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Lithography technique patterns cells and proteins

BioPhotonics
Jun 2008
Hank Hogan

By taking a cue from nature, a group of researchers has developed an inexpensive and robust technique that can pattern proteins and cells into ordered arrays on various substrates. Such arrays could be used in materials science, in synthetic chemistry, and in biology and synthetic biology as well as in medicine.

Research team leader Jane P. Bearinger already has some potential applications in mind. “My initial goals for the work are in tissue engineering and proteomic or genomic arrays towards systems biology work.”

TSLitho_1_3.jpg
Using a patterned transparent mask coated with a photosensitizer, researchers created adhesive substrate regions surrounded by a protein-resistant matrix (top). The technique can be used to generate cell adhesion in discrete areas (middle and bottom), resulting in an ordered pattern of cells. This could be useful in tissue engineering and in building proteomic or genomic arrays. Features as small as 200 nm now can be created with this technique. Courtesy of Jane Bearinger, Lawrence Livermore National Laboratory.

Bearinger is medical technology program leader in the applied physics and biophysics division of Lawrence Livermore National Laboratory in Livermore, Calif. The project was a collaboration between Lawrence Livermore and the Swiss Federal Institute of Technology in Lausanne.

The method is based on photocatalytic lithography and involves exposing a mask coated with photoreactive materials to light. The result is patterned surface chemistry that can be used to form arrays of biological materials. The model for the technique is photosynthesis, in which chemically active singlet oxygen can be produced by light-harvesting chlorophyll.

In a demonstration of the technique, the researchers started with silicon wafers. By using standard photolithography to pattern photoresist and then using reactive ion etching to transfer the pattern into the silicon, they created master masks with a desired pattern of ridges and valleys. This stage was followed by removal of the photoresist and by oxidation of the surface. From the silicon, they created polydimethylsiloxane (PDMS) masks by pouring the PDMS prepolymer over the silicon, curing it and then peeling it off. Because PDMS is transparent, the patterned polymer served as a master through which they could shine light.

They coated the masters with a photosensitizer and brought the PDMS into contact with a substrate fabricated out of multiple layers. The substrates had a silicon base topped by silicon dioxide, with a coating of allyltrichlorosilane over everything. They exposed the structures to light for about 10 s, causing the allyltrichlorosilane in contact with the photosensitizer on the ridges of the master to be oxidized. They used a variety of light sources, including a 480-nm blue Lumex LED, a 660-nm Superbright red LED and a Greenspot UV Source light.

Bearinger characterized the photocatalytic process as being quite fast and easy as well as flexible in terms of photosensitizers and light sources that can be used. “Many photosensitizers work, and the process even works with an LED flashlight purchased at a hardware store, which is both low-energy and inexpensive.”

After the exposure was completed, the researchers removed the master, leaving behind a negative. They grafted a thin acrylamide hydrogel polymer layer to the remaining allyltrichlorosilane, creating a nonfouling interpenetrating network. They noted that a variety of polymers could have been used.

They demonstrated that the patterned substrate was capable of biomolecular adsorption by testing it with fluorescently tagged proteins, with fluorescein isothiocyanate-labeled neutravidin and with HeLa cells. In one case, they created miniature uppercase letter L’s and in another, the word “cell,” spelling it out with 200-μm-wide letters. The HeLa cells were viable for a number of days after adsorption, they noted. The work is described in the May 6, 2008, issue of Langmuir.

They acquired images of these fluorescent patterns using a Nikon camera mounted on a Nikon microscope. With a Veeco atomic force microscope, they measured the linewidth of the logo components to be 4 μm, with a hydrogel height of about 20 nm.

The resolution achievable with the technique is a function of the diffusion of the radical oxygen species in the material being oxidized and of the resolution of the mask. Theoretically, the limit for the former is about 50 nm. However, in practical terms, the current PDMS masks impose a higher limit, Bearinger said.

As for the future, plans call for improving the mask technology so that it becomes possible to do nanoscale array work. Once that is done, the technique may be used for nanoscale ligand research or for proteomics or genomics investigations.

Contact: Jane P. Bearinger, Lawrence Livermore National Laboratory, Livermore, Calif.;
e-mail: bearinger1@llnl.gov.


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