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Fabrication of oligonucleotide and protein arrays on rigid and flexible substrates coated with reactive polymer multilayers.

ABSTRACT: We report a top-down approach to the fabrication of oligonucleotide and protein arrays on surfaces coated with ultrathin, amine-reactive polymer multilayers fabricated by the covalent "layer-by-layer" (LbL) assembly of polyethyleneimine (PEI) and the amine-reactive, azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA). Manual spotting of amine-terminated oligonucleotide probe sequences on planar glass slides coated with PEI/PVDMA multilayers (~35 nm thick) yielded arrays of immobilized probes that hybridized fluorescently labeled complementary sequences with high signal intensities, high signal-to-noise ratios, and high sequence specificity. Treatment of residual azlactone functionality with the nonfouling small-molecule amine d-glucamine resulted in regions between the features of these arrays that resisted adsorption of protein and permitted hybridization in complex media containing up to 10 mg/mL protein. The residual azlactone groups in these films were also exploited to immobilize proteins on film-coated surfaces and fabricate functional arrays of proteins and enzymes. The ability to deposit PEI/PVDMA multilayers on substrates of arbitrary size, shape, and composition permitted the fabrication of arrays of oligonucleotides on the surfaces of multilayer-coated sheets of poly(ethylene terephthalate) and heat-shrinkable polymer film. Arrays fabricated on these flexible plastic substrates can be bent, cut, resized, and manipulated physically in ways that are difficult using more conventional rigid substrates. This approach could thus contribute to the development of new assay formats and new applications of biomolecule arrays. The methods described here are straightforward to implement, do not require access to specialized equipment, and should also be compatible with automated liquid-handling methods used to fabricate higher-density arrays of oligonucleotides and proteins on more traditional surfaces.

SUBMITTER: Broderick AH

PROVIDER: S-EPMC3553252 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Fabrication of oligonucleotide and protein arrays on rigid and flexible substrates coated with reactive polymer multilayers.

Broderick Adam H AH Carter Matthew C D MC Lockett Matthew R MR Smith Lloyd M LM Lynn David M DM

ACS applied materials & interfaces 20121228 2

We report a top-down approach to the fabrication of oligonucleotide and protein arrays on surfaces coated with ultrathin, amine-reactive polymer multilayers fabricated by the covalent "layer-by-layer" (LbL) assembly of polyethyleneimine (PEI) and the amine-reactive, azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA). Manual spotting of amine-terminated oligonucleotide probe sequences on planar glass slides coated with PEI/PVDMA multilayers (~35 nm thick) yielded arrays ...[more]

PMID: 23237360

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Project description:We report on the fabrication of covalently crosslinked and amine-reactive hollow microcapsules using 'reactive' layer-by-layer assembly to deposit thin polymer films on sacrificial microparticle templates. Our approach is based on the alternating deposition of layers of a synthetic polyamine and a polymer containing reactive azlactone functionality. Multilayered films composed of branched poly(ethylene imine) (BPEI) and poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) were fabricated layer-by-layer on the surfaces of calcium carbonate and glass microparticle templates. After fabrication, these films contained residual azlactone functionality that was accessible for reaction with amine-containing molecules. Dissolution of the calcium carbonate or glass cores using aqueous ethylenediamine tetraacetic acid (EDTA) or hydrofluoric acid (HF), respectively, led to the formation of hollow polymer microcapsules. These microcapsules were robust enough to encapsulate and retain a model macromolecule (FITC-dextran) and were stable for at least 22 hours in high ionic strength environments, in low and high pH solutions, and in several common organic solvents. Significant differences in the behaviors of capsules fabricated on CaCO(3) and glass cores were observed and characterized using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Whereas capsules fabricated on CaCO(3) templates collapsed upon drying, capsules fabricated on glass templates remained rigid and spherical. Characterization using EDS suggested that this latter behavior results, at least in part, from the presence of insoluble metal fluoride salts that are trapped or precipitate within the walls of capsules after etching of the glass cores using HF. Our results demonstrate that the assembly of BPEI/PVDMA films on sacrificial templates can be used to fabricate reactive microcapsules of potential use in a wide range of fields, including catalysis, drug and gene delivery, imaging, and biomedical research.

Project description:The number of people aged 12 years and older using illicit drugs reached 59.3 million in 2020, among which 5.2 million are cocaine users based on the national data. In order to fully understand cocaine addiction and develop effective therapies, a tool is needed to reliably measure real-time cocaine concentration and neural activity in different regions of the brain with high spatial and temporal resolution. Integrated biochemical sensing devices based upon flexible microelectrode arrays (MEA) have emerged as a powerful tool for such purposes; however, MEAs suffer from undesired biofouling and inflammatory reactions, while those with immobilized biologic sensing elements experience additional failures due to biomolecule degradation. Aptasensors are powerful tools for building highly selective sensors for analytes that have been difficult to detect. In this work, DNA aptamer-based electrochemical cocaine sensors were integrated on flexible MEAs and protected with an antifouling zwitterionic poly (sulfobetaine methacrylate) (PSB) coating, in order to prevent sensors from biofouling and degradation by the host tissue. In vitro experiments showed that without the PSB coating, both adsorption of plasma protein albumin and exposure to DNase-1 enzyme have detrimental effects on sensor performance, decreasing signal amplitude and the sensitivity of the sensors. Albumin adsorption caused a 44.4% sensitivity loss, and DNase-1 exposure for 24 hr resulted in a 57.2% sensitivity reduction. The PSB coating successfully protected sensors from albumin fouling and DNase-1 enzyme digestion. In vivo tests showed that the PSB coated MEA aptasensors can detect repeated cocaine infusions in the brain for 3 hrs after implantation without sensitivity degradation. Additionally, the same MEAs can record electrophysiological signals at different tissue depths simultaneously. This novel flexible MEA with integrated cocaine sensors can serve as a valuable tool for understanding the mechanisms of cocaine addiction, while the PSB coating technology can be generalized to improve all implantable devices suffering from biofouling and inflammatory host responses.

Dataset Information

Fabrication of oligonucleotide and protein arrays on rigid and flexible substrates coated with reactive polymer multilayers.

Publications

Fabrication of oligonucleotide and protein arrays on rigid and flexible substrates coated with reactive polymer multilayers.

Similar Datasets

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