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 porcelain immunostaining boat for batch-cleaning small coverslips. Treat small coverslips with air plasma at 10 W for 30 sec prior to immersion into the solution.</li>
		<li>Rinse the cleaned samples in crystallization dishes under running deionized (DI) water for 3 min. Blow-dry samples with a nitrogen gun over lint-free towels.</li>
	</ol>
	</li>
	<li>Prepare stencil mask (Method 1: Use this for creating millimeter-scale patterns)
	<ol>
		<li>Punch 250 &mu;m-thick silicone elastomer sheets with biopsy punches and/or incise out regions with a scalpel over a semi-hard plastic surface. Clean the processed elastomer sheets in 70% isopropanol and dry with nitrogen gun.</li>
		<li>Place the punched sheet on a lint-free cleanroom towel and align the piranha-cleaned coverslips over the stencil with the sample surface facing the stencil.</li>
	</ol>
	</li>
	<li>Pattern lift-off photoresist (Method 2: Use this for creating sub-millimeter-scale patterns)
	<ol>
		<li>Place a piranha-cleaned microscope slide on spinning chuck and blow off any particulates with nitrogen gun. Dispense 1.5 ml of adhesion promoter (hexamethyldisilazane) onto the glass slide using a plastic pipette. Spread the promoter by spinning the slide successively at 500 rpm for 5 sec and 1,500 rpm for 30 sec. Bake the slide on a hotplate at 115 &deg;C for 7 min and let it cool for 5 min.</li>
		<li>Dispense 4 ml of photoresist onto the glass slide (3-inch by 1-inch). Spread the photoresist by spinning the slide with the same protocol as for the adhesion promoter. Bake the photoresist on a hotplate at 115 &deg;C for 1.5 min and let it cool for 10 min.</li>
		<li>Expose the photoresist-coated slide with UV light (intensity: 22 mW/cm2) through a transparency mask for 15 sec. Bake the photoresist on a hotplate at 115 &deg;C for 1.5 min and wait for 45 min. Dissolve the exposed photoresist in the developer for at least 3.5 min. Rinse thoroughly with DI water. Inspect the developed patterns under an optical microscope.</li>
	</ol>
	</li>
	<li>Deposit precursor metals to produce np-Au thin films
	<ol>
		<li>Load the samples into a sputtering machine that can independently deposit gold, silver, and chrome. Sputter-clean the samples for 90 sec at 50 W under 25 mTorr argon processing atmosphere before starting metal deposition.</li>
		<li>Sputter chrome for 10 min at 300 W under 10 mTorr argon. Sputter gold for 90 sec at 400 W under 10 mTorr argon. Co-sputter gold and silver for 10 min with silver at 200 W and Au power at 100 W. Turn off gold sputter source approximately 10 sec before turning off the silver sputter source.</li>
	</ol>
	</li>
	<li>Obtain precursor metal micropatterns
	<ol>
		<li>Sonicate photoresist-coated samples in ~180 ml of photoresist stripper for 10 cycles of 20 sec sonication and 2 min pause between the cycles. Rinse the samples with DI water and dry with a nitrogen gun. Inspect the metal patterns under a microscope.</li>
		<li>Peel the elastomer stencil from non-photoresist coated samples using two tweezers to reveal the deposited metal.</li>
	</ol>
	</li>
	<li>Dealloy precursor metal and modify nanostructure via thermal treatment
	<ol>
		<li>Fill a 200 ml glass beaker with 170 ml of nitric acid (70%) and maintain the solution temperature at 55 &deg;C on a hotplate. CAUTION: The nitric acid is extremely corrosive and should be handled with appropriate protective equipment.</li>
		<li>Treat small coverslips with air plasma at 10 W for 30 sec prior to immersion into nitric acid. Place 1-inch by 3-inch microscope slides into the beaker using acid-resistant forceps and dealloy them for 15 min. Use a porcelain immunostaining boat for batch-dealloying small coverslips.</li>
		<li>Rinse the dealloyed samples by successively immersing them in two beakers filled with fresh DI water three times. Store samples in DI water and replace the water with fresh DI water every day for at least a week. Blow-dry samples with a nitrogen gun over lint-free towels before use.</li>
		<li>Load samples onto a clean silicon wafer in a rapid thermal processing equipment. Adjust the temperature to between 200 &deg;C and 450 &deg;C and the ramp rate to 10 &deg;C/sec. Expose the samples to the prescribed temperature for 10 min under nitrogen ambient. Let the chamber cool (&lt;100 &deg;C) and remove the samples. Alternatively, slowly place the samples on the hotplate for thermal treatment.</li>
	</ol>
	</li>
</ol>
2. Cell Culture
<ol>
	<li>Prepare np-Au samples for cell culture
	<ol>
		<li>Place np-Au samples in polystyrene dishes and treat with air plasma at 10 W for 30 sec and transfer the samples to 24-well tissue culture plates.</li>
		<li>Add 500 &mu;l complete culture media (Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin/streptomycin) to each well. Store in a humidified incubator at 37 &deg;C and 5% CO2 until seeding the cells (&lt;1 hr).</li>
	</ol>
	</li>
	<li>Maintain, passage, and seed cells
	<ol>
		<li>Maintain cells of interest (in this case 3T3-NIH fibroblasts or murine astrocytes) in T75 flasks with culture media and passage when cells are 70% confluent.</li>
		<li>For passaging the cells, remove media from the flask, wash twice with 10 ml phosphate buffered saline (PBS), add 2 ml of 1x Trypsin/EDTA and incubate until the cells detach (~5 min). Add 3 ml fresh media and centrifuge at 1,200 rpm for 3 min. Aspirate supernatant and suspend the pellet in 2 ml media.</li>
		<li>Remove the spent media from the wells and seed the cells onto the glass coverslips at a density of 25,000 cells/cm2 with a final volume of 1 ml. Shake the culture plate forward-back and right-left to ensure a uniform coating of cells over the samples. Incubate the cells until analysis while inspecting daily.</li>
	</ol>
	</li>
</ol>
3. Cell and Material Analysis
<ol>
	<li>Stain cells to visualize cytoskeleton and nuclei
	<ol>
		<li>Remove spent media from wells and wash cells twice with PBS. Fix cells in 4% paraformaldehyde in PBS for 15 min.</li>
		<li>Prepare staining solution of 300 nM Alexa Fluor 488 phalloidin in PBS with 1% bovine serum albumin.</li>
		<li>Wash cells twice with PBS, and permeabilize them in 500 &mu;l 0.1% Triton X-100 in PBS for 5 min.</li>
		<li>Wash cells twice with PBS and transfer them to clean wells. Blot 50 - 200 &mu;l staining solution onto the samples and store in dark for 20 min.</li>
		<li>Wash cells with PBS and counter-stain with 3 nM of DAPI in PBS for 5 min.</li>
		<li>Wash cells with PBS, and dip in deionized water before mounting on glass cover slides with mounting media and seal with clear nail polish.</li>
	</ol>
	</li>
	<li>Acquire images of np-Au samples and cell cultures on np-Au surfaces
	<ol>
		<li>Image np-Au surfaces with scanning electron microscope (SEM) with 50,000X magnification at 10 kV electron energy using secondary electron detector.</li>
		<li>Capture composite cell images at different spots on the samples using an inverted fluorescence microscope at 10X magnification with the appropriate filter cubes.</li>
	</ol>
	</li>
	<li>Process images to determine pore and cell morphology
	<ol>
		<li>Open images in ImageJ and split into individual channels if applicable. Convert the images to 8-bit, subtract the background, and smooth them by median filtering. Adjust threshold either manually or by built-in thresholding algorithms to highlight the pores (voids) and cell bodies/nuclei.</li>
		<li>Use watershed command to separate merged pores or cells. Set particle analysis parameters and execute the command to extract number of particles, average area, and percent coverage by particles. Use DAPI-stained cell images for cell counting and phalloidin-stained cell images for quantifying percent cell coverage.</li>
		<li>Modify the included macro files to perform batch analysis of multiple images.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Arico, A. S., Bruce, P., Scrosati, B., Tarascon, J. M., Van Schalkwijk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+materials+for+advanced+energy+conversion+and+storage+devices.">Nanostructured materials for advanced energy conversion and storage devices.</a> Nature Materials. 4, 366-377 (2005).</li><li>Roy, S., Gao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructure-based+electrical+biosensors.">Nanostructure-based electrical biosensors.</a> Nano Today. 4, 318-334 (2009).</li><li>Chen, C. L., 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=DNA-decorated+carbon-nanotube-based+chemical+sensors+on+complementary+metal+oxide+semiconductor+circuitry.">DNA-decorated carbon-nanotube-based chemical sensors on complementary metal oxide semiconductor circuitry.</a> Nanotechnology. 21, 095504 (2010).</li><li>Lu, J., Rao, M. P., MacDonald, N. C., Khang, D., Webster, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+endothelial+cell+adhesion+and+proliferation+on+patterned+titanium+surfaces+with+rationally+designed,+micrometer+to+nanometer+features.">Improved endothelial cell adhesion and proliferation on patterned titanium surfaces with rationally designed, micrometer to nanometer features.</a> Acta Biomaterialia. 4, 192-201 (2008).</li><li>Wagner, V., Dullaart, A., Bock, A. K., Zweck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+nanomedicine+landscape.">The emerging nanomedicine landscape.</a> Nat. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication-Compatible+Nanoporous+Gold+Foams+as+Biomaterials+for+Drug+Delivery.">Microfabrication-Compatible Nanoporous Gold Foams as Biomaterials for Drug Delivery.</a> Advanced Healthcare Materials. 1, 172-176 (2012).</li><li>Okman, O., 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=Microfabrication+of+Nanoporous+Gold.">Microfabrication of Nanoporous Gold.</a> Nanoporous Gold: From an Ancient Technology to a High-Tech Material. 22, 69 (2012).</li><li>Lee, D., 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=Microfabrication+and+mechanical+properties+of+nanoporous+gold+at+the+nanoscale.">Microfabrication and mechanical properties of nanoporous gold at the nanoscale.</a> Scripta Mater. 56, 437-440 (2007).</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+effects+of+post-fabrication+annealing+on+the+mechanical+properties+of+freestanding+nanoporous+gold+structures.">The effects of post-fabrication annealing on the mechanical properties of freestanding nanoporous gold structures.</a> Acta Mater. 55, 4593-4602 (2007).</li><li>Parida, S., 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=Volume+change+during+the+formation+of+nanoporous+gold+by+dealloying.">Volume change during the formation of nanoporous gold by dealloying.</a> Phys. 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

11.3K 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.

Video: Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...