Selective Attachment of Mammalian Cells and Polystyrene Microbeads to Functional Amine Patterns Defined by Perfluoroalkyl Silane Surfaces

Abstract

Surface modification technologies have been widely utilized for the investigation of the effect of surface properties on selective attachment of cells and cell functions. Recently, hydrophobic surfaces modified with organosilanes such as dichlorodimethyl silane and perfluoroalkyl silane have been shown to form a resistant barrier for cell attachment owing to their low surface energy. Moreover, they provide a chemically inert background and prevent the cross-contamination of liquid droplets on different spots. These characteristics have the potential to be utilized in the application of spot synthesis and array-type cell-based assays. To date, there have been a few reports on the formation of amine patterns defined by hydrophobic silane layers for the spatial organization of cells. However, several problems, such as the use of the limited types of organosilane for the cell-adhesive patterns, complicated deep UV irradiation for the patterning, and possible contamination of patterns from organosilane coupling still remain to be overcome. Therefore, it is necessary to develop a new patterning technique that extends the versatile surface chemistry and eliminates the contamination problem. Here, we present a novel and simple surface modification method for preparing amine-spacer patterns defined by perfluoroalkyl silane layers and their feasibility for the directed attachment of human retinal pigment epithelial cell line (ARPE-19) and functional polystyrene microbeads. In particular, we have devoted special efforts to solving the contamination problem, which is generated from the conventional photolithography and organosilane coupling method. As illustrated in Figure 1, our strategy is based on the two-step silanization with a simple photoresist (PR) process and then subsequent diamine coupling. To simplify the PR lift-off process, we have designed a home-made rubber stamp for PR printing directly on the substrate in a short time without any further treatment. Formation of isocyanate layers using isocyanatopropyl triethoxysilane (IPTS) on the substrates makes it possible to introduce a variety of amino functional spacers on the surface via urea linkages. In this study, we employed hydrophilic diamine spacers such as 4,7,10-trioxa-1,13-tridecanediamine (TEGdiamine) and Jeffamine ED-600 (PEGdiamine) for the creation of amino-terminal groups that can serve as charged domains promoting cell adhesion, as well as the starting point for the versatile surface modification. Here, we investigated two methods for preparing the fundamental amine-spacer patterns defined by the perfluoroalkyl silane. As illustrated in Figure 1 (Method 1), the PR was patterned on the acid-treated glass substrate by stamping. Thereafter, perfluoroalkyl silane layers (1H,1H,2H,2H-perfluorodecylmethyldichlorosilane, 17F) were formed on the exposed glass surface of the patterned substrates. Subsequently, after the removal of the PR on the substrates with acetone, the substrates were reacted with IPTS to produce patterned reactive sites. However, we found that the 17F layers were contaminated by IPTS coupling and the following amination process, so that substrates were no longer resistant to cell adhesion. In general, functional silane compounds having