Materials Screening for Sol–Gel-Derived High-Density Multi-Kinase Microarrays

Abstract

Protein microarrays based on pin-printing of sol–gel-entrapped biomolecules have emerged as a potential tool to accelerate drug screening and discovery. However, while materials have recently been identified that are suitable for printing of high-density sol–gel-based microarrays, the ability to print arrays of delicate proteins such as kinases, and to assay their activity and inhibition on-array, has yet to be demonstrated. In this study, we have performed a criteria-based directed screen of sol–gel-based materials to identify compositions that are suitable for the fabrication of high-density, multikinase microarrays. Printable formulations were assessed for their compatibility with a fluorescent, phosphospecific dye used as an end-point indicator for on-array kinase assays, including an assessment of the effects of spot size (100 μm vs 400 μm) and slide surface chemistry on signal reproducibility. The combinations of materials, surfaces, and spot sizes that were found to be compatible with reproducible signal generation were evaluated for their ability to retain the activity of a range of kinases, which were co-entrapped with their respective substrates into the optimal sol–gel materials to produce microarrays. Ultimately, two material/surface combinations, from potentially thousands, were identified, one of which was used to produce a robust, highly active kinase microarray that could be used for qualitative screening as well as quantitative inhibition assays.

Alginate/silica hybrid materials for immobilization of green microalgae Chlorella vulgaris for cell-based sensor arrays

“…13 A few studies exist where microarrays of silica gels were directly printed to immobilize proteins [14][15][16][17] and even living cells. 18,19 Nevertheless, it has been shown that pin printing is difficult to perform using traditional sol-gel processing solutions 14 due to severe deposition problems.…”

Contact Pin-Printing onto Porous Silicon for Creating Microarrays with High Chemical Diversity

“…Contact pin-printing is central to several important technologies. [1][2][3][4][5][6][7][8][9][10][11][12][13] For example, contact pin-printing is used to: (i) create high-density DNA or protein microarrays, 1,5,6,9,14,15 (ii) effect high-throughput materials screening campaigns, 2,4,7,11 and (iii) develop chemical sensor arrays. 3,8,13 Thus, because each pin-printed feature (spot) is spatially isolated from its neighbors, contact pinprinting allows one to create, evaluate, and exploit diverse pageants of chemistries simultaneously in a small footprint.…”

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

“…[2][3][4][5] More recently, microarrays have been developed wherein specific materials are used to immobilize functional proteins, producing a three-dimensional microarray element within which proteins are entrapped, allowing for facile measurement of enzymatic activity and inhibition on the microarray itself using a suitable fluorescence assay that is coupled to substrate turnover. [5][6][7][8][9][10] Importantly, such microarrays can be designed to include all necessary components to screen samples and controls together in a highly paralleled fashion. 5,8 Early examples of protein microarrays were typically prepared using standard methods for protein immobilization onto solid supports, such as covalent attachment, 11 affinity capture 3 and physical adsorption.…”

“…[5][6][7][8][9][10] Importantly, such microarrays can be designed to include all necessary components to screen samples and controls together in a highly paralleled fashion. 5,8 Early examples of protein microarrays were typically prepared using standard methods for protein immobilization onto solid supports, such as covalent attachment, 11 affinity capture 3 and physical adsorption. 12 While these methods of bio-immobilization allow for increased sample concentration and accelerated reaction kinetics required for assay miniaturization, they each suffer drawbacks.…”