Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

The overall goal of this procedure is to fabricate nano porous gold micro patterns and demonstrate their compatibility with cell culture. This is accomplished by first creating stencil or photo lithographic masks. The second step is to co sputter gold and silver through the masks to pattern the precursor alloy for nano porous gold.

Next, the gold silver alloy patterns are de alloyed in nitric acid to produce nano porous gold coatings. The final step is to culture cells over the nano porous gold micro patterns and immunostain them ultimately fluorescence and scanning electron microscopy coupled with image analysis are used to quantify cell material interactions. The main advantage of this technique over other methods for creating nano structured coatings, such as carbon nano tubes, is that nano porous gold benefits from microfabrication compatibility, high effective surface area, ease of functionalization with tile based chemistry, electrical conductivity, and biocompatibility.

This method can answer key questions in material science and bioengineering fields, such as the effect of material nanostructure on electrical, mechanical, biological, and chemical properties. Though this method provides insight in the effect of nano structure on cell morphology. It can also be applied to other systems such as the development of biosensors drug delivery systems and energy storage platforms.

Begin this procedure by cleaning one inch by three inch microscope slides and small cover slips with a piranha solution load the small cover slips into a porcelain immuno staining boat and immerse the boat and the microscope slide in the piranha solution. See the text protocol for details on the cleaning procedure. After cleaning, transfer the samples to crystallization dishes and rinse them with di water for three minutes.

Place the samples on lint-free towels and blow dry them with a nitrogen gun To create millimeter scale patterns. Punch 250 micron thick silicone elastomer sheets with a biopsy punch. Clean the processed elastomer sheets by immersing them in 70%isopropanol and then dry them with a nitrogen gun.

Then place the punch sheet on a lint-free towel and align the cleaned cover slips over the stencil with the sample surface facing the stencil. Transfer the stencil with the cover slips onto a carrier wafer to create sub-millimeter scale patterns. Place a cleaned microscope, slide on a spinning chuck and blow off any particulates with a nitrogen gun.

Dispense 1.5 milliliters of hexa methyl dilane onto the glass slide. Using a plastic pipette, spread the hexa methyl dilane by spinning the slide successively at 500 RPM for five seconds and 1500 RPM for 30 seconds. When finished, bake the slide on a hot plate at 115 degrees Celsius for seven minutes, and then let it cool for five minutes.

Next, dispense four milliliters of a positive photo. Resist onto the glass slide. Spread the photo resist by spinning the slide using the conditions described previously.

Bake the slide on a hot plate at 115 degrees Celsius for 1.5 minutes and remove it following UV exposure and baking. Dissolve the exposed photo. Resist by submerging the slide in a positive photo.

Resist developer for at least 3.5 minutes. When finished, rinse the slide thoroughly with DI water and inspect the developed patterns under an optical microscope. Next, load the samples into a sputtering machine that can independently deposit gold, silver, and chrome sputter.

Clean the samples for 90 seconds at 50 watts under a 25 millitorr Argonne processing atmosphere before starting the metal deposition under 10 millitorr Argonne sputter chrome for 10 minutes at 300 watts. Then sputter gold for 90 seconds at 400 watts, followed by co sputtering gold and silver for 10 minutes with silver at 200 watts and gold at 100 watts. Turn off the gold sputter source approximately 10 seconds before turning off the silver sputter source.

Remove the samples from the sputtering machine after removing the samples from the sputtering machine. Sonicate them in about 180 milliliters of a positive photo. Resist stripper for 10 cycles of 22nd sonication with two minute pauses between the cycles.

When finished, rinse the samples with di water and dry them with a nitrogen gun. Inspect the metal patterns under a microscope. Then peel the elastomer stencil from the non-photo resist coated samples using two tweezers to reveal the deposited metal.

Treat the small cover slips with air plasma at 10 watts for 30 seconds prior to immersing them in nitric acid. De alloy the microscope slides by submerging them into a beaker of 70%nitric acid at 55 degrees Celsius for 15 minutes. Once de alloying is complete, rinse the samples by successively immersing them into beakers of di water three times each.

Place the samples on lint-free towels and blow dry them with a nitrogen gun. Adjust the temperature of a hot plate to between 150 and 300 degrees Celsius. Then lower the samples slowly onto the hot surface to minimize sudden temperature change.

After five minutes, remove the samples from the hot plate to cool. Place the nano porous gold samples in polystyrene dishes and treat them with air plasma at 10 watts for 30 seconds. Then transfer the samples to 24 well tissue culture plates.

Add 500 microliters of complete culture media to each. Well store the plates in a humidified incubator at 37 degrees Celsius and 5%CO2 until seeding the cells. Maintain murine astrocyte cells in T 75 flasks with culture media.

Once the cells are 70%confluent, passage them by first removing the old media and washing the cells with PBS. See the text protocol for details on the passeng conditions. After removing the culture plates from the incubator, aspirate the spent media from the wells and seed the cells onto the glass.

Cover slips at a density of 25, 000 cells per square centimeter with a final volume of one milliliter. Shake the culture plate to ensure a uniform coating of cells over the samples and place the culture plate in the incubator when cells are ready to be analyzed. Remove the spent media from the wells and wash them twice with PBS.

Once the cells have been fixed in 4%para formaldehyde in PBS and wash twice, perme them in 500 microliters of 0.1%Triton X 100 in PBS for five minutes. Repeat the PBS wash two times and transfer the cells to clean wells. Blot 80 microliters of the staining solution onto the samples and store them covered in aluminum foil for 20 minutes.

Following another PBS wash, counterstain the cells with three nanomolar of DPI in PBS for five minutes. Wash cells with PBS once more and dip them in di then mount the cells on glass. Cover slides with mounting media and seal them with clear nail polish image.

The nano porous gold surfaces with a scanning electron microscope with 50, 000 x magnification at 10 kilovolts electron energy using a secondary electron detector. Following this capture composite cell images at different spots on the samples using an inverted fluorescence microscope at 10 x magnification with the appropriate filter cubes. Open the images in image J.Split the images to individual channels.

Convert them to eight bid. Subtract the background and smooth them by median filtering. Then adjust the threshold manually to highlight the pores, cell bodies, and nuclei.

Use the watershed command to separate merged pores or cells. Set particle analysis parameters and execute the command to extract the number of particles, average area and percent coverage by particles. Finally, modify the included macro files to perform batch analysis of multiple images.

The color change of the deposited metal patterns before and after d alloying is displayed here. The silvery finish is due to the silver rich alloy content upon the alloying. The film acquires a visibly orange brown tint.

Shown here are the finer features produced by photo lithographic. Patterning of the metal. Different poor morphologies can be obtained by thermal treatment of the nano porous gold films.

The cross-sectional image reveals whether the films are de alloyed uniformly through the film thickness. The scanning electron microscope images can be segmented into binary images for determining the size and percent coverage by voids. The code to generate this output can be downloaded from the JoVE webpage.

A representative fluorescence image of adherent murine astrocyte cells cultured on a nano porous gold surface is pictured here. The individual channels of the image can be split and segmented to produce binary images for analyzing cell material interaction. Segmented cytoskeleton images can be used for quantifying cell area and surface coverage.

While the segmented cellular nucleus images are useful for cell counting. The codes to generate these outputs can be downloaded from the JoVE webpage. Shown here is a visual summary of failures in the fabrication of nano porous gold structures, including PO film adhesion, absence of porosity, and excessive thermal treatment.

Following this procedure, other methods suggest electrochemical characterization at forest microscopy, cell lysis, and PCR can be performed to further answer additional questions such as the effect of nanostructure cell adhesion and gene expression. After watching this video, you should have a good understanding of how to produce nanopores gold patterns and quantify their interactions with cells by digital image analysis of microscopy.Results. Don't forget that working with nitric acid, sulfur acid and hydrogen peroxide can be extremely hazardous and precautions including growing personal protective equipment should always be taken while performing this procedure.