Patterned Biofunctional Poly(acrylic acid) Brushes on Silicon Surfaces Rong Dong, ²,‡ Sitaraman Krishnan, ‡ Barbara A. Baird, ² Manfred Lindau, § and Christopher K. Ober* ,‡ Departments of Chemistry and Chemical Biology, Materials Science and Engineering, and Applied and Engineering Physics, Cornell University, Ithaca, New York 14853 Received May 6, 2007; Revised Manuscript Received July 11, 2007 Protein patterning was carried out using a simple procedure based on photolithography wherein the protein was not subjected to UV irradiation and high temperatures or contacted with denaturing solvents or strongly acidic or basic solutions. Self-assembled monolayers of poly(ethylene glycol) (PEG) on silicon surfaces were exposed to oxygen plasma through a patterned photoresist. The etched regions were back-filled with an initiator for surface- initiated atom transfer radical polymerization (ATRP). ATRP of sodium acrylate was readily achieved at room temperature in an aqueous medium. Protonation of the polymer resulted in patterned poly(acrylic acid) (PAA) brushes. A variety of biomolecules containing amino groups could be covalently tethered to the dense carboxyl groups of the brush, under relatively mild conditions. The PEG regions surrounding the PAA brush greatly reduced nonspecific adsorption. Avidin was covalently attached to PAA brushes, and biotin-tagged proteins could be immobilized through avidin-biotin interaction. Such an immobilization method, which is based on specific interactions, is expected to better retain protein functionality than direct covalent binding. Using biotin-tagged bovine serum albumin (BSA) as a model, a simple strategy was developed for immobilization of small biological molecules using BSA as linkages, while BSA can simultaneously block nonspecific interactions. 1. Introduction The ability to pattern biomolecules, especially proteins, onto a substrate is important for a variety of biological studies and applications including biosensors, studies of cell-surface interac- tions, cell patterning, and the like. 1,2 Though concentrated efforts have been made on protein patterning, many methods are based on nonspecific physical adsorption on hydrophobic surfaces, wherein the proteins tend to unfold and partially denature 3 upon adsorption. The complexity of creating patterned substrates that combine self-assembled monolayers (SAMs) bearing functional groups to immobilize proteins with SAMs that are resistant to nonspecific protein adsorption leads to difficulties in effective surface construction. Veiseh et al. have created patterned surfaces containing both carboxylic acid groups and poly- (ethylene glycol) (PEG) and used the surface for cell patterning studies. 4,5 In their work, it was necessary to use a gold-patterned silicon substrate and a combination of thiol-gold and silane chemistry to create the chemically patterned substrates. While the formation of SAMs of ω-mercapto carboxylic acids on gold- covered substrates is widely reported, 6-9 reports on direct functionalization of a silicon surface with carboxyl-terminated alkylchlorosilanes (or alkoxysilanes) are relatively rare. Some of the advantages of silicon surfaces are that they are inexpen- sive, molecularly flat, form thermally stable siloxy linkages with organosilanes, and are compatible with the well-established microfabrication techniques of the electronic industry. However, the fact that carboxyl-terminated alkylsilanes (which are required to form SAMs on silicon surfaces) are difficult to synthesize may have limited their use. 10 Moreover, strong interactions between the carboxyl head group of the silane and the silanol groups on the surface may complicate the process of SAM formation. To avoid this complication, vinyl-, carboalkoxy-, or bromo-terminated alkylsilanes have been converted to carboxylic acids after self-assembly and reaction with the surface silanol groups. 11-13 Here we report the synthesis of poly(acrylic acid) (PAA) brushes by the atom transfer radical polymerization (ATRP) of sodium acrylate in aqueous media to generate carboxylic acid groups on a silicon surface. Besides providing a high surface density of -COOH groups for protein immobilization, the PAA brushes are expected to impart biocompatibility 14 and are more robust and self-healing toward defects than carboxyl-terminated SAMs. In comparison to carboxyl-terminated SAMs, PAA brushes have significantly higher protein-binding capacities due to high concentrations of -COOH groups at the brush inter- face. 15 Our synthetic methods enable the direct and efficient formation of PAA brushes in contrast to prior studies 15 that involved hydrolysis of poly(tert-butyl acrylate) brushes by means of strong acid catalysts and the use of potentially undesirable organic solvents. We also describe the patterning of these PAA brushes using optical lithography as an effective alternative to microcontact printing. The creation of a patterned surface using multicom- ponent, microcontact printing (μCP) has become a widely used method. Usually, one compound is printed onto a substrate, and the patterned substrate is back-filled with another compound. The substrate used in μCP is often gold and not silicon, pro- bably because the technique is more suited for thiol chemistry than the moisture-sensitive silane chemistry. In addition, depending on the nature and roughness of the stamp and substrate, defects of the monolayer are difficult to avoid in a large patterned area. * Author to whom correspondence should be addressed. Phone: (607) 255-8417. Fax: (607) 255-2365. E-mail: cober@ccmr.cornell.edu. ² Department of Chemistry and Chemical Biology. ‡ Department of Materials Science and Engineering. § Department of Applied and Engineering Physics. 3082 Biomacromolecules 2007, 8, 3082-3092 10.1021/bm700493v CCC: $37.00 © 2007 American Chemical Society Published on Web 09/20/2007