Patterning of functional antibodies and other proteins by photolithography of silane monolayers.

Abstract

We have demonstrated the assembly of twodimensional patterns of functional antibodies on a surface. In particular, we have selectively adsorbed micrometer-scale regions of biotinylated immunoglobulin that exhibit specific antigen binding after adsorption. The advantage of this technique is its potential adaptability to adsorbing arbitrary proteins in tightly packed monolayers while retaining functionality. The procedure begins with the formation of a self-assembled monolayer of n-octadecyltrimethoxysilane (OTMS) on a silicon dioxide surface. This monolayer can then be selectively removed by UV photolithography. Under appropriate solution conditions, the OTMS regions will adsorb a monolayer of bovine serum albumin (BSA), while the silicon dioxide regions where the OTMS has been removed by UV light will adsorb less than 2% of a monolayer, thus creating high contrast patterned adsorption of BSA. The attachment of the molecule biotin to the BSA allows the pattern to be replicated in a layer of streptavidin, which bonds to the biotinylated BSA and in turn will bond an additional layer of an arbitrary biotinylated protein. In our test case, functionality of the biotinylated goat antibodies raised against mouse immunoglobulin was demonstrated by the specific binding of fluorescently labeled mouse IgG.We are interested in the development and application of techniques for protein patterning, especially for in vitro studies of protein function and for applications outside of the biological context (e.g., for artificial biomineralization and in biochemical sensors). In this paper we describe a general technique for producing two-dimensional patterns of functional protein on silicon dioxide surfaces by using a combination of self-assembled monolayers (SAMs) of alkyl silanes, albuminalkyl adsorption, and biotin-avidin interactions. We illustrate the technique by generating two-dimensional patterns of functional goat antibodies raised against mouse immunoglobulins (GAM).Our procedure begins with the formation of an noctadecyltrimethoxysilane (OTMS) ¶ SAM on a sio2 substrate. SAMs are increasingly important tools in the micro-and nanolithographic patterning of organic and inorganic materials. They have recently been used for selected area polypeptide synthesis (1), as barriers to chemical etching (2, 3), for the formation of microcrystals (4, 5) or microelectrodes (4), for creation of electrically conducting polypyrrole circuitry (6), for patterned water condensation (4), and for adhesion of cells (7-10) and proteins (8,(11)(12)(13)(14)(15)(16)(17). OTMS is one of the most widely used of the alkyl silanes for producing hydrophobic SAMs on SiO2. The SAM can be selectively removed from the surface by ultraviolet (UV) exposure through a lithographic mask. The difference in adsorption of bovine serum albumin (BSA) to the exposed and unexposed regions of the surface creates a two-dimensional pattern of BSA with a surface coverage "50 times greater on the OTMS than on the exposed sio2. If the BSA is tagged with biotin, the p...

Protein Adhesion on SAM Coated Semiconductor Wafers: Hydrophobic versus Hydrophilic Surfaces

“…Good quality SAMs result from a low siloxane polymerization rate in solution relative to SAM formation on the substrate. Trialkoxysilanes, with their relatively slow hydrolysis rates in solution, are excellent reagents for high quality SAM formation on silicon surfaces, exhibiting little sensitivity to the amount of trace water content in solution (35,36).…”

Infrared light induced patterning of proteins on ppNIPAM thermoresponsive thin films: a “protein laser printer”

“…Proteins in micro-arrays are typically deposited with techniques based on robotic contact printing [1], ink-jetting [3], photolithography [4], and dip-pen lithography [5,6]. High speed, high density, large array row and column count, large number of proteins, and easy reconfigurability are all important factors for the preparation of protein arrays.…”

Fabrication of Protein Microarrays for Immunoassay Using the Electrospray Deposition (ESD) Method.

“…Methods enabling active protein film formation are rapidly developing and there are now many different types. For example, proteins have been deposited using photolithography and lift-off techniques (Pritchard et al, 1995;Mooney et al, 1996), a robotic micromanipulation system including ink jetting (Newman et al, 1992), a stamp microcontact printing method (Bernard et al, 1998), and electrospray deposition techniques (Morozov and Morozova, 1999). Among these methods, the electrospray deposition (ESD) method is the most significant as it allows the spontaneous deposition of many identical dots, it has a remarkable spatial resolution and it overcomes limitations like low rates of deposition, low efficiency of substance transfer, and cross-contamination.…”