Patterned Immobilization of Biomolecules
for Study of Cell Behavior

3T3 fibroblast cells cultured on gelatin that has been immobilized in a UMN pattern on an agar surface. The cells have been stained for viability and visualization with fluorescein diacetate (green cells; live) and ethidium bromide (red nuclei; dead). The vertical paths of the letters are approximately 50 microns wide.

Biomolecule Immobilization

Patterned immobilization is a useful method for looking at the effects of biological molecules on cell morphology and migration. In our laboratory, we are actively researching and developing technology towards this goal. We have developed a technique in which molcules of interest are immobilized onto surfaces using hetero-bifunctional photo-reactive crosslinking agents. The crosslinking agents used contain one reactive group that forms a covalent bond through primary amine groups in a protein or peptide. The other group on the crosslinking molecule is photo-reactive, capable of reaction with other molecules upon exposure to light with sufficient energy. We are able to synthesize these crosslinkers such that the spacers between the reactive groups have the desired chemical and physical properties, such as length and hydrophilicity.

Proteins, such as gelatin and collagen, and peptides containing the biologically active sequences RGD or IKVAV are first covalently modified through the amine-reactive group of the crosslinker. Surfaces coated with these modified proteins and peptides are irradiated using laser light and a motorized stage. The photo-reactive groups, when activated with the laser, react with the surface, linking the proteins to the surface through a covalent bond. Patterns are created by moving the sample through the laser beam in the desired pattern. With the motorized stage we can move the sample within the path of the laser beam to produce the desired patterns. Cell attachment is then increased on those areas which have immobilized adhesion proteins or peptides.

FS4 cells inoculated onto a surface on which an RGD-containing peptide has been immobilized in a pattern using the described technique. Cells are well spread and orient along the ladder-like pattern generated. Patterns such as this allow for the confinement of cells to specific regions of the surface because the cells attach poorly to those regions not modified with the peptide.

Pattern Visualization

Gradients are of interest to us because of their possible role in cell haptotaxis. This laser-induced immobilization technique is useful for creating immobilized gradients. By varying the speed of the motorized stage during the laser irradiation, we can control the total amount of laser light which strikes a given area of the surface. Higher laser dosage corresponds to a higher concentration of immobilized biomolecule.

This images shows a fluorescent pattern produced by irradiating a polystyrene surface with the laser for long periods of time. This type of fluorescence study allows us to characterize the photoimmobilization system without the use of a photolinker. The gradient pattern shown is 250 microns x 250 microns square, with a change in laser dosage of one order of magnitude over the horizontal distance.

Line patterns of the type shown below are also of great interest to us. With this pattern-making system, we are able to produce lines of immobilized peptide that are on the order of 10-20 microns wide. The ability to produce these lines allows us to study phenomena like neurite outgrowth in a well controlled environment.

This work was funded by NSF.

We also have the ability to directly image some patterns via antibody labeling. This image shows an IKVAV-containing peptide pattern consisting of two sets of parallel lines. The lines are spaced 50 microns apart and the different sets intersect at a 30 degree angle. DRG neurites grown on such a pattern tend to follow the peptide region, corresponding to the bright green fluoresence.

Selected Publications:

Hypolite, C.L., McLernon, T.L., Adams, D.N., Chapman, K.E., Herbert, C.B., Huang, C.C., Distefano, M.D., and Hu, W.S. Formation of Microscale Gradients of Protein Using Heterobifunctional Photolinkers. Bioconjugate Chemistry, 8(5):658-663

Herbert, C.B., McLernon, T.L., Hypolite, C.L., Adams, D.N., Pikus, L., Huang, C-C., Fields, G.B., Letouneau, P.C., Distefano, M.D., Hu, W-S. Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates. Chemistry and Biology, 4(10):731-737

Researchers:

Collaborating Professors:

Mark D. Distefano
Professor, Department of Chemistry, University of Minnesota

distefan@chem.umn.edu

Paul C. Letourneau
Professor, Department of Neuroscience, University of Minnesota

letour@lenti.med.umn.edu

Current Graduate Students:

Edmund Kao
B. S., Chemical Engineering, Rice University, 1996

kao@cems.umn.edu

Undergraduate Researchers:

Clarice Policarpio
Dennis Royzenfeld

Last updated: November, 2000

Patterned Immobilization of Biomolecules for Study of Cell Behavior