Antigen Binding Specificity of Antibodies Patterned by Microcontact Printing David J. Graber,* ,† Thomas J. Zieziulewicz, ‡ David A. Lawrence, ‡ William Shain, † and James N. Turner † Laboratories of Nervous System Disorders & Clinical and Experimental Endocrinology and Immunology, Wadsworth Center, Albany, New York 12201, and Department of Biomedical Sciences, State University at Albany, Albany, New York 12222 Received February 5, 2003. In Final Form: April 10, 2003 The ability to pattern functional biomolecules onto surfaces at the micrometer scale is critical to a large number of important biological methods. Since devices such as microarrays and biosensors, or surfaces for generation of complex cellular networks, require such “bioselective” surfaces, it is important to verify the functionality of the patterned biomolecules. Patterning of antibodies onto substrates by microcontact printing or soft lithography has been previously demonstrated, but the ability of these antibodies to selectively bind antigens has not been fully explored. This work re-examines the transfer of antibodies from elastomer stamps onto glass coverslips and documents the antigen-binding capabilities of these antibodies. Various microcontact print (MCP) applied antibodies are tested for selectivity in binding fluorescence-conjugated antigens or antigens detected with fluorescently labeled molecules, which are quantified by wide-field fluorescent microscopy. The fluorescence intensity of bound antigens is compared to that of MCP-applied controls, as well as to that of antibodies that were directly adsorbed to the glass substrates. The results indicate that MCP-applied antibodies, although they are less functional immunologically than are those that have been directly bath-adsorbed onto substrates, maintain their selective binding properties. I. Introduction The patterning of surfaces with biomolecules that provide highly specific cell-surface interactions on the micrometer scale is a powerful tool being applied to the study of cells. 1-5 Antibodies are an important class of molecules for many biological studies, and they have been patterned onto substrates by several methods, including hydrogel stamping, 6 direct microcontact printing, 7-11 and microfluidic devices. 12 Direct microcontact printing or soft lithography is an ideal patterning method, due to its ability to deposit proteins in unlimited patterns and to permit the printing of multiple proteins in individual patterns. 11 There have been numerous investigations of the ability of antibodies to be patterned onto a variety of substrates, including silanized and unsilanized glass, 7,11 chemically modified or gold-coated silicon, 9,10 polystyrene, 7 and silicon 8 via microcontact printing. Unmodified glass may be pre- ferable because some cellular experiments are performed on this substrate. Elastomer stamp properties have also been examined. Tan and co-workers found that the most efficient transfer occurred when the stamp was relatively more hydrophobic and the substrate was relatively more hydrophilic. 10 Unmodified elastomer stamps are hydro- phobic, whereas clean glass is hydrophilic and anionic. Antibodies have been printed in a wide variety of pat- terns, using variations of the microcontact technique, and their deposition has been evaluated by use of antibodies prelabeled with a fluorescent marker, 10 a labeled antibody after printing that binds specifically to the patterned antibody, 7-9,11 or atomic force microscopy. 7-9,11 The antigen- binding properties of microcontact print (MCP) applied antibodies have not been fully explored, although bacteria have been shown to bind to patterns of printed anti- Escherichia coli antibodies, 8,9 and Cornish and co-work- ers 13 recently reported specific antigen binding to MCP- applied antibodies on amine-reactive glass substrates. In the present study, antibodies have been analyzed for transferability and for their antigen-binding properties, after being MCP-applied onto glass substrates without prior chemical modification. These patterned antibodies are also compared to antibodies that were bath-adsorbed directly to a similar substrate, to determine whether loss of function occurs as a result of microcontact printing. II. Materials and Methods Stamp Production. Poly(dimethylsiloxane) (PDMS) elas- tomer stamps were fabricated by curing Sylgard 184 (Dow Corning) on a silicon wafer that contained a pattern generated in photoresist using standard photolithography. The cured elastomer was peeled off the master and cut into 1 × 1 cm squares. * To whom correspondence should be addressed. Tel: (518) 474- 2463. E-mail: djgraber7@aol.com. † Laboratory of Nervous System Disorders. ‡ Laboratory of Clinical and Experimental Endocrinology and Immunology. (1) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. (2) St. John, P. M.; Kam, L.; Turner, S. W.; Craighead, H. G.; Issacson, M.; Turner, J. N.; Shain, W. J. Neurosci. Methods 1997, 75, 171-177. 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