Direct Protein Microarray Fabrication Using a Hydrogel “Stamper”

Abstract

Micropatterned arrays of active proteins are vital to the next generation of high-throughput multiplexed biosensors and advanced medical diagnostics. We have developed a simple method for fabricating antibody arrays using a micromolded hydrogel “stamper” and an aminosilylated receiving surface. The stamping procedure permits direct protein deposition and micropatterning while avoiding cross-contamination of separate patterned regions. Three different antibodies were stamped in adjacent arrays of 50−80 μm circular areas with retention of activity. 125I labeling and atomic force microscopy studies showed that the stamper deposited protein as a submonolayer. The fluorescent signal-to-background ratio of labeled bound antigen was greater than 25:1.

Bio-Microarray Fabrication Techniques—A Review

“…Hydrogels have also been used as a stamp material for protein patterning. 49 One limitation of elastomeric microstamp fabrication procedure is the necessity of using photolithog-FIGURE 13 Microstamp fabrication. The master is fabricated by photolithography to define the feature of the stamp.…”

“…Likewise, if microstamps are to be reused, the washing process is more tedious than for pins because of non-specific adsorption. 59 Microstamping techniques for biomolecules can be categorized into indirect stamping 40,49,69,80 and the more recently developed direct stamping. 13,30,41,43,70 Both categories utilize similar tools but different stamping procedures.…”

“…The capability of microstamping to yield multiple arrays from a single sample load has also been demonstrated. 36 Martin et al 49 introduced hydrogel polymer poly(6-acryloyl-β -O-methylgalactopyranoside-co-methylenebis-acrylamide) as a stamp material for protein microarray fabrication. The stamp consisted of a narrow capillary with hydrogel inserted at the end of the capillary.…”

“…3,40,49,69,80 Patterned SAMs are generally formed by printing alkanethiol on a gold substrate, 35 which is typically followed by coating the rest of the exposed surface with other SAMs to prevent nonspecific sample binding, as shown in Figure 18. Active chemical groups can also be introduced onto a patterned stamp surface, instead of the substrate, to selectively bind molecules from the ink (i.e., a sample solution).…”

“…49 When forming multiplex microarrays, the entire surface is exposed to a series of solutions containing different molecules/cells to be patterned. Very 84 often, undesired nonspecific molecule adsorption or cross-reaction into a previously patterned region occurs.…”