O. Kurtulus and D. Dimlioglu are supported by a University of California Laboratory Fees Research Program Award 12-LR-237197. P. Daggumati is supported by a University of California Davis Research Investments in the Sciences &amp; Engineering (RISE) Award. C.A. Chapman is supported by a Department of Education Graduate Assistance Areas of National Need Fellowship. This work was supported by UC Lab Fees Research Program, UC Davis RISE, and UC Davis College of Engineering start-up funds.

1. Nanoporous Gold Fabrication
<ol>
	<li>Clean substrates in Piranha solution
	<ol>
		<li>Add 25 ml hydrogen peroxide (30%) to 100 ml sulfuric acid (96%) in a crystallization dish and heat the mixture to 65 &deg;C on a hotplate. CAUTION: The liquids are extremely corrosive and must be handled with care. The spent solution should not be stored in a sealed container, as it may explode.</li>
		<li>Place 1-inch by 3-inch microscope slides into the mixture using acid-resistant forceps and clean them for 10 min. Use a porcel...

<ol>
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Biotechnol. 24, 1211-1218 (2006).</li><li>Weissm&#252;ller, J., Newman, R., Jin, H., Hodge, A., Kysar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theme+Article+-+Nanoporous+Metals+by+Alloy+Corrosion:+Formation+and+Mechanical+Properties.">Theme Article - Nanoporous Metals by Alloy Corrosion: Formation and Mechanical Properties.</a> Materials Research Society Bulletin. 34, 577-586 (2009).</li><li>Erlebacher, J., Aziz, M., Karma, A., Dimitrov, N., Sieradzki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+nanoporosity+in+dealloying.">Evolution of nanoporosity in dealloying.</a> Nature. 410, 450-453 (2001).</li><li>Okman, O., Lee, D., Kysar, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+crack-free+nanoporous+gold+blanket+thin+films+by+potentiostatic+dealloying.">Fabrication of crack-free nanoporous gold blanket thin films by potentiostatic dealloying.</a> Scripta Mater. 63, 1005-1008 (2010).</li><li>Seker, E., Reed, M., Begley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoporous+Gold:+Fabrication,+Characterization,+and+Applications.">Nanoporous Gold: Fabrication, Characterization, and Applications.</a> Materials. 2, 2188-2215 (2009).</li><li>Biener, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+effects+on+the+mechanical+behavior+of+nanoporous+Au.">Size effects on the mechanical behavior of nanoporous Au.</a> Nano Lett. 6, 2379-2382 (2006).</li><li>Senior, N., Newman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+tough+nanoporous+metals+by+controlled+electrolytic+dealloying.">Synthesis of tough nanoporous metals by controlled electrolytic dealloying.</a> Nanotechnology. 17, 2311-2316 (2006).</li><li>Zielasek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+catalysts:+Nanoporous+gold+foams.">Gold catalysts: Nanoporous gold foams.</a> Angew. Chem. Int. Ed. 45, 8241-8244 (2006).</li><li>Wittstock, A., Biener, J., B&#228;umer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoporous+gold:+a+new+material+for+catalytic+and+sensor+applications.">Nanoporous gold: a new material for catalytic and sensor applications.</a> PCCP. 12, 12919-12930 (2010).</li><li>Shulga, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+porous+gold+and+its+application+as+a+platform+for+immobilization+of+acetylcholine+esterase.">Preparation and characterization of porous gold and its application as a platform for immobilization of acetylcholine esterase.</a> Chem. Mater. 19, 3902 (2007).</li><li>Shulga, O., Zhou, D., Demchenko, A., Stine, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+free+prostate+specific+antigen+(fPSA)+on+a+nanoporous+gold+platform.">Detection of free prostate specific antigen (fPSA) on a nanoporous gold platform.</a> The Analyst. 133, 319-322 (2008).</li><li>Seker, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fabrication+of+low-impedance+nanoporous+gold+multiple-electrode+arrays+for+neural+electrophysiology+studies.">The fabrication of low-impedance nanoporous gold multiple-electrode arrays for neural electrophysiology studies.</a> Nanotechnology. 21, 125504 (2010).</li><li>Seker, E., Berdichevsky, Y., Staley, K. J., Yarmush, M. 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Rev. Lett. 97, 35504-35506 (2006).</li></ol>

Authors have no conflicting financial interest.

We demonstrate two different techniques to micropattern np-Au films for expanding use of these films in microsystems and biological studies. Sputter-coating gold and silver is a versatile method to create np-Au patterns, as sputtering is compatible with conventional microfabrication processes and the alloy composition and thickness can be easily controlled by varying the individual sputtering gun powers (for gold and silver targets) and the deposition time respectively. Typical np-Au film thicknesses range from 200 nm to...

Materials with nano-scale features have shown promise in enhancing various applications, including fuel cells1, sensors2,3, and biomedical devices4,5. A relatively new material is nanoporous gold (np-Au), which is produced by a nano-scale self-assembly process. The precursor to np-Au is a gold alloy that most commonly consists of silver at 60% to 80% by atomic percentage. Briefly, the characteristic open-pore nanostructure is the result of rearrangement of gold atoms in clusters as silver is dissolved by a strong acid (e.g. nitric acid 70%) or under an electrochemical potential6-8. Np-Au benefits from several desirable attributes, including large effective surface area, high electrical conductivity, well-established surface functionalization techniques, and biocompatibility9. Even though there has been a rapid expansion of studies on np-Au, most of them focus on np-Au's mechanical properties10,11, catalytic activity12, and biomolecular sensing performance13-15. While the desirable attributes are highly useful for several biomedical tools16,17, the applications in this area have been limited. One possible reason for this is that most studies have predominantly used macro-scale specimens (e.g. sheets, foils, and ingots) and the techniques for incorporating np-Au in miniaturized systems have remained inadequate. In fact there are only a handful of examples of using conventional microfabrication techniques that employ np-Au films16-20. With the advent of miniaturization technology and the need for novel biomedical tools, it has become crucial to be able to integrate new materials into devices. This typically requires that the materials can be deposited and patterned with conventional microfabrication techniques. In addition, rapid quantification of cell-material interactions is commonly necessary to evaluate the biocompatibility of a new material. The goal of this paper is to demonstrate basic techniques to micropattern np-Au films and quantify both nanostructure and cell-material interactions via digital image processing.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Gold target</td>
 <td>Lesker</td>
 <td>EJTAUXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Chrome target</td>
 <td>Lesker</td>
 <td>EJTCRXX353A2</td>
 <td >Adhesive layer</td>
 </tr>
 <tr>
 <td>Silver target</td>
 <td>Lesker</td>
 <td>EJTAGXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Porcelain boat</td>
 <td>Thomas Scientific </td>
 <td>8542E40</td>
 <td >Used for processing small samples</td>
 </tr>
 <tr>
 <td>Nitric acid</td>
 <td>Sigma-Aldrich</td>
 <td>43873</td>
 <td >Used at 70% for dealloying</td>
 </tr>
 <tr>
 <td>Sulfuric acid</td>
 <td>J.T Baker </td>
 <td>7664-93-9</td>
 <td >Used at 96% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide</td>
 <td>J.T Baker </td>
 <td>7722-84-1</td>
 <td >Used at 30% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Biopsy punches</td>
 <td>Ted Pella</td>
 <td>150xx</td>
 <td >Available in several sizes</td>
 </tr>
 <tr>
 <td>Silicone elastomer sheets</td>
 <td>Rogers Corporation </td>
 <td>HT 6240</td>
 <td >Available in several thicknesses</td>
 </tr>
 <tr>
 <td>Hexamethyldisilazane</td>
 <td>Sigma-Aldrich</td>
 <td>440191-100ML</td>
 <td >Used as adhesion promoter for positive resist</td>
 </tr>
 <tr>
 <td>Microposit MF CD26</td>
 <td>Shipley</td>
 <td>38490</td>
 <td >Positive photoresist developer</td>
 </tr>
 <tr>
 <td>PRS 3000</td>
 <td>J.T Baker</td>
 <td>JT6403-5</td>
 <td >Positive photoresist stripper</td>
 </tr>
 <tr>
 <td>Circular glass coverslips (12 mm)</td>
 <td>Ted Pella</td>
 <td>26023</td>
 <td >Used as substrate for metal patterns and cell culture</td>
 </tr>
 <tr>
 <td>Glass slides (1 x 3 inch)</td>
 <td>Ted Pella</td>
 <td>26007</td>
 <td >Used as substrate for metal patterns</td>
 </tr>
 <tr>
 <td>Kapton polyimide tape</td>
 <td>VWR</td>
 <td>82030-950</td>
 <td >Used for securing elastomer</td>
 </tr>
 <tr>
 <td>Transparency masks</td>
 <td>Output City</td>
 <td></td>
 <td>Used in photolithography <a href="http://www.outputcity.com/" target="_blank">http://www.outputcity.com/</a></td>
 </tr>
 <tr>
 <td>Plasma cleaner</td>
 <td>Harrick Plasma</td>
 <td>PDC-32G</td>
 <td >Used for activating glass surfaces</td>
 </tr>
 <tr>
 <td>Sputtering machine</td>
 <td>Kurt J. Lesker</td>
 <td>LAB18</td>
 <td >Used for depositing metals</td>
 </tr>
 </tbody>
 </table>

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.

Figure 1 outlines the major procedural steps, including creating the np-Au patterns, culturing cells, quantifying the nanostructure, and characterizing cell morphologies. The elastomer stencil shown in Figure 2a (top) is used for creating the np-Au patterns shown in the images underneath. Figure 2b is a photograph of the porcelain boat for batch processing specimens. Figure 2c displays the color change of the deposited metal patterns before and afte...

Watch this Scientific Journal Video about Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies at JoVE.com

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

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, ...

microfabrication-nanoporous-gold-patterns-for-cell-material

Research

JoVE Journal

Bioengineering

11.2K Views.  University of California, Davis. 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.