Multifunctional Surfaces with Discrete Functionalized Regions for Biological Applications

Abstract

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.

Sialyl Lewisx-dependent binding of human monocyte-derived dendritic cells to selectins

“…Octadecyltrichlorosilane treated glass slides were first incubated with anti-human IgG Fc and then with the appropriate P-, L- or E-selectin IgG Fc chimera, and blocked with 1% BSA, as described in [19]. Mo-DCs (1×10 6 cells/mL) suspended in D-PBS buffer containing Ca 2+ /Mg 2+ and 0.1% BSA were perfused over immobilized P-, L- or E-selectin-coated slides [20] at 1 dyn/cm 2 using a microfluidic channel affixed to the selectin-coated slides.…”

Multicolor microcontact printing of proteins on nanoporous surface for patterned immunoassay

“…Since then, lCP has been extended to incorporate many applications, including the patterning of functional proteins for immunoassays and biosensors (Bernard et al 2000;Pattani et al 2008). Furthermore, to enhance the diagnostic power of immunoassays, multiple proteins can be patterned on the same substrate and has been demonstrated in a variety of ways (Inerowicz et al 2002;Crozatier et al 2006;Ghosh et al 2008). The ability to print multiple proteins on a single substrate and on such a small scale is important in creating an efficient point of care detection and analysis systems.…”

Time correlated fluorescence characterization of an asymmetrically focused flow in a microfluidic device

“…Flow cytometry in general (Ghosh et al 2008;Srisa-Art et al 2009), and hydrodynamic focusing methods in particular, are efficient techniques to divide, count, or even sort microparticles or different cell subpopulations (Liu et al 2009;Chen et al 2009). Cells and microparticles manipulation and separation is an essential requirement in many biological ) and analytical applications.…”