Microcontact Printing of Proteins for Cell Biology

Published 12/05/2008
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Summary

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

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Shen, K., Qi, J., Kam, L. C. Microcontact Printing of Proteins for Cell Biology. J. Vis. Exp. (22), e1065, doi:10.3791/1065 (2008).

Abstract

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (http://gmwgroup.harvard.edu/) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning. An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Protocol

1. Preparation of solutions and materials

These steps should be carried out several days in advance.

  1. Glass coverslips. Coverslips were cleaned by immersion for 10 minutes into a solution of Linbro 7X detergent : water, mixed at a 1 : 3 ratio and heated, with stirring, until clear. Coverslips were rinsed extensively with deionized water, and then baked at 450°C for 6 hours. Loading coverslips into ceramic staining racks (see Reagents) simplified this process.
  2. Protein solution for stamping. Reconstitute fibronectin following the manufacturers instructions to a stock solution of 1 mg/ml concentration.

2. Cast stamps from the topological master

These steps can be carried out several days in advance. Store stamps pattern-up in a covered dish, such as a tissue culture dish.

  1. Remove loose dust from master using a stream of compressed, filtered air or inert gas.
  2. Place the master, patterned side up, in the bottom of a plastic dish just larger than the master. 60- or 100-mm tissue culture dishes are well suited for this purpose.
  3. In a polystyrene 50 ml centrifuge tube, combine the Sylgard components at a ratio of curing agent : elastomer base of 1 : 10 by weight. Mix thoroughly using a plastic device, such as a disposable pipette Prepare at least 0.2 ml of elastomer per square centimeter of dish area.
  4. Centrifuge the 50 ml tube at 300 G for 5 min to remove air bubbles.
  5. Pour the elastomer over the master, then place in a desiccator, under vacuum, for 30 minutes.
  6. Cure the elastomer in an oven at 65°C for at least 2 hours. Curing at higher temperatures and for longer times results in stiffer elastomer. Let cool to room temperature.
  7. Separate the sheet of stamps from the master.

3. Microcontact printing of fibronectin onto glass

  1. Cut out a single stamp. Stamps measuring 4 mm X 4 mm to 1 cm X 1 cm in area and 1 – 2 mm thick are easiest to start with. Place pattern side up on a glass slide or small plastic dish.
  2. Place stamp in a plasma cleaner, and process, under vacuum, for 30 seconds. A Harrick Scientific plasma cleaner (see Equipment), set at its highest output setting will render the PDMS surface hydrophilic. Longer times result in cracking of the elastomer.
  3. Dilute the fibronectin solution with deionized water to a stamping concentration of 50 µg/ml.
  4. Place a small drop (10 – 50 µl) of stamping solution on the stamp. It will spread across the hydrophilic surface. Add only enough solution that the drop covers the stamp, but does not run over the edges. Let protein adsorb to stamp for 5 minutes.
  5. Using a Kimwipe® or other clean paper tissue, wick off most of the protein solution from the stamp, without touching the patterned region.
  6. Dry the remaining solution from the stamp under a stream of clean, dry, inert gas, such as nitrogen.
  7. Using tweezers, remove the stamp from the glass slide, invert, and place in contact with the cleaned glass coverslip (the surface to be patterned). Place a weight on top to promote good contact. The specific weight that provides the best patterning is dependent on the stamp size and pattern; start with a 5 g weight, and adjust between stampings. Leave stamp in contact with surface for 1 minutes.
  8. Carefully disassemble the stack, and separate stamp from coverslip.
  9. Vigorously rinse the patterned coverslip in PBS, followed by deionized water, to remove protein that is not adsorbed to the surface. Dry coverslip under a nitrogen stream.

 Representative Results

Microcontact printing is a powerful process for patterning molecules on surfaces. This process has the ability to create features with dimensions ranging from tens of micrometers to hundreds of nanometers; in Fig. 1A, the logos on the left are each 200 µm in height, while the green spots illustrated in Fig. 1B are 1 µm in diameter and spaced at 4 µm intervals, measured center-to-center. Fig. 1B also illustrates a powerful property of microcontact printing, namely that it is and additive process. Multiple rounds of microcontact printing can be applied to a single surface to create multicomponent systems3.

Figure A Figure B

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Discussion

The microcontact printing process is conceptually simple and very robust, having been applied to patterning a wide range of molecules on a variety of substrates. However, this process remains something of an art. The specific geometry of the pattern to be created, protein to be patterned, applied weight, and coating/stamping conditions all affect the stamping quality. For example, too little weight, applied to large features, often results in gaps in the pattern as can be seen in the upper right logo of Fig. 1A. Conversely, too much weight will cause sagging and collapse of the stamp, resulting in unintended deposition of protein in regions between patterned features.As a second example, specific proteins (such as antibodies) pattern with better fidelity if the plasma treatment step is omitted, leaving the PDMS hydrophilic. The stamping of fibronectin onto glass is presented here as a starting point for such modifications and optimizations, demonstrating the basic techniques of this process.

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Disclosures

The authors have nothing to disclose.

Materials

Name Type Company Catalog Number Comments
Plasma Cleaner Harrick Scientific Products, Inc. PDC-32G
Desiccator Nalge Nunc international 5315-0150
PBS Reagent Invitrogen 10010-072
Protein labeling kit Reagent Invitrogen A30006
Fibronectin Reagent Sigma-Aldrich F2006
Staining rack Reagent Thomas Scientific 8542E40
Coverslips Reagent Fisher Scientific 12-544-12
Sylgard 184 Reagent Ellsworth Adhesives 184 Sil Elast Kit
Diffraction Grating Reagent Edmund Scientific 3040267

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References

  1. Chen, C. S. Geometric control of cell life and death. Science. 276, 1425-1425 (1997).
  2. Kumar, A., Whitesides, G. M. Features of Gold Having Micrometer to Centimeter Dimensions can be Formed Through a Combination of Stamping with an Elastomeric Stamp and an Alkanethiol "Ink" Followed by Chemical Etching. Applied Physics Letters. 63, 4-4 (1993).
  3. St. John, P. M. Preferential Glial Cell Attachment to Microcontact-printed Surfaces. Journal of Neuroscience Methods. 75, 171-171 (1997).
  4. Kam, L., Boxer, S. G. Cell adhesion to protein-micropatterned-supported lipid bilayer membranes. Journal of Biomedical Materials Research. 55, 487-487 (2001).
  5. Kung, L. A., Kam, L., Hovis, J. S., Boxer, S. G. Patterning Hybrid Surfaces of Proteins and Supported Lipid Bilayers. Langmuir. 16, 6773-6773 (2000).
  6. Shen, K., Thomas, V. K., Dustin, M. L., Kam, L. C. Micropatterning of costimulatory ligands enhances CD4+ T cell function. Proceedings of the National Academy of Sciences. 105, 7791-7791 (2008).
  7. Shi, P., Shen, K., Kam, L. C. Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro. Developmental Neurobiology. 67, 1765-1765 (2007).

Comments

6 Comments

  1. Excellent presentation, very useful technique thank you very much for posting. regards Swetha

    Reply
    Posted by: Anonymous
    December 10, 2008 - 6:44 AM
  2. Good work and excellent way of presenting the work.. Jatin

    Reply
    Posted by: Anonymous
    December 11, 2008 - 1:54 PM
  3. Can you mention the details about how you obtained the images in fig 1A? I am attempting to microstamp using a biotinylated thiol and hydroxyl thiol followed by labeling with Streptavidin Alexa 488.

    Thanks,
    Celso

    Reply
    Posted by: Celso P.
    June 24, 2013 - 6:30 PM
  4. Could you please clarify which temperature at which the coverslips should be baked after cleaning? The text reads 450 °C but the video and the video subtitles indicate 45 °C, though the furnace certainly looks capable of the higher temperature. What is the rationale behind the baking step?

    Reply
    Posted by: Tim S.
    November 30, 2013 - 2:16 AM
  5. Hi Tim-
    It should be 450 C. The goal of this step is to burn off any organic contaminants that are still on the substrate without melting the glass; don't go much higher than 450C for most coverslip material. This was done in the context of making barriers for supported lipid bilayers (please see work of Steve Boxer's lab at Stanford (Chemistry) for this background) which requires a very clean glass surface. Simple detergent wash or organic solvent wash would leave contaminants on the glass, including detergent that would not rinse off. The bake gets rid of this. Certainly, less stringent cleaning will work for other purposes, it really depends on your downstream application.

    Reply
    Posted by: Lance K.
    November 30, 2013 - 2:33 AM
  6. Excellent demonstration! One thing I am not sure is whether the surface of the coverslip need to be oxygen plasma treated to enhance the protein transfer efficiency. I was wondering do you have an answer to that. Thanks a lot!

    Reply
    Posted by: Yusheng S.
    February 11, 2014 - 12:47 AM

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