The overall goal of the following experiment is to develop a two dimensionally patterned substrate for the control of protein absorption and cell growth. This is achieved by using photolithography to fabricate a patterned master, which is used to produce A-P-D-M-S stamp for micro contact printing. As a second step, the patterned gold substrate is prepared, which involves micro contact printing to produce a pattern with regions that absorb and resist protein.
Next protein and cells are added to the substrate in order to visualize the pattern and study. Cellular growth and behavior results are obtained that show good protein and cell confinement on these pattern substrates based on fluorescence and face contrast microscopy. The main advantage of this technique over other existing methods, such as MicroCon printing of proteins, is that the glycol terminated monolayer resist non-specific protein and cell absorption in the background of the pattern.
Generally, individuals new to this method will struggle because proper stamping technique must be optimized for each pattern printed. To begin preparation of the patterned master center, the silicon wafer on the spin coder, and during the initial step of the two cycle spin program, rinse the wafer with acetone. During the second step of the spin program, the acetone will evaporate leaving a clean, dry wafer at the beginning of the spin, apply photoresist to the wafer and then spin coat using the conditions described previously.
Soft bake the photo resist coated wafer at 110 degrees Celsius for two minutes using a high uniformity hot plate. Next photo pattern. The photo resist coated wafer using a mask aligner system and an appropriate mask.
First, place the substrate onto the vacuum truck of the mask aligner. Then insert the mask into the holder tray and turn on the vacuum to hold the mask in place to confirm good contact between the mask and substrate. Raise the substrate until it meets the mask.
Use the optics of the aligner to confirm that good contact has been made. Finally, turn on the lamp to expose the substrate to UV light through the lithographic mask. Develop the patterned wafer in developer for one minute 45 seconds with gentle agitation.
Rinse thoroughly with semiconductor grade, deionized water, and dry with a stream of nitrogen gas check pattern development using a microscope with a UV filter. Begin this procedure by preparing a 10 to one by weight resin hardener. Mixture of cigar 180 2 completely.
Cover the photo patterned wafer with the mixture in a disposable Petri dish in a vacuum desiccate Degas, the PDMS covered master until no bubbles are visible. After making sure that the master is at the bottom of the dish, allow the stamp to cure in an oven at 65 degrees Celsius for 1.5 hours. When the PDMS stamp is cured, cut the stamp out of the master and trim it to the appropriate size.
Store the stamp in a covered container feature side up to protect it from dust and debris. To prepare the substrates for metal deposition first treat round glass cover slips with oxygen plasma for 10 minutes. Then rinse the cover slips twice with deionized water drying with the stream of nitrogen gas between the rinses after rinsing with water, rinse twice with ethanol again drying with the stream of nitrogen gas between rinses.
Using a multip pocket electron beam deposition system, deposit 50 angstroms titanium followed by 150 angstroms gold. Do not fent the evaporator between deposition of the titanium layer and gold layer. Rinse the PDMS stamp with ethanol and dry thoroughly.
With nitrogen gas, apply stamping solution to the stamp dropwise until fully coated. Dry the stamp thoroughly with nitrogen gas. The micro contact printing of HEXA Decane file depends on the features of the PDMS stamp and is one of the most difficult aspects of this procedure.
Successful stamping requires development of a good technique that applies even pressure. During stamping. For conventional micro contact printing in air, gently press the stamp onto the gold substrate and allow the monolayer to form for 15 seconds.
Alternatively, for patterns comprised of very small features and high aspect ratios, place the gold substrate into a Petri dish containing 18.2 mega om deionized water, ensuring the substrate is submerged. Then gently press the stamp onto the gold substrate and allow the mono a layer to form for 15 seconds. Next, rinse the stamped substrate twice with ethanol drying with nitrogen gas.
After each rinse, place the substrate in a Petri dish and cover the substrate with back filling solution. Seal the dish with param to prevent evaporation. Allow the background monolayer to form in the dark for 12 to 14 hours.
Finally, remove the patterned cover slip from the back filling solution and rinse twice with ethanol drying with nitrogen gas after each rinse. To apply protein to the patterned cover slip, place the cover slip in either a small Petri dish or a cell culture chamber Cover with 500 microliters to one milliliter of delcos phosphate buffered saline. The DPBS must completely cover the substrate during protein incubation.
To ensure even protein coverage, add a concentrated solution of protein to the DPBS and mix the solution by pipetting several times. Incubate the protein mixture with the substrate at 37 degrees Celsius for one hour. After the incubation, rinse the substrate thoroughly with DPBS to remove unbound protein, taking care not to dry the substrate or bring it through the air water interface.
After three rinses aspirate the DPBS. Then add approximately 500 microliters of complete cell growth media to maintain a wet substrate. Replace the growth media used to rinse the substrate.
With fresh media, the substrate is now ready for plating with cells. Typically 30 to 200 cells per square millimeter are plated onto the substrate. This image shows A-P-D-M-S stamp visualized under a microscope by placing the stamp feature side down on a glass cover slip, the scale bar is 100 micrometers.
Proper stamping results in a sharp, clear protein pattern that can be visualized by the application of a fluorescently labeled protein such as LOR 6 47 labeled fibronectin. The confinement of chk one cells to the protein pattern is shown here. Scale bars are 100 micrometers in these images.
Alternatively, immunohistochemistry may be used to visualize the protein pattern after SU fixation. Here, LOR three 50 conjugated anti laminate antibody is used to visualize patterned laminate seeded with E 18 mouse hippocampal neurons shown at four days in vitro E 18 mouse hippocampal neurons stained with MIT tracker red five 80 show that cell growth is well confined to the protein pattern. While this technique is easily mastered, several common problems may arise as illustrated by these pattern substrate preparations visualized by LOR 6 47 conjugated fibronectin absorption.
The application of protein without sufficient mixing of the concentrated protein solution in the DPBS can lead to uneven protein patterns. Improper stamping can lead to partial pattern transfer or stamp collapse. In addition, exposing the pattern substrate containing absorbed protein to air can disrupt the monolayer causing decreased resistance.
In the background, submerged patterning can produce patterns with small features that are difficult to print by conventional micro contact printing and air. These two images show different regions of the same pattern printed with the same PDMS stamp in air or deionized water support lines are shown in the image on the left to demonstrate that only the small features of the pattern have failed to print successfully. Scale bars are 20 micrometers Once mastered, this technique can be done in 18 to 24 hours if it is performed properly.
After watching this video, you should have a good understanding of how to fabricate a pattern substrate. Produce A-P-D-M-S stamp. Utilize micro contact printing to prepare a pattern gold substrate and visualize this pattern using protein and cells.
