By changing the surface area to volume ratio, the created hb-NPG electrode offers tremendous potential for biosensor development. The manuscript discusses a scalable method to create hb-NPG surface structures, as they offer a large surface area for the immobilization of small molecules and improved transport pathways for faster reactions.

The ligament size and inter-ligament gap adjustments are of utmost significance for the manufactured electrode. Creating a structure with dual-sized pores by optimizing the Au/Ag ratios is the first step in this study, along with the characterization utilizing surface morphology, roughness factor, and loading capacity. Compared to conventional NPG, the bimodal pore structure has demonstrated a higher electrochemical surface area, roughness factor, and protein loading capacity15.
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Watch this Scientific Journal Video about Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold at JoVE.com

Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold

Nanoporous gold with a hierarchical and bimodal pore size distribution can be produced by combining electrochemical and chemical dealloying. The composition of the alloy can be monitored via EDS-SEM examination as the dealloying process advances. The material's loading capacity can be determined by studying protein adsorption onto the material.

The potential to generate variable pore sizes, simplistic surface modification, and a breadth of commercial uses in the fields of biosensors, actuators, ...

versatile-technique-to-produce-hierarchical-design-nanoporous

Research

JoVE Journal

Chemistry

1.5K Views.  University of Missouri-Saint Louis. Nanoporous gold with a hierarchical and bimodal pore size distribution can be produced by combining electrochemical and chemical dealloying. The composition of the alloy can be monitored via EDS-SEM examination as the dealloying process advances. The material's loading capacity can be determined by studying protein adsorption onto the material.

Video: Versatile Technique to Produce a Hierarchical Design in Nanoporous Gold

Synthesis and Catalytic Performance of Gold Intercalated in the Walls of Mesoporous Silica

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties of the materials were investigated. We used a one pot sol-gel approach to intercalate gold nano particles in the walls of mesoporous silica. To start with the synthesis, P123 was used as template to form micelles. Then TESPTS was used as a surface modification agent to intercalate gold nano particles. Following this process, TEOS was added in as a silica source which underwent a polymerization process in acid environment. After hydrothermal processing and calcination, the final product was acquired. Several techniques were utilized to characterize the porosity, morphology and structure of the gold intercalated mesoporous silica. The results showed a stable structure of mesoporous silica after gold intercalation. Through the oxidation of benzyl alcohol as a benchmark reaction, the GMS materials showed high selectivity and recyclability.

Here, we present a protocol with a sol-gel process to synthesize gold intercalated in the walls of mesoporous materials (GMS), which is confirmed to possess a mesoporous matrix with gold intercalated in the walls imparting great stability and recyclability.

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

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 ...

Synthesis of Hierarchical ZnO/CdSSe Heterostructure Nanotrees

A two-step chemical vapor deposition procedure is here employed to prepare tree-like hierarchical ZnO/CdSSe hetero-nanostructures. The structures are composed of CdSSe branches grown on ZnO nanowires that are vertically aligned on a transparent sapphire substrate. The morphology was measured via scanning electron microscopy. The crystal structure was determined by X-ray powder diffraction analysis. Both the ZnO stem and CdSSe branches have a predominantly wurtzite crystal structure. The mole ratio of S and Se in the CdSSe branches was measured by energy dispersive X-ray spectroscopy. The CdSSe branches result in strong visible light absorption. Photoluminescence (PL) spectroscopy showed that the stem and branches form a type-II heterojunction. PL lifetime measurements showed a decrease in the lifetime of emission from the trees when compared to emission from individual ZnO stems or CdSSe branches and indicate fast charge transfer between CdSSe and ZnO. The vertically aligned ZnO stems provide a direct electron transport pathway to the substrate and allow for efficient charge separation after photoexcitation by visible light. The combination of the abovementioned properties makes ZnO/CdSSe nanotrees promising candidates for applications in solar cells, photocatalysis, and opto-electronic devices.

Here, we prepare and characterize novel tree-like hierarchical ZnO/CdSSe nanostructures, where CdSSe branches are grown on vertically aligned ZnO nanowires. The resulting nanotrees are a potential material for solar energy conversion and other opto-electronic devices.

A two-step chemical vapor deposition procedure is here employed to prepare tree-like hierarchical ZnO/CdSSe hetero-nanostructures. The structures are ...

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 ...

Surface Properties of Synthesized Nanoporous Carbon and Silica Matrices

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) with a 4.6 nm pore size, and ordered silica porous matrix, SBA-15, with a 5.3 nm pore size. This work describes the surface properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the differently ordered porous materials with similar pore sizes. For this purpose, OMC and SBA-15 with highly ordered nanoporous structures are synthesized via impregnation of the silica matrix by applying a carbon precursor and by the sol-gel method, respectively. The porous structure of investigated systems is characterized by an N2 adsorption-desorption analysis at 77 K. To determine the electrochemical character of the surface of synthesized materials, potentiometric titration measurements are conducted; the obtained results for OMC shows a significant pHpzc shift toward the higher values of pH, relative to SBA-15. This suggests that investigated OMC has surface properties related to oxygen-based functional groups. To describe the surface properties of the materials, the contact angles of liquids penetrating the studied porous beds are also determined. The capillary rise method has confirmed the increased wettability of the silica walls relative to the carbon walls and an influence of the pore roughness on the fluid/wall interactions, which is much more pronounced for silica than for carbon mesopores. We have also studied the melting behavior of D2O confined in OMC and SBA-15 by applying the dielectric method. The results show that the depression of the melting temperature of D2O in the pores of OMC is about 15 K higher relative to the depression of the melting temperature in SBA-15 pores with a comparable 5 nm size. This is caused by the influence of adsorbate/adsorbent interactions of the studied matrices.

Here we report the synthesis and characterization of ordered nanoporous carbon (with a 4.6 nm pore size) and SBA-15 (with a 5.3 nm pore size). The work describes the surface and textural properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the materials.

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and &#62;50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.

This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination&#160;experiments and one-pot glycosylation of SSEA-4.

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for ...

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products and notably to generate chiral carbon centers. The crucial first step consists in the selective double hydroboration of CO2 catalyzed by an iron hydride complex. The product obtained with this 4 e- reduction is a rare bis(boryl)acetal, compound 1, which is subjected in situ to three different reactions in a second step. The first reaction concerns a condensation reaction with (diisopropyl)phenylamine affording the corresponding imine 2. In the second and third reaction, intermediate 1 reacts with triazol-5-ylidene (Enders&#39; carbene) to afford compounds 3 or 4, depending on the reaction conditions. In both compounds, C-C bonds are formed, and chiral centers are generated from CO2 as the only source of carbon. Compound 4 exhibits two chiral centers obtained in a diastereoselective manner in a formose-type mechanism. We proved that the remaining boryl fragment plays a key role in this unprecedented stereocontrol. The interest of the method stands on the reactive and versatile nature of 1, giving rise to various complex molecules from a single intermediate. The complexity of a two-step method is compensated by the overall short reaction time (2 h for the larger reaction time), and mild reaction conditions (25 &#176;C to 80 &#176;C and 1 to 3 atm of CO2).

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations are conducted in a one-pot two-step procedure for the synthesis of complex molecules. The selective 4 e- reduction of CO2 with a hydroborane reductant affords a reactive and versatile bis(boryl)acetal intermediate which is subsequently involved in condensation reaction or carbene-mediated C-C coupling generation.

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products ...

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.