A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.
The overall goal of this procedure is to produce a large area of nanopatterned substrate from small nanopatterned molds by using a simple and affordable yet versatile stitch technique. This method can help study substrate nanotopography modulation of cell phenotype and function at cellular and molecular levels, such as expression of focal adhesion proteins. The main advantage of this technique is that it is simple and cost effective.
To begin, prepare the silanized silicon mold in the PDMS prepolymer, as described in the text protocol. Put the silanized silicon mold in a 60 millimeter Petri dish. Pour the PDMS prepolymer on the silicon mold in the Petri dish.
Place the Petri dish in a plastic desiccator and degas for about 10 minutes, until all bubbles disappear. Transfer the Petri dish to a hot plate and cure the PDMS prepolymer at 70 degrees Celsius for four hours. Carefully peel off the PDMS mold from the silicon mold by using tweezers.
Determine the orientation of anisotropic PDMS nanopatterns, such as nanogratings, under an optical microscope, and mark it on the backside of the PDMS molds with a marker pen. Clean the silicon substrate with ethanol in a fume hood, and then dry it with compressed air. Trim off the unpatterned areas of each PDMS mold with a blade.
The PDMS molds should be aligned as close as possible, but not touching the surrounding mold, to minimize the unpatterned area and to avoid touching to the nanopattern deformation when the compressor force is applied. Place the trimmed PDMS mold with the nanopattern face-down on the mirror side of the silicon substrate, and then align other molds close to but not touching the surrounding molds. It is critical to try to cure the PDMS at the basal layer because uncured PDMS may flow through the gap between the multiple molds and damage the nanopatterns.
However, the completely cured PDMS cannot bond the multiple molds together. To prepare a PDMS adhesive layer, cast one gram of degassed PDMS prepolymer on a clean glass slide to form a 0.5 millimeter thick layer. Bake the PDMS layer at 100 degrees Celsius on a hot plate for three to five minutes.
Use a needle to touch the layer and ensure that the layer is partially but not completely cured. Place the PDMS layer on the backside of the aligned PDMS molds, quickly invert this assembly, and transfer it to the hot plate. Apply a compressive force using a metal block on top of the assembly to ensure good contact between the PDMS adhesive layer and the backside of the PDMS molds.
Cure the PDMS adhesive layer at 100 degrees Celsius for one hour. Turn off the hot plate, remove the metal block, and peel off the single-stiched PDMS mold from the silicon substrate. To nanoimprint the stitched PDMS mold into the PS plate, place the PS plate in an aluminum spacer set on a three inch silicon wafer.
Heat the PS plate on a hot plate at 250 degrees Celsius for 30 minutes. Then, place the stitched PDMS mold, with nanopatterns face-down, on the molten PS plate. Next, place an aluminum plate on the glass slide of the stitched PDMS mold.
Apply a compressive pressure by using metal blocks on the aluminum plate, and wait for three minutes. Lift and replace the metal block from the aluminum plate, and increase the compressive pressure to 25 kilopascals. Then, repeat this step with the pressure increased to 50 kilopascals.
Maintain the temperature of the hot plate at between 240 and 250 degrees Celsius under consistent pressure of 50 kilopascals for 15 minutes. Turn off the hot plate, and cool down the whole setup. Remove the metal blocks after the temperature is below 50 degrees Celsius, and carefully peel off the stitched PDMS mold from the PS plate.
To nanoimprint the PDMS mold on a PS thin film, place the stitched PDMS mold with nanotopography face-down on the PS thin film, which is set on a hot plate. Apply compressive pressure of 12 kilopascals on the PDMS mold by using metal blocks on the glass slide of the PDMS mold. Increase the temperature of the hot plate to 180 degrees Celsius, and maintain it for 15 minutes.
Turn off the hot plate, and cool down the whole setup. Remove the metal blocks after the temperature drops below 50 degrees Celsius, and carefully peel off the stitched PDMS mold from the PS film. Using a hollow steel arch punch, cut the nanopatterned PDMS substrates into discs to fit the configuration of a specific multi-well plate.
Then, use tweezers to place the PDMS discs into the wells of the multi-well plate. Sterilize the PDMS substrates by using 70%ethanol for 30 minutes. Afterwards, aspirate the ethanol, and follow this by UV exposure for 30 minutes.
Wash the PDMS substrates with 1X sterile phosphate-buffered saline three times. Next, coat the PDMS substrates with extracellular matrix protein for 30 minutes at room temperature. Rinse the PDMS substrates three times with sterile PBS, each for five minutes.
Suspend human A549 lung cancer cells in Dulbecco's Modified Eagle Medium with 10%fetal bovine serum, and count the cells using a hemocytometer. Plate the cells at a seeding density of 2, 000 cells per square centimeter on the PDMS substrates. Culture the cells at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide for one day, and observe them afterwards.
The generated nanopattern on the PS plate and the thin film are shown here. The arrows indicate the polymer raise in the interstices of the stitched PDMS molds. These SEM images demonstrate that PDMS-working substrates are faithfully transferred from the electron beam lithography-written nanopattern by applying the stitch technique.
Representative SEM images of A549 cells grown on nanogratings and nanopillars are shown here. The cells show aligned cell morphology on the nanogratings, while the cells spread randomly on the nanopillars. After watching this video, you should have a good understanding of how to generate a large area of nanopatterned substrate by stitching multiple, small defined nanopatterned molds.
Don't forget that working with toluene and paraformaldehyde can be extremely hazardous, and always wear appropriate personal protection equipment while performing the procedures.
