Functional Antibody Immobilization on 3-Dimensional Polymeric Surfaces Generated by Reactive Ion Etching Victor C. Rucker, †,# Karen L. Havenstrite, †,⊥ Blake A. Simmons, ‡ Shane M. Sickafoose, § Amy E. Herr, † and Rene ´e Shediac* ,| Biosystems Research Department, Nanoscale Science and Technology Department, Analytical Materials Science Department, and Microfluidics Department, Sandia National Laboratories, Livermore, California 94551 Received January 27, 2005. In Final Form: June 2, 2005 Reactive ion etching (RIE) was used to pattern antibodies onto the surfaces of polymer substrates. A low pressure, inductively coupled oxygen plasma was used to anisotropically etch 25-30 µm deep features into poly(methyl methacrylate) (PMMA), Zeonex, and polycarbonate (PC). Scanning electron microscopy and contact angle measurements show that the resulting surfaces exhibit significant microroughness and enhanced hydrophilicity. Fourier transform infrared spectroscopy suggests that, in addition to enhanced surface area, chemical modifications may contribute to antibody immobilization. Polyclonal antibodies preferentially bind to the etched areas in RIE-patterned PMMA and Zeonex substrates but localize in unetched regions of RIE-patterned PC surfaces. Simple immunoassays were performed to demonstrate a potential application for RIE-modified polymer surfaces. Antibodies specific for the capture of fluorescently labeled cholera toxin, S. aureus enterotoxin B, and B. anthracis protective antigen were immobilized onto etched PMMA surfaces and shown to specifically capture their labeled antigen from solution. This work demonstrates a potentially useful fabrication methodology for constructing antibody microarrays on plastic substrates. Introduction A powerful tool within the field of proteomics is the protein microarray. An extension of DNA microarrays used in genetic analyses, protein microarrays offer the possibility of rapid and reliable profiling of the expression levels and interactions between proteins involved in signaling and disease pathways 1,2 or other events of biological significance. 3 Advances within the area of protein microarray technology have generally focused on the development of novel substrates for immobilizing functional proteins, the miniaturization of the size of functional features one can print and interrogate on a slide surface, and strategies for detecting specific protein- protein interactions while inhibiting nonspecific protein- protein or protein-substrate interactions. 4 Proteins are commonly arrayed (printed) onto planar glass substrates that have been surface-derivatized with chemical functionalities such as epoxides or aldehydes to immobilize the protein through covalent bonds formed between nucleophiles on the protein and electrophiles on the slide surface. Detection of an interaction between the immobilized protein, commonly an antibody, and its partner in solution, e.g., capture of a fluorescently labeled antigen from solution, is usually achieved by spatially correlating the fluorescent signal with the known location of the printed capture reagent. Although printed protein (micro)arrays can be quickly and easily generated, suc- cessful implementation necessitates modified glass slides, a contact or piezoelectric dispenser capable of depositing small volumes, and robust handling protocols to ensure good signal-to-noise ratios. 5 Polymeric supports may provide a suitable and versatile alternative to the use of chemically sophisticated glass substrates. 6 Although often unsuitable for short-wave- length fluorescence-based detection strategies due to intrinsic fluorescence or opaqueness, many polymers, such as Zeonex, poly(methyl methacrylate), and polycarbonate, do not suffer from these detractions and are suitable for use in longer-wavelength (λ > 520 nm) fluorescence-based detection schemes. Previous applications of polymeric supports for protein immobilization have relied on surface functionalization to enhance the protein capture and retention properties of the polymer, most notably by polymer grafting 7 or other modification to generate surface-localized carboxylic acid groups that can be activated and coupled to the amines of proteins for immobilization. 8,9 The enzyme-linked immunosorbent assay (ELISA), in which antibodies or antigens are chemisorbed onto polystyrene surfaces, is perhaps the most common application of polymeric supports in this regard. An important trend in protein microarray development is the move from planar substrates to 3-dimensional supports. 10-12 Rather than immobilize only in the two dimensions available on a planar substrate, the addition * To whom correspondence should be addressed. Address: Microfluidics Department, Sandia National Laboratories, PO Box 969, MS 9292, Livermore, CA 94551. E-mail: rshedia@sandia.gov. Phone: 925-294-4504. † Biosystems Research Department. ‡ Nanoscale Science and Technology Department. § Analytical Materials Science Department. | Microfluidics Department. ⊥ Present address: Stanford University, Stanford, CA 94539. # Present address: Gilead Sciences, Foster City, CA 94404. (1) Kramer, A.; Feilner, T.; Possling, A.; Radchuk, V.; Weschke, W.; Burkle, L.; Kersten, B. Phytochemistry 2004, 65, 1777-1784. (2) Zhu, H.; Bilgin, M.; Snyder, M.; Richardson, C. C. Annu. Rev. Biochem. 2003, 72, 783-812. (3) Haab, B. B. Proteomics 2003, 3, 2116-2122. (4) Angenendt, P.; Glokler, J.; Murphy, D.; Lehrach, H.; Cahill, D. J. Anal. Biochem. 2002, 309, 253-260. (5) Heller, M. J.; Yarmush, M. L.; Diller, K. R.; Toner, M. Annu. Rev. Biomed. Eng. 2002, 4, 129-153. (6) Vaidya, A. A.; Norton, M. L. Langmuir 2004, 20, 11100-11107. (7) Lee, S.-D.; Hsiue, G.-H.; Chang, P. C.-T.; Kao, C.-Y. Biomaterials 1996, 17, 1599-1608. (8) Kato, K.; Ikada, Y. Biotechnol. Bioeng. 1995, 47, 557-566. (9) McCarley, R. L.; Vaidya, B.; Wei, S.; Smith, A. F.; Patel, A. B.; Feng, J.; Murphy, M. C.; Soper, S. A. J. Am. Chem. Soc. 2005, 127, 842-843. (10) Charles, P. T.; Goldman, E. R.; Rangasammy, J. G.; Schauer, C. L.; Chen, M. S.; Taitt, C. R. Biosens. Bioelectron. 2004, 20, 753-764. 7621 Langmuir 2005, 21, 7621-7625 10.1021/la050251r CCC: $30.25 © 2005 American Chemical Society Published on Web 07/22/2005