Hydrogel-Based Protein Microchips: Manufacturing, Properties, and Applications

Abstract

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.

Hydrogel-based protein and oligonucleotide microchips on metal-coated surfaces: Enhancement of fluorescence and optimization of immunoassay

“…In our work we used microchips with three-dimensional hydrogel pads produced via technology based on the photoinduced polymerization immobilization of molecular probes simultaneously with the formation of gel pads on a microchip Rubina et al, 2003Rubina et al, , 2004Rubina et al, , 2005. For the experiments on the assessment of fluorescence enhancement, several sets of biochips were prepared.…”

“…Gel-based microchips were fabricated by copolymerization immobilization method developed earlier Rubina et al, 2003Rubina et al, , 2004Rubina et al, , 2005. Solutions containing gelforming monomers methacrylamide and N-substituted amino sugars together with immobilized proteins or oligonucleotides were spotted onto substrates using a QArray pin robot (Genetix, New Milton, UK).…”

High-throughput and multiplexed protein array technology: protein–DNA and protein–protein interactions

“…Moreover, the low threshold of fluorescence of this type of support leads to a greater sensitivity of detection. The proteins can be placed in solution with polyacrylamide and co-polymerized by UV irradiation [82] or spotted directly onto a layer of commercial hydrogel (http://www.perkin.elmer.com). The immobilized enzymes retain almost 70% of their activity.…”

“…However, porous supports, like nitrocellulose membranes or hydrogel, are equally compatible with crude cell extracts containing non-purified overexpressed proteins [82,87].…”

Quantification of target proteins using hydrogel antibody arrays and MALDI time-of-flight mass spectrometry (A2M2S)

“…Another is nonspecific binding that results in background both on the array itself and the feature. Solutions to remedy this have been the use of PVDF membranes [23] or three-dimensional structures such as agarose [24] or polyacrylamide-gel-based arrays on glass surfaces [20,[25][26][27]. Gel substrates for microarrays have some advantages over twodimensional surfaces such as very high binding capacity, homogeneous aqueous environment of immobilized molecules and absence of contact of immobilized substances with the hydrophobic glass surface frequently used for arrays, which is especially important for proteins.…”