Micropatterning of Nanoengineered Surfaces to Study Neuronal Cell Attachment in Vitro J. Shaikh Mohammed, † M. A. DeCoster, ‡ and M. J. McShane* ,†,§ Institute for Micromanufacturing, Louisiana Tech University, Ruston, Louisiana, Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana, and Biomedical Engineering Program, Louisiana Tech University, Ruston, Louisiana Received March 7, 2004; Revised Manuscript Received May 17, 2004 Methods for producing protein patterns with defined spatial arrangement and micro- and nanoscale features are important for studying cellular-level interactions, including basic cell-cell communications, cell signaling, and mechanisms of drug action. Toward this end, a straightforward, versatile procedure for fabricating micropatterns of bioactive nanofilm coatings as multifunctional biological testbeds is demonstrated. The method, based on a combination of photolithography and layer-by-layer self-assembly (LbL), allows for precise construction of nanocomposite films of potentially complex architecture, and patterning of these films on substrates using a modified lift-off (LO) procedure. As a first step in evaluating nanostructures made with this process, “comparison chips,” comprising two coexisting regions of square patterns with relevant proteins/polypeptides on a single substrate, were fabricated with poly(diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Using neuronal cells as a model biological system, comparison chips were produced with secreted phospholipase A 2 (sPLA 2 ), a known membrane-active enzyme for neurons, for direct comparison with gelatin, poly-l-lysine (PLL), or bovine serum albumin (BSA). Fluorescence microscopy, surface profilometry, and atomic force microscopy techniques were used to evaluate the structural properties of the patterns on these chips and show that the patterning technique was successful. Preliminary cell culture studies show that neurons respond and bind specifically to the sPLA 2 enzyme embedded in the polyelectrolyte thin films and present as the outermost layer. These findings point to the potential for this method to be applied in developing test substrates for a broad array of studies aimed at identifying important biological structure-function relationships. Introduction Bio-active surfaces are continuously being investigated to use their applications for a vast range of scientific fields. The ability to engineer and control the interactions of cells with biomaterials is critical for fundamental cell biology studies, 1 medical implants, and functional biomaterial scaf- folds for tissue engineering, as well as for the development of cell integrated biochips used in cell-based sensors and “lab-on-a-chip” bioanalytical systems. 2 Physicochemical parameters such as hydrophobicity, surface charge, molecular and elemental composition, and roughness are known to affect protein adsorption and, consequently, cellular adhe- sion. 3 The controlled attachment of desired cell populations using specific cell-signaling molecules or adhesion ligands in precisely engineered geometries will enable production of truly bioactive systems with a broad spectrum of applica- tions. 2,4,5 The primary goal of this work is to develop a versatile yet precise process for engineering multiprotein micropatterns that can be used as biological testbeds for basic biological studies in cell signaling. As a model, a system allowing investigation into the differential role of proteins in signaling for neuronal cells was selected. To be able to create substrates, it is desirable to be able to place organic thin films with differing functionality next to each other on the surface. For example, true tissue engineering often requires patterning of multiple cell types on different areas of a substrate in order to build defined architecture into multi- functional tissues. The cartoon in Figure 1 illustrates the lateral definition of micropatterns with varying functionality placed next to each other. The micropatterns also have a varied vertical configuration. Organic thin films have been exploited for biomaterial applications due to their useful properties, including their light weight, ease of functionalization, processability, and flexibility. 6 Self-assembled monolayers (SAMs) and Lang- muir-Blodgett (LB) films are well-studied for these ap- plications. The ionic LbL assembly technique, introduced to practice by Decher in 1991, is a recent development in this field. 7,8 This versatile technique, based on the alternate deposition of polyanions and polycations from dilute aqueous solutions on surfaces of any size, shape, or material, produces nanoscale films with highly tunable architectures and proper- ties, including film thickness, uniformity, composition, * To whom correspondence should be addressed. Mailing Address: Institute for Micromanufacturing, 911 Hergot St., Ruston, LA 71272. Tel: 318-257-5112. Fax: 318-257-5104. E-mail: mcshane@coes.latech.edu. † Institute for Micromanufacturing, Louisiana Tech University. ‡ Neuroscience Center, Louisiana State University Health Sciences Center. § Biomedical Engineering Program, Louisiana Tech University. 1745 Biomacromolecules 2004, 5, 1745-1755 10.1021/bm0498631 CCC: $27.50 © 2004 American Chemical Society Published on Web 07/03/2004