In this paper we describe a method for creating multifunctional glass surfaces presenting discrete patches of different proteins on an inert PEG-functionalized background. Microcontact printing is used to stamp the substrate with octadecyltrichlorosilane to define the active regions. The substrate is then back-filled with PEG-silane {[[2-methoxypoly(ethyleneoxy)]propyl]trimethoxysilane} to define passive regions. A microfluidics device is subsequently affixed to the substrate to deliver proteins to the active regions, with as many channels as there are proteins to be patterned. Examples of trifunctional surfaces are given which present three terminating functional groups, i.e., protein 1, protein 2, and PEG. These surfaces should be broadly useful in biological studies, as patch size is well established to influence cell viability, growth, and differentiation. Three examples of cellular interactions with the surfaces are demonstrated, including the capture of cells from a single cell suspension, the selective sorting of cells from a mixed suspension, and the adhesion of cells to ligand micropatches at critical shear stresses. Within these examples, we demonstrate that the patterned immobilized proteins are active, as they retain their ability to interact with either antibodies in solution or receptors presented by cells. When appropriate (e.g., for E-selectin), proteins are patterned in their physiological orientations using a sandwich immobilization technique, which is readily accommodated within our method. The protein surface densities are highly reproducible in the patches, as supported by fluorescence intensity measurements. Potential applications include biosensors based on the interaction of cells or of marker proteins with protein patches, fundamental studies of cell adhesion as a function of patch size and shear stress, and studies of cell differentiation as a function of surface cues.
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