Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Summary

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.

Abstract

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.

Introduction

Materials with nano-scale features have shown promise in enhancing various applications, including fuel cells¹, sensors^2,3, and biomedical devices^4,5. 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 potential^6-8. 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 properties^10,11, catalytic activity¹², and biomolecular sensing performance^13-15. While the desirable attributes are highly useful for several biomedical tools^16,17, 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 films^16-20. 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.

Protocol

1. Nanoporous Gold Fabrication

1. Clean substrates in Piranha solution
   1. Add 25 ml hydrogen peroxide (30%) to 100 ml sulfuric acid (96%) in a crystallization dish and heat the mixture to 65 °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.
   2. 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.
   3. 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.
2. Prepare stencil mask (Method 1: Use this for creating millimeter-scale patterns)
   1. Punch 250 μm-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.
   2. 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.
3. Pattern lift-off photoresist (Method 2: Use this for creating sub-millimeter-scale patterns)
   1. 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 °C for 7 min and let it cool for 5 min.
   2. 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 °C for 1.5 min and let it cool for 10 min.
   3. 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 °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.
4. Deposit precursor metals to produce np-Au thin films
   1. 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.
   2. 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.
5. Obtain precursor metal micropatterns
   1. 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.
   2. Peel the elastomer stencil from non-photoresist coated samples using two tweezers to reveal the deposited metal.
6. Dealloy precursor metal and modify nanostructure via thermal treatment
   1. Fill a 200 ml glass beaker with 170 ml of nitric acid (70%) and maintain the solution temperature at 55 °C on a hotplate. CAUTION: The nitric acid is extremely corrosive and should be handled with appropriate protective equipment.
   2. 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.
   3. 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.
   4. Load samples onto a clean silicon wafer in a rapid thermal processing equipment. Adjust the temperature to between 200 °C and 450 °C and the ramp rate to 10 °C/sec. Expose the samples to the prescribed temperature for 10 min under nitrogen ambient. Let the chamber cool (<100 °C) and remove the samples. Alternatively, slowly place the samples on the hotplate for thermal treatment.

2. Cell Culture

1. Prepare np-Au samples for cell culture
   1. 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.
   2. Add 500 μl 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 °C and 5% CO₂ until seeding the cells (<1 hr).
2. Maintain, passage, and seed cells
   1. 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.
   2. 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.
   3. 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.

3. Cell and Material Analysis

1. Stain cells to visualize cytoskeleton and nuclei
   1. Remove spent media from wells and wash cells twice with PBS. Fix cells in 4% paraformaldehyde in PBS for 15 min.
   2. Prepare staining solution of 300 nM Alexa Fluor 488 phalloidin in PBS with 1% bovine serum albumin.
   3. Wash cells twice with PBS, and permeabilize them in 500 μl 0.1% Triton X-100 in PBS for 5 min.
   4. Wash cells twice with PBS and transfer them to clean wells. Blot 50 - 200 μl staining solution onto the samples and store in dark for 20 min.
   5. Wash cells with PBS and counter-stain with 3 nM of DAPI in PBS for 5 min.
   6. Wash cells with PBS, and dip in deionized water before mounting on glass cover slides with mounting media and seal with clear nail polish.
2. Acquire images of np-Au samples and cell cultures on np-Au surfaces
   1. Image np-Au surfaces with scanning electron microscope (SEM) with 50,000X magnification at 10 kV electron energy using secondary electron detector.
   2. Capture composite cell images at different spots on the samples using an inverted fluorescence microscope at 10X magnification with the appropriate filter cubes.
3. Process images to determine pore and cell morphology
   1. 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.
   2. 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.
   3. Modify the included macro files to perform batch analysis of multiple images.

Results

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...

Discussion

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

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