In situ Synthesis of Oligonucleotide Arrays on Surfaces Coated with Crosslinked Polymer Multilayers

Abstract

We report an approach to the in situ synthesis of oligonucleotide arrays on surfaces coated with crosslinked polymer multilayers. Our approach makes use of methods for the ‘reactive’ layer-by-layer assembly of thin, amine-reactive multilayers using branched polyethyleneimine (PEI) and the azlactone-functionalized polymer poly(2-vinyl–4,4’-dimethylazlactone) (PVDMA). Post-fabrication treatment of film-coated glass substrates with d-glucamine or 4-amino-1-butanol yielded hydroxyl-functionalized films suitable for the Maskless Array Synthesis (MAS) of oligonucleotide arrays. Glucamine-functionalized films yielded arrays of oligonucleotides with fluorescence intensities and signal-to-noise ratios (after hybridization with fluorescently labeled complementary strands) comparable to those of arrays fabricated on conventional silanized glass substrates. These arrays could be exposed to multiple hybridization-dehybridization cycles with only moderate loss of hybridization density. The versatility of the layer-by-layer approach also permitted synthesis directly on thin sheets of film-coated poly(ethylene terephthalate) (PET) to yield flexible oligonucleotide arrays that could be readily manipulated (e.g., bent) and cut into smaller arrays. To our knowledge, this work presents the first use of polymer multilayers as a substrate for the multi-step synthesis of complex molecules. Our results demonstrate that these films are robust and able to withstand the ~450 individual chemical processing steps associated with MAS (as well as manipulations required to hybridize, image, and dehybridize the arrays) without large-scale cracking, peeling, or delamination of the thin films. The combination of layer-by-layer assembly and MAS provides a means of fabricating functional oligonucleotide arrays on a range of different materials and substrates. This approach may also prove useful for the fabrication of supports for the solid-phase synthesis and screening of other macromolecular or small-molecule agents.

Covalent Layer‐by‐Layer Assembly Using Reactive Polymers

“…Subsequent work demonstrated the versatility of this azlactone-based approach as a means to coat and modify the interfacial properties of a variety of different materials and more complex substrates, including the surfaces of glass [54][55][56][57][58][59][60]143], soft materials (e.g., polymer-based substrates) [59,61], paper [62], cotton thread and commercial wound dressings (Figure 15.12e,f) [62], protein-based surfaces (e.g., horsehair) [62], inorganic microparticles (for the fabrication of hollow microcapsules Figure 15.12g,h) [63], and, owing to the ability to perform covalent assembly in organic solvents that are immiscible with water, liquid/liquid interfaces formed between organic and aqueous phases (for the fabrication of suspended membranes; Figure 15.12i) [64]. In each of these studies, the presence of amine-reactive azlactone functionality on the surface (or within the bulk) of the films provided a convenient and straightforward platform for further postfabrication modification of surface properties by exposure to a broad range of different amine-functionalized molecules (including amine-functionalized small molecules [54,55,[57][58][59][60][61][62][63], proteins [55,61], peptides [65], functional polymers [61], and amine-functionalized droplets of thermotropic liquid crystals [56]).…”

“…Recently, we demonstrated that cross-linked PEI/PVDMA multilayers can be used as platforms for the multistep maskless array synthesis (MAS) of oligonucleotide arrays (an iterative and automated process based on phosphoramidite chemistry that requires reactive hydroxyl-functionalized surfaces for the initial covalent attachment of nucleoside bases) (Figure 15.12q) [59]. Glucamine-functionalized films fabricated on aminosilane-treated glass substrates supported the synthesis of arrays of oligonucleotides (Figure 15.12r) with fluorescence intensities and S/N ratios (after hybridization with fluorescent complementary strands) that were comparable to those of arrays fabricated on conventional silanized glass.…”

“…We emphasize, however, that this work also serves to highlight again the stable nature of the cross-linked multilayers that arise from this covalent assembly approach. For example, during the MAS array synthesis process, substrates are exposed to over 450 individual chemical processing steps, including both flow-based and static exposure to different organic solvents, base-pair solutions, photoirradiation steps, and oxidation procedures [59]. The results of this study demonstrated that these films did not crack, peel, or delaminate from their underlying substrates significantly during this process or during manipulations required to hybridize and dehybridize the arrays (including prolonged exposure to 8 M urea).…”

“…For example, because the polymers that are used for these (and other) covalent LbL processes are reactive, they can be used to form covalent linkages to appropriately designed substrates to improve film adhesion and prevent film delamination. The resistance to delamination during oligonucleotide synthesis and exposure to solutions of 8 M urea in the MAS-based example [59] above is likely a consequence of bond-forming reactions between PVDMA (deposited as a first layer during LbL assembly) and amine groups on the aminosilane-treated glass substrates used in those experiments. This level of stability is, of course, likely to be useful in a range of contexts and is, in general, something that is more difficult to achieve using ionically cross-linked PEMs fabricated using conventional (noncovalent) LbL methods.…”

“…(b) Post-polymerization reaction of the pendant azlactone functionality with a nucleophilic species (here, a primary amine) yielding a side-chain-functionalized poly(acrylamide)-type polymer. (Reprinted with permission from Ref [59]. (copyright 2012 American Chemical Society) and[143] (reproduced by permission of The Royal Society of Chemistry).…”