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.
The following half-cell reactions17 depict the electrochemical dissolution of gold and silver cyanide salt solutions:
Au (CN)2- + 1e- <img alt="Equation 1" src="/files/ftp_upload/65065/65065eq01v2.jpg" style="margin: auto;" />
E0 = -1060 mV vs. SCE
Ag (CN)32- + 1e- <img alt="Equation 2" src="/files/ftp_upload/65065/65065eq02v2.jpg" style="margin: auto;" />
E0 = -1198 mV vs. SCE
The following parallel processes mostly determine how silver is oxidized by nitric acid in the dealloying step18.
4HNO3 + 3Ag -&#62; 3AgNO3 + NO + 2H2O
2HNO3 + Ag -&#62; AgNO3 + NO2 + H2O
The impact of changes in the process parameters on the electrode&#39;s electrochemical performance is well discussed. It was discovered that an NPG electrode with a bimodal pore structure, made from a gold and silver alloy with a high atomic percentage of silver and annealed at 600 &#176;C for 3 h, contain dual-sized pores that allow for a greater protein loading than a conventional electrode with nanopores. Compared to other nanoporous electrodes, the structure&#39;s interconnected network also offers high mass transfer efficiency, improving performance in terms of activity and sensitivity15.
The interconnecting ligaments that connect the highly active, low-coordinated atoms that make the upper hierarchy of the hierarchical electrode have a dimension of 938 &#177; 285 nm. The electrode&#39;s application in the field of catalysis is improved by the dimensions of the ligament it produces. A ligament width of 51 &#177; 5 nm, a characteristic of lower hierarchical structures, enhances the electrode&#39;s capacity to immobilize bioactive compounds. The material created has a lot of potential for applications like catalysis and sensing, that need speedy detection of larger molecules as well as easy passage for those larger molecules15.
The large surface area and hierarchical structure of the electrodes have an impact on the protein loading. A greater protein loading capacity than conventional NPG has been shown for hb-NPG. Bimodal architecture provides a platform for protein interaction, communication with the substrate, and passage for the facile flow of protein molecules. The type of material and its morphology impact how many bioactive molecules can be loaded into an electrode. Because of the hierarchy, which speeds up the binding of most proteins through electrostatic and physical forces, the hb-NPG has a higher concentration of immobilized proteins15.
Even though the development of materials with complex structural designs has advanced significantly, there are still some challenges. Commercialization calls for new preparation techniques with fewer processes and cheaper production. Future research that tackles the issue of mass output and carefully analyzes the in situ development process of hierarchical structures will be interesting to work on.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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><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>&gt; 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&amp;amp;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&amp;nbsp;</td><td>1/32&quot; Inner Diameter, 5/32&quot; Outer Diameter, 1/16&quot; Wall Thickness, 25&#039; 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&amp;amp;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&quot; wide 600&quot; long</td></tr><tr><td>Reference electrode (Ag/AgCl)</td><td>Princeton Applied Research&amp;nbsp;</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&amp;Omega; cm at 25 &amp;deg;C)&amp;nbsp;</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, &gt;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.
hb-NPG has demonstrated an open, linked network of ligaments and pores following chemical dealloying. Here, larger holes are indicated by an upper hierarchy, and a lower hierarchy indicates smaller pores. Figure 3 shows SEM images of the electrode with a hierarchical bimodal structure. Figure 4 illustrates color-coded elemental mapping for each step of the creation of hb-NPG, with red and yellow designating silver and gold, respectively. SEM&#39;s ability to assign distinct colors to various sections is a useful feature in the instrument.
The proportion of electrochemically active surface area (ECSA) to geometric surface area yields a roughness factor for each electrode. Utilizing the charge beneath the gold oxide reduction peak, CV was employed to assess the ECSA, and 7.64 cm2 is discovered to be the hb-NPG ECSA15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65065/65065fig01.jpg" /> 
Figure 1:&#160;Hierarchical bimodal electrode (hb-NPG) after the multistep fabrication process. The hb-NPG coating on the gold wire after the completion of alloying-dealloying-annealing-dealloying is depicted here. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65065/65065fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65065/65065fig02.jpg" /> 
Figure 2:&#160;Sample preparation for SEM. Electrodes are mounted on carbon tape that is placed on aluminum stubs. The sample is then loaded into the imaging chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65065/65065fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65065/65065fig03.jpg" /> 
Figure 3:&#160;SEM micrograph showing the bimodal structure comprising of bigger and smaller pore sizes. Images are taken at 15 kV and a spot size of 10. (A) Upper hierarchy in the form of larger pores at 20,000x (scale bar: 5 &#181;m). (B) Lower hierarchy depicting nanopores at a higher magnification of 80,000x (scale bar: 2 &#181;m). This figure has been reproduced with permission from Sondhi et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65065/65065fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65065/65065fig04.jpg" /> 
Figure 4:&#160;SEM micrographs showing the color-coded elemental mapping done after every fabrication step. SEM of the following structures with color-coded elements (Au, yellow; and Ag, red): (A) Au10:Ag90 alloy, (B) electrochemically dealloyed, (C) annealed, (D) hierarchical nanoporous structure following the last chemical dealloying step. Aux:Ag100-x stands for the alloy of gold and silver, where x is the atomic percentage of gold in the alloy. This figure has been reproduced with permission from Sondhi et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65065/65065fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65065/65065fig05.jpg" /> 
Figure 5:&#160;Cyclic voltammograms comparing the electroactive surface area of the electrodes. The curve shown as an inset (blue) depicts the CV of the Au10:Ag90 alloy. The structure created via chemical dealloying shows a small gold oxide reduction (red curve) peak. The bimodal structure incorporating chemical and electrochemical dealloying shows a much more pronounced gold oxide reduction peak (green), indicating an increase in the surface area. CV was performed using a potential scan from -0.2-1.6 V. This figure has been reproduced with permission from Sondhi et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65065/65065fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65065/65065fig06.jpg" /> 
Figure 6:&#160;Absorbance versus time graph showing real-time protein loading. Real-time immobilization of BSA, fetuin, and HRP on NPG electrodes is depicted in (A), (C), and (E), while on hb-NPG is depicted in (B), (D), and (F). Change in absorbance and the number of immobilized molecules is monitored over 120 min. Absorbance was recorded after every 60 s. The average of the three readings is depicted in the graph. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65065/65065fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File&#160;1: User guide for the easy operation of two pieces of software used in the fabrication and characterization process of the hb-NPG. Screenshots explaning the step-by-step procedure to use &#34;PowerSuite&#34; and &#34;XT microscope&#34;. Arrows used in the flowchart are pointing toward the next step of the operation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65065/user guide-screenshots.pdf" target="_blank">Please click here to view a larger version of this figure.</a>

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, ...

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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

Fabricating Nanogaps by Nanoskiving

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two electrodes-and generating them in practical quantities-is still challenging. The preparation of nanogap electrodes using nanoskiving, which is a form of edge lithography, is a fast, simple and powerful technique. This method is an entirely mechanical process which does not include any photo- or electron-beam lithographic steps and does not require any special equipment or infrastructure such as clean rooms. Nanoskiving is used to fabricate electrically addressable nanogaps with control over all three dimensions; the smallest dimension of these structures is defined by the thickness of the sacrificial layer (Al or Ag) or self-assembled monolayers. These wires can be manually positioned by transporting them on drops of water and are directly electrically-addressable; no further lithography is required to connect them to an electrometer.

The fabrication of electrically addressable, high-aspect-ratio (> 1000:1) metal nanowires separated by gaps of single nanometers using either sacrificial layers of aluminum and silver or self-assembled monolayers as templates is described. These nanogap structures are fabricated without a clean room or any photo- or electron-beam lithographic processes by a form of edge lithography known as nanoskiving.

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two ...

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates

In recent years &pi;-conjugated organic semiconductors have emerged as the active material in a number of diverse applications including large-area, low-cost displays, photovoltaics, printable and flexible electronics and organic spin valves. Organics allow (a) low-cost, low-temperature processing and (b) molecular-level design of electronic, optical and spin transport characteristics. Such features are not readily available for mainstream inorganic semiconductors, which have enabled organics to carve a niche in the silicon-dominated electronics market. The first generation of organic-based devices has focused on thin film geometries, grown by physical vapor deposition or solution processing. However, it has been realized that organic nanostructures can be used to enhance performance of above-mentioned applications and significant effort has been invested in exploring methods for organic nanostructure fabrication.	
A particularly interesting class of organic nanostructures is the one in which vertically oriented organic nanowires, nanorods or nanotubes are organized in a well-regimented, high-density array. Such structures are highly versatile and are ideal morphological architectures for various applications such as chemical sensors, split-dipole nanoantennas, photovoltaic devices with radially heterostructured "core-shell" nanowires, and memory devices with a cross-point geometry. Such architecture is generally realized by a template-directed approach. In the past this method has been used to grow metal and inorganic semiconductor nanowire arrays. More recently &pi;-conjugated polymer nanowires have been grown within nanoporous templates. However, these approaches have had limited success in growing nanowires of technologically important &pi;-conjugated small molecular weight organics, such as tris-8-hydroxyquinoline aluminum (Alq3), rubrene and methanofullerenes, which are commonly used in diverse areas including organic displays, photovoltaics, thin film transistors and spintronics.	
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We report a simple method for fabricating an ultrahigh density array of vertically ordered small-molecular organic nanowires. This method allows for synthesis of complex heterostructured hybrid nanowire geometries, which can be inexpensively grown on arbitrary substrates. These structures have potential applications in organic electronics, optoelectronics, chemical sensing, photovoltaics and spintronics.

In  recent years &pi;-conjugated  organic semiconductors have emerged as the active material in a number of  diverse applications including large-area, ...

Engineering

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 ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Bioengineering

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing in combination with new nanofabrication techniques. A plasmon is a quantum of charge density oscillation that lends nanoscale metals such as gold and silver unique optical properties. In particular, gold and silver nanoparticles exhibit localized surface plasmon resonances-collective charge density oscillations on the surface of the nanoparticle-in the visible spectrum. Here, we focus on the fabrication of periodic arrays of anisotropic plasmonic nanostructures. These half-shell (or nanocup) structures can exhibit additional unique light-bending and polarization-dependent optical properties that simple isotropic nanostructures cannot. Researchers are interested in the fabrication of periodic arrays of nanocups for a wide variety of applications such as low-cost optical devices, surface-enhanced Raman scattering, and tamper indication. We present a scalable technique based on colloidal lithography in which it is possible to easily fabricate large periodic arrays of nanocups using spin-coating and self-assembled commercially available polymeric nanospheres. Electron microscopy and optical spectroscopy from the visible to near-infrared (near-IR) was performed to confirm successful nanocup fabrication. We conclude with a demonstration of the transfer of nanocups to a flexible, conformal adhesive film.

We demonstrate the fabrication of periodic gold nanocup arrays using colloidal lithographic techniques and discuss the importance of nanoplasmonic films.

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing ...

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and high-density data storage have demonstrated the potential benefit of their use in industrial applications. These applications require precise control over nanoparticle size, spacing, and sometimes shape. These requirements have resulted in the use of time and cost intensive processing steps to produce nanoparticles, thus making the transition to industrial application unrealistic. This protocol will resolve this issue by providing a scalable and affordable method for the large-area production of nanoparticle films with improved nanoparticle control compared to the current techniques. In this article, the process will be demonstrated with gold, but other metals can also be used.

This protocol details a novel nano-manufacturing technique that can be used to make controllable and customizable nanoparticle films over large areas based on the self-assembly of dewetting of capped metal films.

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and ...

Growth of Gold Dendritic Nanoforests on Titanium Nitride-coated Silicon Substrates

In this study, a high-power impulse magnetron sputtering system is used to coat a flat and firm titanium nitride (TiN) film on silicon (Si) wafers, and a fluoride-assisted galvanic replacement reaction (FAGRR) is employed for the rapid and easy deposition of gold dendritic nanoforests (Au DNFs) on the TiN/Si substrates. Scanning electron microscopy (SEM) images and energy-dispersive X-ray spectroscopy patterns of TiN/Si and Au DNFs/TiN/Si samples validate that the synthesis process is accurately controlled. Under the reaction conditions in this study, the thickness of the Au DNFs increases linearly to 5.10 &#177; 0.20 &#181;m within 15 min of the reaction. Therefore, the employed synthesis procedure is a simple and rapid approach for preparing Au DNFs/TiN/Si composites.

This study presents a feasible procedure for synthesizing gold dendritic nanoforests on titanium nitride/silicon substrates. The thickness of gold dendritic nanoforests increases linearly within 15 min of a synthesis reaction.

In this study, a high-power impulse magnetron sputtering system is used to coat a flat and firm titanium nitride (TiN) film on silicon (Si) wafers, and a ...

Microscopic Visualization of Porous Nanographenes Synthesized through a Combination of Solution and On-Surface Chemistry

On-surface synthesis has recently been regarded as a promising approach for the generation of new molecular structures. It has been particularly successful in the synthesis of graphene nanoribbons, nanographenes and intrinsically reactive and instable, yet attractive species. It is based on the combination of solution chemistry aimed at preparation of appropriate molecular precursors for further ultra-high vacuum surface assisted transformations. This approach also owes its success to an incredible development of characterization techniques, such as scanning tunneling/atomic force microscopy and related methods, which allow detailed, local characterization at atomic scale. While the surface-assisted synthesis can provide molecular nanostructures with outstanding precision, down to single atoms, it suffers from basing on metallic surfaces and often limited yield. Therefore, the extension of the approach away from metals and the struggle to increase productivity seem to be significant challenges toward wider applications. Herein, we demonstrate the on-surface synthesis approach for generation of non-planar nanographenes, which are synthesized through a combination of solution chemistry and sequential surface-assisted processes, together with the detailed characterization by scanning probe microscopy methods.

A combination of solution and surface assisted synthesis opens new directions in the atomically precise synthesis of nanostructures. Scanning tunneling microscopy (STM) supplemented by non-contact atomic force microscopy (nc-AFM) enables detailed characterization of newly designed and generated carbon-based nano-objects.